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A mutually induced conformational fit underlies Ca2+-directed interactions between calmodulin and the proximal C terminus of KCNQ4 K+ channels

  • Crystal R. Archer
    Affiliations
    From the Departments of Cell and Integrative Physiology, University of Texas Health San Antonio, San Antonio, Texas 78229

    Departments of Biochemistry and Structural Biology, University of Texas Health San Antonio, San Antonio, Texas 78229
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  • Benjamin T. Enslow
    Affiliations
    the Long School of Medicine, University of Texas Health San Antonio, San Antonio, Texas 78229
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  • Alexander B. Taylor
    Affiliations
    Departments of Biochemistry and Structural Biology, University of Texas Health San Antonio, San Antonio, Texas 78229
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  • Author Footnotes
    1 Present address: CONACYT, Facultad de Medicina, Universidad Autónoma de San Luis Potosí 78210, México.
    Victor De la Rosa
    Footnotes
    1 Present address: CONACYT, Facultad de Medicina, Universidad Autónoma de San Luis Potosí 78210, México.
    Affiliations
    From the Departments of Cell and Integrative Physiology, University of Texas Health San Antonio, San Antonio, Texas 78229
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  • Author Footnotes
    2 Present address: Beckman Coulter Life Sciences, 5600 Lindbergh Dr., Loveland, CO 80538.
    Akash Bhattacharya
    Footnotes
    2 Present address: Beckman Coulter Life Sciences, 5600 Lindbergh Dr., Loveland, CO 80538.
    Affiliations
    Departments of Biochemistry and Structural Biology, University of Texas Health San Antonio, San Antonio, Texas 78229
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  • Mark S. Shapiro
    Correspondence
    To whom correspondence should be addressed: Dept. of Cell and Integrative Physiology, University of Texas Health at San Antonio, STRF, MC 8253, 8403 Floyd Curl Dr., San Antonio, TX 78229. Tel.:210-562-4092; Fax:210-562-4060
    Affiliations
    From the Departments of Cell and Integrative Physiology, University of Texas Health San Antonio, San Antonio, Texas 78229
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  • Author Footnotes
    1 Present address: CONACYT, Facultad de Medicina, Universidad Autónoma de San Luis Potosí 78210, México.
    2 Present address: Beckman Coulter Life Sciences, 5600 Lindbergh Dr., Loveland, CO 80538.
    4 The abbreviations used are: CaMcalmodulinHSQCheteronuclear single-quantum coherenceTROSYtransverse relaxation optimized spectroscopyMΩmegaohmsapoCaMnon-Ca2+-loaded CaMVGCCvoltage-gated Ca2+ channelRDregulatory domainPIP2phosphatidylinositol 4,5-bisposphatePDBProtein Data BankHBSHepes-buffered salineRMSDroot mean square deviationITCisothermal titration calorimetryMSTmicroscale thermophoresisCHOChinese hamster ovaryLBLuria broth.
Open AccessPublished:February 26, 2019DOI:https://doi.org/10.1074/jbc.RA118.006857
      Calmodulin (CaM) conveys intracellular Ca2+ signals to KCNQ (Kv7, “M-type”) K+ channels and many other ion channels. Whether this “calmodulation” involves a dramatic structural rearrangement or only slight perturbations of the CaM/KCNQ complex is as yet unclear. A consensus structural model of conformational shifts occurring between low nanomolar and physiologically high intracellular [Ca2+] is still under debate. Here, we used various techniques of biophysical chemical analyses to investigate the interactions between CaM and synthetic peptides corresponding to the A and B domains of the KCNQ4 subtype. We found that in the absence of CaM, the peptides are disordered, whereas Ca2+/CaM imposed helical structure on both KCNQ A and B domains. Isothermal titration calorimetry revealed that Ca2+/CaM has higher affinity for the B domain than for the A domain of KCNQ2–4 and much higher affinity for the B domain when prebound with the A domain. X-ray crystallography confirmed that these discrete peptides spontaneously form a complex with Ca2+/CaM, similar to previous reports of CaM binding KCNQ-AB domains that are linked together. Microscale thermophoresis and heteronuclear single-quantum coherence NMR spectroscopy indicated the C-lobe of Ca2+-free CaM to interact with the KCNQ4 B domain (Kd ∼10–20 μm), with increasing Ca2+ molar ratios shifting the CaM-B domain interactions via only the CaM C-lobe to also include the N-lobe. Our findings suggest that in response to increased Ca2+, CaM undergoes lobe switching that imposes a dramatic mutually induced conformational fit to both the proximal C terminus of KCNQ4 channels and CaM, likely underlying Ca2+-dependent regulation of KCNQ gating.

      Introduction

      Calmodulin (CaM)
      The abbreviations used are: CaM
      calmodulin
      HSQC
      heteronuclear single-quantum coherence
      TROSY
      transverse relaxation optimized spectroscopy
      megaohms
      apoCaM
      non-Ca2+-loaded CaM
      VGCC
      voltage-gated Ca2+ channel
      RD
      regulatory domain
      PIP2
      phosphatidylinositol 4,5-bisposphate
      PDB
      Protein Data Bank
      HBS
      Hepes-buffered saline
      RMSD
      root mean square deviation
      ITC
      isothermal titration calorimetry
      MST
      microscale thermophoresis
      CHO
      Chinese hamster ovary
      LB
      Luria broth.
      is a highly conserved Ca2+ sensor among vertebrates involved in a variety of physiological roles, with at least 300 known binding targets (
      • Faas G.C.
      • Raghavachari S.
      • Lisman J.E.
      • Mody I.
      Calmodulin as a direct detector of Ca2+ signals.
      ,
      • Westerlund A.M.
      • Delemotte L.
      Effect of Ca2+ on the promiscuous target-protein binding of calmodulin.
      ). The N and C termini of CaM form globular clusters called the N-lobe and C-lobe, respectively, which are connected by a flexible linker. Two Ca2+-binding sites localize to each lobe via “EF-hand” motifs with the N-lobe containing EF-I and -II, and the C-lobe EF-III and -IV (
      • Tjandra N.
      • Kuboniwa H.
      • Ren H.
      • Bax A.
      Rotational dynamics of calcium-free calmodulin studied by 15N-NMR relaxation measurements.
      ). For free CaM protein (i.e. CaM not bound to a target protein), the estimated affinity of Ca2+ for the N-lobe is Kd ∼10 μm, and for the C-lobe it is Kd ∼1 μm, affinities that often change when CaM is bound to target proteins (
      • Linse S.
      • Helmersson A.
      • Forsén S.
      Calcium binding to calmodulin and its globular domains.
      ,
      • Zhang M.
      • Tanaka T.
      • Ikura M.
      Calcium-induced conformational transition revealed by the solution structure of apo calmodulin.
      • Zhang P.
      • Tripathi S.
      • Trinh H.
      • Cheung M.S.
      Opposing intermolecular tuning of Ca2+ affinity for calmodulin by neurogranin and CaMKII peptides.
      ). In neurons and other cells, in which global resting (tonic) [Ca2+]i is estimated to be 30–150 nm (
      • Gamper N.
      • Shapiro M.S.
      Calmodulin mediates Ca2+-dependent modulation of M-type K+ channels.
      ,
      • Beech D.J.
      • Bernheim L.
      • Mathie A.
      • Hille B.
      Intracellular Ca2+ buffers disrupt muscarinic suppression of Ca2+ current and M current in rat sympathetic neurons.
      ), non-Ca2+-loaded CaM (apoCaM) is thought to exist in its “off state.” During physiological cytoplasmic increases of [Ca2+]i up to ∼5 μm globally and ∼100 μm in localized nanodomains (
      • Neher E.
      Vesicle pools and Ca2+ microdomains: new tools for understanding their roles in neurotransmitter release.
      ), CaM transitions to an “on-state” that modifies the function of bound target proteins. Solution NMR studies indicate that free apoCaM adopts a semi-closed conformation, in which the N- and C-lobes are usually folded toward each other (
      • Crivici A.
      • Ikura M.
      Molecular and structural basis of target recognition by calmodulin.
      ). Upon Ca2+ loading, the lobes extend in an open conformation, exposing distinct motifs that often direct CaM to wrap around its target proteins to initiate signaling cascades and regulate physiological function in response to Ca2+ signals (
      • Geiser J.R.
      • van Tuinen D.
      • Brockerhoff S.E.
      • Neff M.M.
      • Davis T.N.
      Can calmodulin function without binding calcium?.
      ).
      Since the unexpected revelation that the well-known Ca2+ sensitivity of voltage-gated Ca2+ channels (VGCCs) and SK-type Ca2+-activated K+ channels are due to direct interactions with CaM, without the need for any kinases (
      • Keen J.E.
      • Khawaled R.
      • Farrens D.L.
      • Neelands T.
      • Rivard A.
      • Bond C.T.
      • Janowsky A.
      • Fakler B.
      • Adelman J.P.
      • Maylie J.
      Domains responsible for constitutive and Ca2+-dependent interactions between calmodulin and small conductance Ca2+-activated potassium channels.
      ,
      • Peterson B.Z.
      • DeMaria C.D.
      • Adelman J.P.
      • Yue D.T.
      Calmodulin is the Ca2+ sensor for Ca2+ -dependent inactivation of L-type calcium channels.
      • Zühlke R.D.
      • Pitt G.S.
      • Tsien R.W.
      • Reuter H.
      Ca2+-sensitive inactivation and facilitation of L-type Ca2+ channels both depend on specific amino acid residues in a consensus calmodulin-binding motif in the α1C subunit.
      ), a number of other channels have been shown to be similarly regulated by Ca2+ ions (
      • Clapham D.E.
      Calcium signaling.
      ). Particularly studied for CaM actions on VGCCs is the issue of apoCaM pre-association with the channels and the complex dynamic changes of the configuration of CaM between metal-free and Ca2+-loaded states. These changing configurations often involve “lobe switching” and have proved surprisingly distinct between the CaV1 (L-type) and CaV2 (N- and P/Q-type) channels (
      • Ben-Johny M.
      • Yue D.T.
      Calmodulin regulation (calmodulation) of voltage-gated calcium channels.
      • DeMaria C.D.
      • Soong T.W.
      • Alseikhan B.A.
      • Alvania R.S.
      • Yue D.T.
      Calmodulin bifurcates the local Ca2+ signal that modulates P/Q-type Ca2+ channels.
      ,
      • Erickson M.G.
      • Alseikhan B.A.
      • Peterson B.Z.
      • Yue D.T.
      Preassociation of calmodulin with voltage-gated Ca2+ channels revealed by FRET in single living cells.
      ,
      • Halling D.B.
      • Aracena-Parks P.
      • Hamilton S.L.
      Regulation of voltage-gated Ca2+ channels by calmodulin.
      ,
      • Lee A.
      • Scheuer T.
      • Catterall W.A.
      Ca2+/calmodulin-dependent facilitation and inactivation of P/Q-type Ca2+ channels.
      ,
      • Liang H.
      • DeMaria C.D.
      • Erickson M.G.
      • Mori M.X.
      • Alseikhan B.A.
      • Yue D.T.
      Unified mechanisms of Ca2+ regulation across the Ca2+ channel family.
      • Zamponi G.W.
      Calmodulin lobotomized: novel insights into calcium regulation of voltage-gated calcium channels.
      ). For SK channels, similar issues are being studied, stemming from the surprising early conclusion that the high-affinity C-lobe of CaM is involved in pre-association with the channels (with neither EF-hand in the C-lobe occupied by Ca2+ and the lower-affinity N-lobe acting as the Ca2+ sensor, or “Ca2+ switch,” for gating). Consistent with that earlier structural hypothesis (
      • Keen J.E.
      • Khawaled R.
      • Farrens D.L.
      • Neelands T.
      • Rivard A.
      • Bond C.T.
      • Janowsky A.
      • Fakler B.
      • Adelman J.P.
      • Maylie J.
      Domains responsible for constitutive and Ca2+-dependent interactions between calmodulin and small conductance Ca2+-activated potassium channels.
      ,
      • Xia X.M.
      • Fakler B.
      • Rivard A.
      • Wayman G.
      • Johnson-Pais T.
      • Keen J.E.
      • Ishii T.
      • Hirschberg B.
      • Bond C.T.
      • Lutsenko S.
      • Maylie J.
      • Adelman J.P.
      Mechanism of calcium gating in small-conductance calcium-activated potassium channels.
      ,
      • Schumacher M.A.
      • Rivard A.F.
      • Bächinger H.P.
      • Adelman J.P.
      Structure of the gating domain of a Ca2+-activated K+ channel complexed with Ca2+/calmodulin.
      • Maylie J.
      • Bond C.T.
      • Herson P.S.
      • Lee W.S.
      • Adelman J.P.
      Small conductance Ca2+-activated K+ channels and calmodulin.
      ) is a recent cryo-EM structure of a Ca2+/CaM-bound SK4 channel (
      • Lee C.H.
      • MacKinnon R.
      Activation mechanism of a human SK-calmodulin channel complex elucidated by cryo-EM structures.
      ).
      In neurons, heart, and smooth muscle, tetrameric voltage-gated KCNQ (Kv7, “M-type”) K+ channels play critical roles in regulating cellular excitability (
      • Gamper N.
      • Shapiro M.S.
      KCNQ Channels.
      ). CaM has been shown to regulate the trafficking and expression of KCNQ channels as well as their gating (
      • Etxeberria A.
      • Aivar P.
      • Rodriguez-Alfaro J.A.
      • Alaimo A.
      • Villacé P.
      • Gómez-Posada J.C.
      • Areso P.
      • Villarroel A.
      Calmodulin regulates the trafficking of KCNQ2 potassium channels.
      • Gao Y.
      • Yechikov S.
      • Vázquez A.E.
      • Chen D.
      • Nie L.
      Impaired surface expression and conductance of the KCNQ4 channel lead to sensorineural hearing loss.
      ,
      • Kosenko A.
      • Hoshi N.
      A change in configuration of the calmodulin-KCNQ channel complex underlies Ca2+-dependent modulation of KCNQ channel activity.
      • Liu W.
      • Devaux J.J.
      Calmodulin orchestrates the heteromeric assembly and the trafficking of KCNQ2/3 (Kv7.2/3) channels in neurons.
      ). CaM acts as the Ca2+ sensor for KCNQ channels via direct interactions with the proximal C terminus, thereby mediating the Ca2+-dependent modulatory action of several types of receptors linked to phospholipase C (
      • Gamper N.
      • Shapiro M.S.
      Calmodulin mediates Ca2+-dependent modulation of M-type K+ channels.
      ,
      • Kosenko A.
      • Hoshi N.
      A change in configuration of the calmodulin-KCNQ channel complex underlies Ca2+-dependent modulation of KCNQ channel activity.
      ,
      • Zaika O.
      • Zhang J.
      • Shapiro M.S.
      Combined phosphoinositide and Ca2+ signals mediating receptor specificity toward neuronal Ca2+ channels.
      ,
      • Tobelaim W.S.
      • Dvir M.
      • Lebel G.
      • Cui M.
      • Buki T.
      • Peretz A.
      • Marom M.
      • Haitin Y.
      • Logothetis D.E.
      • Hirsch J.A.
      • Attali B.
      Competition of calcified calmodulin N lobe and PIP2 to an LQT mutation site in Kv7.1 channel.
      ). It has been presumed for KCNQ channels that Ca2+ loading of certain EF-hands of CaM induces a conformational change that inhibits channel opening. For KCNQ1-containing channels, however, Ca2+ loading of CaM augments opening (
      • Shamgar L.
      • Ma L.
      • Schmitt N.
      • Haitin Y.
      • Peretz A.
      • Wiener R.
      • Hirsch J.
      • Pongs O.
      • Attali B.
      Calmodulin is essential for cardiac IKS channel gating and assembly: impaired function in long-QT mutations.
      ), perhaps in accord with their role in cardiomyocytes, inner ear, and epithelia in which KCNQ1 almost always is expressed together with KCNE β-subunits (
      • Peroz D.
      • Rodriguez N.
      • Choveau F.
      • Baró I.
      • Mérot J.
      • Loussouarn G.
      Kv7.1 (KCNQ1) properties and channelopathies.
      ,
      • Rocheleau J.M.
      • Kobertz W.R.
      KCNE peptides differently affect voltage sensor equilibrium and equilibration rates in KCNQ1 K+ channels.
      ). In all cases, the extent and manner of pre-association of apoCaM with the channels and the nature of that conformational change are vigorously under debate.
      It has been argued that apoCaM is required for KCNQ channels to properly function (
      • Etxeberria A.
      • Aivar P.
      • Rodriguez-Alfaro J.A.
      • Alaimo A.
      • Villacé P.
      • Gómez-Posada J.C.
      • Areso P.
      • Villarroel A.
      Calmodulin regulates the trafficking of KCNQ2 potassium channels.
      ,
      • Liu W.
      • Devaux J.J.
      Calmodulin orchestrates the heteromeric assembly and the trafficking of KCNQ2/3 (Kv7.2/3) channels in neurons.
      ,
      • Shamgar L.
      • Ma L.
      • Schmitt N.
      • Haitin Y.
      • Peretz A.
      • Wiener R.
      • Hirsch J.
      • Pongs O.
      • Attali B.
      Calmodulin is essential for cardiac IKS channel gating and assembly: impaired function in long-QT mutations.
      ,
      • Alaimo A.
      • Gómez-Posada J.C.
      • Aivar P.
      • Etxeberría A.
      • Rodriguez-Alfaro J.A.
      • Areso P.
      • Villarroel A.
      Calmodulin activation limits the rate of KCNQ2 K+ channel exit from the endoplasmic reticulum.
      ,
      • Haitin Y.
      • Attali B.
      The C-terminus of Kv7 channels: a multifunctional module.
      • Wen H.
      • Levitan I.B.
      Calmodulin is an auxiliary subunit of KCNQ2/3 potassium channels.
      ), but it is still unclear whether truly metal-free CaM pre-associates with the channels. All studies have shown two highly conserved domains in the proximal C terminus, the A and B domains, as the loci of CaM actions (Fig. 1, A and B). This proximal half of the C terminus, which we call the regulatory domain (RD), besides containing highly conserved A and B domains involved in CaM interactions (
      • Wen H.
      • Levitan I.B.
      Calmodulin is an auxiliary subunit of KCNQ2/3 potassium channels.
      ,
      • Yus-Najera E.
      • Santana-Castro I.
      • Villarroel A.
      The identification and characterization of a noncontinuous calmodulin-binding site in noninactivating voltage-dependent KCNQ potassium channels.
      ), also contains sites of regulation of opening by phosphatidylinositol 4,5-bisphosphate (PIP2) (
      • Hernandez C.C.
      • Zaika O.
      • Shapiro M.S.
      A carboxy-terminal inter-helix linker as the site of phosphatidylinositol 4,5-bisphosphate action on Kv7 (M-type) K+ channels.
      ) and protein kinase C, the regulatory site of the latter being just after the B domain (
      • Higashida H.
      • Hoshi N.
      • Zhang J.S.
      • Yokoyama S.
      • Hashii M.
      • Jin D.
      • Noda M.
      • Robbins J.
      Protein kinase C bound with A-kinase anchoring protein is involved in muscarinic receptor-activated modulation of M-type KCNQ potassium channels.
      ,
      • Hoshi N.
      • Zhang J.S.
      • Omaki M.
      • Takeuchi T.
      • Yokoyama S.
      • Wanaverbecq N.
      • Langeberg L.K.
      • Yoneda Y.
      • Scott J.D.
      • Brown D.A.
      • Higashida H.
      AKaP150 signaling complex promotes suppression of the M current by muscarinic agonists.
      • Kosenko A.
      • Kang S.
      • Smith I.M.
      • Greene D.L.
      • Langeberg L.K.
      • Scott J.D.
      • Hoshi N.
      Coordinated signal integration at the M-type potassium channel upon muscarinic stimulation.
      ). Rich interplay between these molecules at the RD is proposed to exquisitely regulate KCNQ channels.
      Figure thumbnail gr1
      Figure 1KCNQ1–5 contain conserved A and B domains, which are disordered in the absence of CaM. A, representative schematic of a KCNQ protein subunit depicting the S1–S6 transmembrane helices, the intracellular N and C termini, and the A domain (teal) and B domain (gold) within the proximal half of the C terminus, referred to as the RD. The schematic does not show the likely intimate proximity of the RD to the plasma membrane. For reference, the PIP2 interaction sites are shown as gray circles, and the protein kinase C phosphorylation site conserved between KCNQ2–5 is represented by a black circle. B, sequence alignments of the A domain (left) and B domain (right) of human KCNQ1–5 subunits taken from Uniprot. All sequences represent “isoform 1” of each subunit, with the exception of the KCNQ2 sequences that represent the universally used isoform 4 and those of KCNQ4a. Alignments were performed using the PRALINE alignment tool, and the colors were adjusted using Photoshop Elements. The dots above the alignments indicate the interactions with Ca2+/CaM shown in and . Black dots indicate the residues with strong interactions between the KCNQ4 peptides and CaM that are different from those of KCNQ1, and gray dots indicate strong interactions for which the interacting residues are conserved. Open circles indicate those KCNQ4 residues having nonbonded contacts with CaM, and the star indicates the residues of KCNQ1 that may cause changes of the CaM backbone compared with KCNQ4. C, CD spectra of the peptides used in this study and of CaM. Two dashed vertical lines at 208 and 222 nm indicate the points of deflection typical of proteins such as CaM (black) with high helical content. All of the Q2-4 A and B peptides appear to lack helical content in the absence of CaM.
      Recent structural investigations suggest either that Ca2+/CaM embraces both the A and B domains of KCNQ1, -4, and -5 and KCNQ2/3 hybrids (
      • Strulovich R.
      • Tobelaim W.S.
      • Attali B.
      • Hirsch J.A.
      Structural insights into the M channel proximal C-terminus/calmodulin complex.
      ,
      • Sachyani D.
      • Dvir M.
      • Strulovich R.
      • Tria G.
      • Tobelaim W.
      • Peretz A.
      • Pongs O.
      • Svergun D.
      • Attali B.
      • Hirsch J.A.
      Structural basis of a Kv7.1 potassium channel gating module: studies of the intracellular C-terminal domain in complex with calmodulin.
      • Sun J.
      • MacKinnon R.
      Cryo-EM Structure of a KCNQ1/CaM complex reveals insights into congenital long-QT syndrome.
      ) or that Ca2+-loading of CaM induces the A domain to be released from the trimeric complex, leaving Ca2+-loaded CaM to wrap tightly around the B domain alone (
      • Chang A.
      • Abderemane-Ali F.
      • Hura G.L.
      • Rossen N.D.
      • Gate R.E.
      • Minor Jr., D.L.
      A calmodulin C-lobe Ca2+-dependent switch governs Kv7 channel function.
      ,
      • Xu Q.
      • Chang A.
      • Tolia A.
      • Minor Jr., D.L.
      Structure of a Ca2+/CaM:Kv7.4 (KCNQ4) B-helix complex provides insight into M-current modulation.
      ). In contrast, a recent solution NMR study of a similar complex of the A and B domains of KCNQ2 and CaM suggested only minor changes in the structure of the complex between low and high [Ca2+], arguing against a dramatic structural change in KCNQ channels in response to intracellular rises in [Ca2+] (
      • Bernardo-Seisdedos G.
      • Nuñez E.
      • Gomis-Perez C.
      • Malo C.
      • Villarroel Á.
      • Millet O.
      Structural basis and energy landscape for the Ca2+ gating and calmodulation of the Kv7.2 K+ channel.
      ).
      We investigated this issue for KCNQ4, as this isoform is expressed in cells and tissues mainly as homomeric channels, simplifying our interpretations. We used a gamut of biophysical chemical and structural analyses, such as heteronuclear single-quantum coherence NMR spectroscopy (HSQC-NMR) and X-ray crystallography. In our investigations, we used separate synthetic peptides corresponding to the A and B domains of KCNQ2–4 to gain better insight into CaM interactions with the domains independent from each other. We were scrupulously careful to know both the free [Ca2+] and the stoichiometric ratios of [Ca2+], CaM, and A and/or B domains in all of our experiments. The goal of this inquiry was to build a stepwise model of the mechanism of CaM binding to the KCNQ4 C terminus from <10 nm to physiologically high [Ca2+].

      Results

      The A and B domains appear intrinsically disordered in the absence of CaM

      The amino acid sequences of the A and B domains of KCNQ channels are conserved within the KCNQ family (Fig. 1B). The A domain, also known as the “A helix,” contains a highly conserved IQXXR amino acid sequence characteristic of IQ CaM-binding motifs found in a multitude of other CaM target proteins (
      • Houdusse A.
      • Silver M.
      • Cohen C.
      A model of Ca2+-free calmodulin binding to unconventional myosins reveals how calmodulin acts as a regulatory switch.
      ). The A domain also contains 1–12 and 1–16 motifs. The B domain, also called the “B helix,” contains multiple canonical and noncanonical CaM-binding motifs, including 1-5-10 and 1–14 hydrophobic anchoring residues (
      • Mruk K.
      • Farley B.M.
      • Ritacco A.W.
      • Kobertz W.R.
      Calmodulation meta-analysis: predicting calmodulin binding via canonical motif clustering.
      ). Having multiple CaM motifs within the RD suggests that CaM may adopt several different orientations when binding to KCNQ channels, as recently suggested (
      • Bernardo-Seisdedos G.
      • Nuñez E.
      • Gomis-Perez C.
      • Malo C.
      • Villarroel Á.
      • Millet O.
      Structural basis and energy landscape for the Ca2+ gating and calmodulation of the Kv7.2 K+ channel.
      ).
      The existing co-crystal and cryo-EM structures of CaM with purified full channel and purified fragments containing the A and/or B domains show the A and B domains adopting an α-helix secondary structure or assembled in coiled-coil arrangements when embraced by CaM (
      • Strulovich R.
      • Tobelaim W.S.
      • Attali B.
      • Hirsch J.A.
      Structural insights into the M channel proximal C-terminus/calmodulin complex.
      ,
      • Sachyani D.
      • Dvir M.
      • Strulovich R.
      • Tria G.
      • Tobelaim W.
      • Peretz A.
      • Pongs O.
      • Svergun D.
      • Attali B.
      • Hirsch J.A.
      Structural basis of a Kv7.1 potassium channel gating module: studies of the intracellular C-terminal domain in complex with calmodulin.
      • Sun J.
      • MacKinnon R.
      Cryo-EM Structure of a KCNQ1/CaM complex reveals insights into congenital long-QT syndrome.
      ,
      • Xu Q.
      • Chang A.
      • Tolia A.
      • Minor Jr., D.L.
      Structure of a Ca2+/CaM:Kv7.4 (KCNQ4) B-helix complex provides insight into M-current modulation.
      ). However, it is not known whether the free A and B domains of KCNQ channels adopt helical structure in the absence of CaM, despite their common reference as the “A helix” or “B helix.” We used CD spectroscopy to analyze the secondary structure of synthesized peptides corresponding to the A or B domains of KCNQ2–4 subunits. Although peptides typically appear disordered in the absence of a binding partner, CD can indicate whether they adopt secondary structure on their own, such as that corresponding to the NSCate domain of L-type Ca2+ channels, which has α-helical structure in the absence of CaM (
      • Liu Z.
      • Vogel H.J.
      Structural basis for the regulation of L-type voltage-gated calcium channels: interactions between the N-terminal cytoplasmic domain and Ca2+-calmodulin.
      ). Each of the KCNQ peptides displayed single peaks at 205 or 220 nm (Fig. 1C), suggesting that the A and B domains may be intrinsically disordered in the absence of CaM and require CaM to adopt their coiled-coil nature in reported structures. In contrast, CaM displayed dual peaks at 208 and 222 nm, which is the signature for proteins with α-helices. Because the CD buffer contained Ca2+ in equimolar ratio to protein, the CaM in this experiment is expected to only be partially Ca2+-bound. This experiment also suggests that Ca2+ does not directly induce the secondary structure of the A and B domains. To highlight the malleable nature of these CaM-binding domains, we generally call these regions the “A and B domains” in this study. Under “Discussion,” we comment on the importance of these observations in the role of CaM in proper channel expression.

      The independent Q4A and Q4B peptides adopt an anti-parallel α-helical conformation enveloped by CaM in the presence of Ca2+

      To determine with high resolution how CaM and the independent A and B domains of KCNQ4 spontaneously assemble under conditions of high [Ca2+], compared with existing structures of CaM that have been co-expressed with proteins of the connected KCNQ-AB fragments, we obtained the X-ray crystal structure of Ca2+/CaM in complex with KCNQ4 A domain (Q4A) and KCNQ4 B domain (Q4B) peptides. We refer to this structure as Ca2+/CaM:Q4A:Q4B (PDB entry 6N5W) (Fig. 2A). Elongated, hexameric crystals grew to full size after 8 days at room temperature in the presence of 2 mm free [Ca2+]. No crystals were observed in Ca2+-free conditions (in a formation buffer of HBS plus 2 mm EGTA) in our extensive array of crystallization screens. The X-ray structure of Ca2+/CaM:Q4A:Q4B was determined by molecular replacement using the structure of Ca2+/CaM:KCNQ1-AB (PDB entry 4UMO) as a model and refined to a resolution of 2.15 Å (crystallographic statistics found in Table S1). The trimeric structure includes residues 4–147 of CaM, Arg338–Asp356 of Q4A, and Asp525–Phe549 of Q4B. The schematic in Fig. 2B illustrates our understanding of this complex as it exists within the entire KCNQ4 subunit. A common observation of existing structures of Ca2+/CaM with the A and B domains of KCNQ1 and KCNQ4 and of the frog oocyte KCNQXem channel is that Ca2+ is shown coordinated by both EF-hands in the N-lobe but is only occasionally present in EF-hands of the C-lobe. This has been a surprising finding, because, as mentioned above, for free CaM in isolation, the EF-hands in the C-lobe have a higher affinity for Ca2+ than does the N-lobe, although this discrepancy was also seen previously for VGCCs and SK channels (
      • Evans T.I.
      • Hell J.W.
      • Shea M.A.
      Thermodynamic linkage between calmodulin domains binding calcium and contiguous sites in the C-terminal tail of CaV1.2.
      ). Our Ca2+/CaM:Q4A:Q4B crystal structure also shows EF-hands I and II of the CaM N-lobe to each coordinate a Ca2+ ion. EF-III and -IV are empty, despite this complex being formed in a buffer containing an excess of Ca2+ (albeit modest) relative to the number of EF-hands (222 μm CaM and 2 mm Ca2+). The crystallization buffer contained citrate, which also binds Ca2+ but with much lower affinity than CaM (Ka = 103.5 m−1) (
      • Keowmaneechai E.
      • McClements D.J.
      Influence of EDTA and citrate on physicochemical properties of whey protein-stabilized oil-in-water emulsions containing CaCl2.
      ). Citrate has also been shown to directly interact with the C-lobe of CaM, but we do not observe such an interaction in this structure (
      • Neufeld T.
      • Eisenstein M.
      • Muszkat K.A.
      • Fleminger G.
      A citrate-binding site in calmodulin.
      ). We also point out that the Ca2+/CaM:Q1AB structure was not obtained in a high-citrate buffer (
      • Chang A.
      • Abderemane-Ali F.
      • Hura G.L.
      • Rossen N.D.
      • Gate R.E.
      • Minor Jr., D.L.
      A calmodulin C-lobe Ca2+-dependent switch governs Kv7 channel function.
      ) but is very similar to this structure (RMSD = 1.68 Å) and was also found to lack Ca2+ ion density in the C-lobe EF-hands, suggesting this to be more related to [Ca2+] or that Ca2+ ions dissociate from the C-lobe upon complex formation. Contrary to these findings, the Ca2+/CaM:Q3AQ2B structure (PDB entry 5J03) found Ca2+ coordinated in all four EF-hands formed in a crystallization buffer containing a stoichiometric ratio of [Ca2+] to [CaM] of 125:1. Thus, we must admit the likely possibility that a greater stoichiometric excess of Ca2+ to CaM than what we and others have used is required to fully load all “loadable” C-lobe EF-hands under crystallization conditions (
      • Strulovich R.
      • Tobelaim W.S.
      • Attali B.
      • Hirsch J.A.
      Structural insights into the M channel proximal C-terminus/calmodulin complex.
      ). A color plot of crystallographic B-factors of the crystal structure shows the relative level of disorder, and we observe that the EF-hands III and IV map the highest disorder (Fig. S1). This higher potential for disorder in the C-lobe may arise from this trimeric configuration, a result of the C-lobe releasing previously bound Ca2+ ions and rendering EF-hands III and IV less important for stabilizing the structure. The preference of Ca2+ for the N-lobe in this CaM-ion channel co-crystal structure is consistent with the N-lobe, not the C-lobe, being obligatory for CaM function as the Ca2+ sensor that regulates channel opening (
      • Gamper N.
      • Li Y.
      • Shapiro M.S.
      Structural requirements for differential sensitivity of KCNQ K+ channels to modulation by Ca2+/calmodulin.
      ), but as our model in Fig. 10 illustrates, does not mean that the C-lobe is not the first lobe to load Ca2+ ions.
      Figure thumbnail gr2
      Figure 2The X-ray crystal structure of the Ca2+/CaM:Q4A:Q4B complex involves antiparallel A and B helices enveloped by CaM with Ca2+ ions in the N-lobe. A, front view (left) and 90° side view (right) of the trimeric co-crystal X-ray structure of Ca2+/CaM with the Q4A and Q4B peptides. CaM is shown in pink, with the C-lobe facing the bottom, lacking Ca2+ ions, and the N lobe on top, bound by two Ca2+ ions, colored dark gray. Q4B (gold) and Q4A (teal) are embraced together by CaM. B, cartoon schematic depicting the crystal structure representing the overall conformation of Ca2+/CaM bound to a full KCNQ4 subunit. C and D, the expanded views of the CaM:Q4A:Q4B structure show the interior of CaM (gray) interacting with the side chains of the Q4A and Q4B peptides. The peptide residues are colored to match the conserved color plot shown in . These interactions are clarified in the plot in , and dashed circles in the inset highlight the loci of the interactions. CaM residues are labeled in maroon, Q4B is labeled in black, and Q4A is labeled in teal. E, the backbone C-α alignment of the Ca2+/CaM:Q4A:Q4B complex with the Ca2+/CaM:KCNQ1AB complex (light gray) from PDB entry 4UMO, in which one of the two asymmetric units of the domain-swapped pair was truncated for clarity. F, the expanded view of the overlaid structures shows the difference in position of the CaM linker as it interacts with Ile539 (pink CaM) or Arg519 (gray CaM). These structures were rendered using PyMOL.
      Figure thumbnail gr10
      Figure 10Proposed “lobe-switching model” for CaM regulation of neuronal KCNQ channels. Shown schematically is our proposed model of how Ca2+ ions direct CaM in interactions with, and regulation of, neuronal KCNQ channels. We here exclude the likely role of Mg2+ or other ions, as discussed under “Discussion.” 1, under very low (<10 nm) cytosolic [Ca2+] (a physiological state that we cannot determine), apoCaM is prebound to the B domain (gold section of KCNQ subunit), and the NMR and MST data derive a Kd of ∼10–20 μm. During this state, the PIP2 interaction sites within the proximal C terminus at the S6Jx (pre-A helix) and the A-B domain linker are available to interact with PIP2. Under such conditions, the A and B domains are likely disordered, not in a helical conformation, and it is still unclear whether this conformational state would represent a functional channel at the plasma membrane, where PIP2 is located. 2, when [Ca2+]i is in the range of that in cytoplasm in neurons at rest, Ca2+ first binds the EF-hands of the C-lobe (indicated by the change in color from orange to pink), displacing CaM from the B domain. 3, upon a rise in [Ca2+] in the proximity of the channel, the Ca2+-bound C-lobe binds to the A domain with a Kd of ∼400 nm, inducing an α-helical conformation to the A domain (cyan, now shown as a helix). This twisting motion may impose torque on the PIP2 interaction sites in the proximal C terminus, partially weakening their interactions with PIP2. 4, in the final step, under a strong [Ca2+] signal (such as strong stimulation of certain Gq/11-coupled receptors), the EF-hands of the N-lobe become occupied by Ca2+ ions, enhancing its affinity for the B domain, inducing it into a helical formation, retaining C-lobe binding to the A domain (still a helix). This final twisting motion may completely twist or pull away the PIP2 interaction sites from the inner leaflet of the membrane, severely hindering the ability of the C terminus to bind PIP2, resulting in maximal Ca2+/CaM-mediated inhibition of neuronal M channels. The subnanomolar affinity of the CaM:A+B trimeric complex may allow Ca2+ ions to rapidly move in and out of EF-III and -IV while maintaining a stable complex during such elevated [Ca2+] conditions; thus, accounting for crystals variably observed to contain Ca2+ ions in the C-lobe EF-hands.
      Similar to the other published Ca2+/CaM:KCNQ structures, our crystal structure shows the A and B domains adopting helical conformations in the presence of Ca2+-loaded CaM. Taking the CD spectra into account, we suggest that CaM imposes α-helical structure on the A and B domains of KCNQ channels. The structure shows CaM wrapped around the A and B domains, assembled into a coiled-coil configuration in anti-parallel orientation, with the N-lobe bending around the B domain and the C-lobe folded around the A domain (Fig. 2, A–E). Comparison of the CaM backbone alignment between this structure (Protein Data Bank entry 6N5W) and the Ca2+/CaM:KCNQ-AB structures published to date (PDB entries 4UMO, 4V0C, 5J03, 6FEG, 6FEH, 6B8M, 6B8N, and 6B8P) yields an RMSD range of 0.98–1.68 Å overall, with 1196–1226 atoms included in the calculation using PyMOL. An interaction plot summarizes multiple van der Waals (nonbonded contacts) in addition to several hydrogen bonds and salt bridges that hold CaM together with the A and B domains (Fig. S2). The residues involved in these interactions are indicated by circles above the residues in the conserved alignment panel in Fig. 1B. This interaction profile is almost identical, as shown, with that reported previously (
      • Chang A.
      • Abderemane-Ali F.
      • Hura G.L.
      • Rossen N.D.
      • Gate R.E.
      • Minor Jr., D.L.
      A calmodulin C-lobe Ca2+-dependent switch governs Kv7 channel function.
      ) and suggests a high degree of similarity with most of the other structures of Ca2+/CaM interacting with KCNQ1–5. A notable exception is the reported structure of Ca2+/CaM interacting only with the KCNQ4 B domain (
      • Chang A.
      • Abderemane-Ali F.
      • Hura G.L.
      • Rossen N.D.
      • Gate R.E.
      • Minor Jr., D.L.
      A calmodulin C-lobe Ca2+-dependent switch governs Kv7 channel function.
      ,
      • Xu Q.
      • Chang A.
      • Tolia A.
      • Minor Jr., D.L.
      Structure of a Ca2+/CaM:Kv7.4 (KCNQ4) B-helix complex provides insight into M-current modulation.
      ). Expanded representations of the interior of the Ca2+/CaM:Q4A:Q4B co-crystal structure obtained here reveal that these interactions of the A and B domains occur with the two loops within the N- and C-lobes and the N-lobe–C-lobe linker region of CaM (Fig. 2, C and D). We also observed that the more highly conserved residues of A and B domains (pink and orange residues, colored according to the scale in Fig. 1B) tend to face the interior of CaM rather than facing toward each other.
      An overlay of this structure with the KCNQ1-AB domain-swapped complex (PDB entry 4UMO) (Fig. 2E) (RMSD = 1.68 Å) indicates that the only noticeable difference in these interactions localizes to the N-lobe–C-lobe linker of CaM. The expanded view reveals that Ile539 of Q4B allows the CaM linker to have closer contact with the B domain (Fig. 2F). The bulker side chain of the KCNQ1 homolog, Arg519, appears to push the linker further away from the B domain. Whether this small structural difference translates to explaining the functional differences between CaM actions on KCNQ1 and KCNQ4 channels remains to be seen. Overall, these data, featuring the assembly of independent proteins, confirm that the peptides interact with Ca2+/CaM similarly to the co-expressed pre-assembled AB proteins with CaM.

      Ca2+/CaM binds the B domain with higher affinity than the A domain

      We wondered whether there might be an obligate “stepwise” mechanism of complex formation between CaM and the A and B domains. The direct biochemical binding affinity of full-length CaM for each KCNQ domain individually has not been reported, although indirect measurements of apparent affinities indicate that Ca2+/CaM binds the A domain of KCNQ2 in the nanomolar range (
      • Wen H.
      • Levitan I.B.
      Calmodulin is an auxiliary subunit of KCNQ2/3 potassium channels.
      ,
      • Alaimo A.
      • Alberdi A.
      • Gomis-Perez C.
      • Fernández-Orth J.
      • Gómez-Posada J.C.
      • Areso P.
      • Villarroel A.
      Cooperativity between calmodulin-binding sites in Kv7.2 channels.
      ). We used isothermal titration calorimetry (ITC) to assess the thermodynamic parameters of Ca2+/CaM interactions with the A and B domains of KCNQ2–4. Peptides corresponding to the A domains each displayed a moderate binding affinity for Ca2+/CaM, with Kd values of ∼0.44–1.2 μm (Fig. 3 (top) and Table 1). The B domain peptide of KCNQ2 also displayed a moderate affinity for Ca2+/CaM (Kd ∼0.24 μm). The B domains of KCNQ3 and KCNQ4, however, bound to Ca2+/CaM very tightly, with Kd values between 4 and 13 nm (Fig. 3, bottom). The stoichiometry for Ca2+/CaM binding to each peptide was near 1:1, with any differences explainable by remaining uncertainty in the precise peptide concentrations. Although we find here that fully Ca2+-loaded CaM has far greater affinity for the B domain versus the A domain when present alone, when together, Ca2+-loaded CaM must strongly interact with both the A and B domains. Because the crystal structures show that a single CaM embraces the A and B domains together, the very different affinities of the two domains are consistent with there being a specific order to how CaM induces formation of this trimeric complex. This is one of the central advances of this work.
      Figure thumbnail gr3
      Figure 3The B domain of KCNQ2–4 has a very high affinity for Ca2+/CaM, whereas that of the A domain is modest. Isotherms are shown for the peptides (50–100 μm) titrated into 5 mm CaM in the presence of 5 μm Ca2+. The A domain peptides are represented in the top row, and the B domain peptides are shown in the bottom row, representing KCNQ2, KCNQ3, and KCNQ4 isoforms. Analysis was performed using the one-site binding model in MicroCal Origin version 7.
      Table 1Summary of ITC results of KCNQ A and B domain peptides binding Ca2+/CaM
      Figure thumbnail fx1

      Formation of the Ca2+/CaM:Q4A:Q4B complex necessitates a highly ordered mechanism

      Additional ITC experiments were performed to further characterize the interactions between Ca2+/CaM and peptides of the KCNQ4 A and B domains (Q4A and Q4B peptides) simultaneously. First, we tested whether the A and B domains have the ability to interact in the absence of CaM. We did not observe any evidence of binding between the A and B domains using ITC (Fig. 4A), and we also did not observe any crystals formed of only Q4A and Q4B peptides. Both suggest that CaM is necessary to bring these two domains together. Next, we tested whether Ca2+/CaM must bind the A domain or the B domain first to form a stable trimeric complex. We found that Q4A did not display a measurable interaction with a preformed complex of Ca2+/CaM+Q4B (Fig. 4B). Reversing this order, however, showed Q4B to bind to the preformed complex of Ca2+/CaM+Q4A with very high affinity (Kd = 0.5 ± 0.2 nm) (Fig. 4C), even higher than of Q4B binding to Ca2+/CaM alone. This is congruent with the data of Xu et al. (
      • Xu Q.
      • Chang A.
      • Tolia A.
      • Minor Jr., D.L.
      Structure of a Ca2+/CaM:Kv7.4 (KCNQ4) B-helix complex provides insight into M-current modulation.
      ), who suggested that CaM may be required to interact with the A domain first to form the trimeric complex with the B domain and who reported a putative structure of Ca2+/CaM wrapped around the B domain only. Although we conclude that the ultimate configuration of Ca2+-loaded CaM is wrapped around both domains, we cannot rule out the possibility that Ca2+/CaM is wrapped around solely the B domain, under certain conditions that we do not yet know. Combining these data with our above ITC experiments, we suggest that an “energy barrier” must be overcome to form the trimeric complex: Ca2+/CaM first binds the “lower affinity” A domain before binding the “higher affinity” B domain to form the trimeric complex. This would imply that for Ca2+/CaM to form a fully functional complex with both the A and B domains of KCNQ4 channels, a mechanism must be in place to ensure the proper order of binding.
      Figure thumbnail gr4
      Figure 4ITC reveals that the A domain must bind CaM first to form a stable Ca2+/CaM:Q4A:Q4B trimeric complex. A, the shown isotherm demonstrates no detectable binding between the A and B domains in the absence of CaM. B, the plot of Q4A to the preformed complex of Ca2+/CaM+Q4B indicates no detectable interaction. C, the isotherm showing the addition of Q4B to the preformed complex of Ca2+/CaM+Q4A revealed a Kd = 0.5 ± 0.2 nm (mean ± S.D., n = 2). Curve fitting was performed using the competitive model in Origin version 7.

      ApoCaM binds the B domain but not the A domain of KCNQ4

      We were not able to obtain a crystal of apoCaM with the separate KCNQ4 A and B domains in our screens, as mentioned above. Additionally, our ITC data showed the Q4A to display negligible interaction with apoCaM or a preformed complex of apoCaM+Q4B in buffer containing 1 mm EGTA (Fig. S3, A and B). However, we noticed weak interactions between Q4B and apoCaM, characterized by an increase in endothermic heat exchange followed by a decrease in the endothermic profile (Fig. S3C). A similar isotherm was observed for Q4B binding to apoCaM+Q4A (Fig. S3D), indicating that apoCaM might weakly interact only with the B domain and not the A domain. Because we were unable to accurately determine a binding constant from these ITC data due to limited amounts of these proteins, we turned to microscale thermophoresis (MST), which requires a much smaller quantity of protein. We examined the interaction of fluorescently labeled apoCaM (200 nm) with Q4A or Q4B in chelexed buffer (ChHBS) supplemented with 0.5 mm EGTA, ensuring that this low concentration of CaM was wholly free of Ca2+. These experiments revealed that apoCaM interacts with the B domain of KCNQ4 with a Kd of ∼10 μm (confidence interval 6–17 μm; Fig. 5A), which is within the range of free [CaM] in cytoplasm under very low [Ca2+] conditions. Similar to our ITC results, the MST experiments suggest that the A domain does not interact with apoCaM (Fig. 5B).
      Figure thumbnail gr5
      Figure 5MST analysis of apoCaM affinity for Q4B and Q4A peptides. Titration plots are shown at the bottom for Alexa Fluor 594–tagged apoCaM (200 nm), titrated with Q4B (A), up to 220 μm, which displayed a Kd = 10 μm (confidence interval 6–17 μm, n = 3), compared with Q4A (B), which was too weak to determine an accurate equilibrium constant (n = 2). The normalized fluorograms are shown at the top, with the analyzed time points highlighted in light blue and light red, including Tjump + thermophoresis activity in the analysis. Five traces exhibiting high levels of aggregation were excluded. Data were analyzed using PALMIST software, and the figures were created using GUSSI software. Error bars, S.D.

      The C-lobe of the apoCaM is loosely associated with the KCNQ4 B domain

      We used solution HSQC-NMR to track interactions of apoCaM with Q4A and Q4B. Spectroscopy was performed using [15N]apoCaM and Q4A and Q4B in 1 mm EGTA. Peptides were added to a slight excess of 150 μm CaM based on the 1:1 stoichiometry we had observed by ITC under high-[Ca2+] conditions. Fig. 6A shows the NMR spectrum of isolated apoCaM, with expanded regions (Fig. 6, B–D) comparing critical spectral peaks of apoCaM before and after the addition of Q4A and Q4B. This apoCaM spectrum closely matches the assignments of the vertebrate apoCaM spectrum shared by John Putkey (UT Health, Houston TX), further confirming that the starting CaM was indeed in the Ca2+-free state. The addition of Q4A did not cause any chemical shifts or peak alterations to those of isolated apoCaM (Fig. 6B). In contrast, the addition of Q4B induced obvious changes to the apoCaM spectrum, revealing the apoCaM residues affected by interaction with the B domain. We observed significant changes in peaks corresponding to residues Ala88, Asn97, Ile100, and Ser101 and between Asp131 and Glu139, as shown in the expanded region in Fig. 6C and the full spectrum in Fig. S4. The addition of Q4B to the solution containing apoCaM+Q4A (Fig. 6D) caused nearly identical chemical shifts or alterations to the spectral peaks of apoCaM+Q4B, further suggesting that the B domain alone is responsible for interactions with apoCaM. To determine the loci of apoCaM interactions with the B domain, we mapped the residues displaying significant chemical peak alterations observed from the above experiment onto an existing solution NMR structure of apoCaM (PDB entry 1DMO) (
      • Zhang M.
      • Tanaka T.
      • Ikura M.
      Calcium-induced conformational transition revealed by the solution structure of apo calmodulin.
      ). That analysis unequivocally revealed the C-lobe of apoCaM to interact with the B domain of KCNQ4 (Fig. 6E). Specifically, the residues that were most affected by the Q4B titrations localized to EF-hands III and IV. We plotted those significant changes in peak height, or line broadening, to Q4B titrated over a range of concentrations, which yielded Kd = 16 ± 5 μm (Fig. S5). Although peak height is not a widely accepted method to determine equilibrium constants, as line broadening is not a direct report of binding, this result is very similar to that obtained using our MST experiments in Fig. 5, thus confirming by two distinct approaches that apoCaM binds the KCNQ4 B domain with a moderate affinity consistent with known cellular physiology. Based on these results, the schematic in Fig. 6F represents our interpretation of the structural interaction between apoCaM and a single KCNQ4 subunit at resting cytoplasmic [Ca2+]. It suggests a conformation of the apoCaM:KCNQ4 complex quite distinct from our Ca2+-loaded crystal structure (Fig. 2) and others (
      • Strulovich R.
      • Tobelaim W.S.
      • Attali B.
      • Hirsch J.A.
      Structural insights into the M channel proximal C-terminus/calmodulin complex.
      ,
      • Sachyani D.
      • Dvir M.
      • Strulovich R.
      • Tria G.
      • Tobelaim W.
      • Peretz A.
      • Pongs O.
      • Svergun D.
      • Attali B.
      • Hirsch J.A.
      Structural basis of a Kv7.1 potassium channel gating module: studies of the intracellular C-terminal domain in complex with calmodulin.
      ,
      • Bernardo-Seisdedos G.
      • Nuñez E.
      • Gomis-Perez C.
      • Malo C.
      • Villarroel Á.
      • Millet O.
      Structural basis and energy landscape for the Ca2+ gating and calmodulation of the Kv7.2 K+ channel.
      ), of Ca2+/CaM in complex with the A and B domains of KCNQ1–4, and the cryo-EM structure of Ca2+/CaM:KCNQXem (
      • Sun J.
      • MacKinnon R.
      Cryo-EM Structure of a KCNQ1/CaM complex reveals insights into congenital long-QT syndrome.
      ), which all show the B domain interacting with the N-lobe of CaM and the A domain embraced by the C-lobe. These results challenge our initial supposition that apoCaM would be constitutively bound to the A domain and suggest a much more dynamic mechanism of Ca2+ directing CaM interactions with KCNQ4 channels. The implications of these results are discussed below.
      Figure thumbnail gr6
      Figure 6HSQC-NMR analysis shows changes in the apoCaM spectrum when combined with Q4B, but not Q4A. The full spectrum representing 150 μm 15N-labeled apoCaM (in 1 mm EGTA) is shown in A. The spectra in B–D are expanded regions of the boxed region of the full spectrum, comparing apoCaM before (orange spectrum) and after titration with Q4A, Q4B, or both peptides (blue spectra) at a ratio of 1:1.2. E, solution NMR structure of apoCaM (PDB entry 1DMO, conformation 27) with an expanded view of the C lobe. The green, labeled regions represent residues with peak changes greater than 2 S.D. values above the mean peak height after addition of Q4B to apoCaM. F, schematic depicting a possible model of the C lobe of apoCaM (orange), interacting with only the B domain (gold) of a single KCNQ4 subunit, with A and B domains in a nonhelical state.

      Lobe switching of CaM occurs with half-loaded EF-hands

      The above findings imply that a rise in [Ca2+] causes a dramatic lobe switching between CaM and the KCNQ4 A and B domains. To test this hypothesis, we performed HSQC-TROSY experiments over a range of [Ca2+] designed to cover <1% loading of the four EF-hands of CaM to that maximal. To avoid competition between EGTA and CaM for Ca2+, we carefully controlled for [Ca2+], using ChHBS in the absence of EGTA as our starting “zero-Ca2+” buffer. This allows a molar stoichiometry of [Ca2+]/[CaM] of no more than 1:100, resulting in an apoCaM spectrum almost identical to the spectrum of apoCaM in HBS buffer + 1 mm EGTA (Fig. S6). We monitored changes of the emission spectrum of double-labeled, deuterated CaM ([2H-15N]apoCaM) (50 μm), which allows better resolution of the spectrum, requiring lower protein concentration than the single-labeled [15N]apoCaM used in the earlier experiments. The better resolution was necessary to track changes in the CaM and CaM+Q4B spectra over increasing Ca2+ titrations.
      With the first addition of Ca2+ at 1:4 stoichiometry (1 Ca2+ ion/4 EF-hands), we observed obvious changes in the peaks of isolated CaM as the spectra displayed little overlap (Fig. 7 (A–C), green and purple peaks). At higher ratios of Ca2+/EF-hands, more peaks began to overlap due to fewer peak shifts or changes, indicating less response to increased [Ca2+] as the EF-hands became more loaded (Fig. 7 (A–C), purple and orange peaks). This suggests that CaM on its own is very responsive to small rises in [Ca2+]. In contrast, CaM+Q4B displayed much spectral overlap at a 1:4 ratio of Ca2+ to EF-hands, observed as very few changes in the peaks (Fig. 7 (D–F), green and purple peaks). At the point of half-loading of Ca2+ to the EF-hands (2:4 stoichiometry), the CaM+Q4B spectrum displayed a sudden shift, observed by the separation of orange peaks from the purple peaks. A graphical plot is shown summarizing the significant peak changes of CaM and CaM+Q4B with each addition of Ca2+ (Fig. 7G). Overall, we suggest that Q4B changes the relationship between CaM and [Ca2+]. When apoCaM is bound to Q4B, more Ca2+ is needed to cause a change in the configuration of CaM.
      Figure thumbnail gr7
      Figure 7TROSY-HSQC-NMR analysis of the relationship between molar ratio of Ca2+/CaM or Ca2+/CaM+Q4B and alterations in apoCaM residues. Shown is the full NMR spectrum of 50 μm [1H-15N]CaM (A) or 50 μm [1H-15N]CaM + 62.5 μm Q4B (D), in which the green peaks are from residues of the metal-free protein in ChHBS, purple peaks are those upon the addition of 50 μm Ca2+, and orange peaks are from residues upon the addition of 100 μm Ca2+. The labeled boxes in the full spectrum images refer to the expanded regions in which B and C correspond to Ca2+ titrated to CaM only, and E and F are expanded regions of the Ca2+ titrations to CaM+Q4B. Those peaks showing overlapping residues are those that were unaffected by the added Ca2+, indicated in color. In contrast, peaks that do not overlap in color indicate a change in the spectral peak of the corresponding residue with the addition of Ca2+. G, graphical representation of the total number of peak height changes of the single NMR titration >55% above the mean peak height after each titration of Ca2+. Because we could no longer track peaks from the previous titrations at 200 μm, we included all peak changes counted by visual inspection for 200 and 400 μm Ca2+. The box at the bottom right shows the expected molar ratios of Ca2+/CaM EF-hands in each case.
      We were able to track many of the amino acid residues from “zero” Ca2+ (0:4) up to a ratio of 2:4 or 3:4 Ca2+/CaM EF-hands. Although more shifts were observed in the spectra, as previously noted in Fig. 7, only those residues that changed in position >55% from the previous titration, and that we could track from the initial zero [Ca2+] spectrum, are plotted. Fig. 8A shows that those residues localize to both lobes of apoCaM changed over the range of 0:4 to 1:4 Ca2+/CaM EF-hands, and even more residues localized throughout the protein changed at a 2:4 stoichiometry (Fig. 8B).
      Figure thumbnail gr8
      Figure 8Graphical plot of CaM residues displaying changes in spectral peaks over the range [Ca2+] titration series of molar ratios of Ca2+. HSQC-NMR peak heights that changed >55% from the previous titration of Ca2+ to 50 μm [2H-15N]CaM or 50 μm [2H-15N]CaM + 62.5 μm Q4B starting in ChHBS, are represented by the gray or black bars. Only peaks that could be tracked from the original apoCaM position are shown. A and B, significant peak changes of peptide-free CaM in response to Ca2+ at a ratio of 1:4 EF-hands (i.e. 50 μm Ca2+ to 50 μm total CaM in A or 2:4 EF-hands in B). The same analysis was performed for Ca2+ titration into CaM + Q4B ranging up to 150 μm Ca2 (C, D, and E). Cartoon schematics are shown with each plot, to show the estimated stoichiometry of Ca2+ ions (green ovals) with respect to the EF-hands and our speculated movement of CaM (orange dumbbell) with respect to the Q4B peptide (thick gold line). The schematics on the right side of the graph represent metal-free CaM and CaM + Q4B before the addition of Ca2+.
      Interestingly, in the presence of Q4B, only the C-lobe residues of CaM+Q4B displayed peak alterations over a range from 0:4 to 1:4 Ca2+/CaM EF-hands (Fig. 8C). This suggests that Ca2+ binds CaM first to the C-lobe of Q4B-bound CaM. As shown in Fig. 6, the C-lobe interacts with Q4B in the absence of Ca2+, so this finding also suggests that Ca2+ may displace Q4B from the C-lobe. At a 2:4 stoichiometry, residue emissions shifted throughout the CaM protein (Fig. 8D), followed by changes mostly within the N-lobe upon further increases of Ca2+ molar ratio (Fig. 8E). Fig. 8 illustrates our interpretation of the changes of CaM structure in relation to the B domain, based on these NMR data. The coordinated response of CaM to Ca2+, in which responding residues shift from the C-lobe to the N-lobe when Q4B is prebound to apoCaM, again contrasts with the behavior of isolated CaM in response to [Ca2+]. These findings suggest that Ca2+ induces CaM to dissociate completely (although probably extremely briefly) from the KCNQ4 B domain to allow the C-lobe to bind to the KCNQ4 A domain. If true, then this dissociation explains the intermediate step involving the C-lobe of CaM translocating from the B domain under “zero” [Ca2+] to the A domain of KCNQ4 under higher [Ca2+]. We conclude that 1) Ca2+ ions interfere with Q4B interactions with apoCaM; 2) Ca2+ ions initially load the C-lobe of the apoCaM:Q4B complex, instead of the N-lobe; and 3) a highly coordinated sequence of binding-unbinding-rebinding steps occurs over a physiological range of very low to high [Ca2+], which we here refer to as a “lobe-switching mechanism.” We elaborate on this in Fig. 10.

      Ca2+/CaM-mediated inhibition of KCNQ4 channels is not associated with shifts in voltage dependence

      Regarding the CaM-mediated inhibition of KCNQ channel gating by rises in [Ca2+]i, there have been conflicting reports of this action involving shifts of the voltage dependence of activation or changes in gating kinetics. Studying endogenous M current in sympathetic neurons or cloned KCNQ2/3 heteromers or KCNQ2–5 homomers, our group has not ever observed any changes in activation or deactivation kinetics induced by any form of CaM over the full range of physiological [Ca2+]i (
      • Gamper N.
      • Shapiro M.S.
      Calmodulin mediates Ca2+-dependent modulation of M-type K+ channels.
      ,
      • Gamper N.
      • Li Y.
      • Shapiro M.S.
      Structural requirements for differential sensitivity of KCNQ K+ channels to modulation by Ca2+/calmodulin.
      ). There are two alternatively spliced isoforms of human KCNQ4, called KCNQ4a and KCNQ4b, displaying very distinct activation kinetics. Most laboratories, including ours, have used KCNQ4a, which we and others call “KCNQ4.” Xu et al. (
      • Xu T.
      • Nie L.
      • Zhang Y.
      • Mo J.
      • Feng W.
      • Wei D.
      • Petrov E.
      • Calisto L.E.
      • Kachar B.
      • Beisel K.W.
      • Vazquez A.E.
      • Yamoah E.N.
      Roles of alternative splicing in the functional properties of inner ear-specific KCNQ4 channels.
      ) found overexpression with KCNQ4a of a “dominant-negative” CaM, in which all four EF-hands have been mutated to be incapable of loading Ca2+ ions, shifted the voltage dependence of activation by ∼−36 mV. Other laboratories have reported much more modest changes of voltage dependence of ∼10 mV for KCNQ2 or KCNQ3 homomers or KCNQ2/3 heteromers, with only minor changes in kinetics (
      • Kosenko A.
      • Hoshi N.
      A change in configuration of the calmodulin-KCNQ channel complex underlies Ca2+-dependent modulation of KCNQ channel activity.
      ,
      • Shahidullah M.
      • Santarelli L.C.
      • Wen H.
      • Levitan I.B.
      Expression of a calmodulin-binding KCNQ2 potassium channel fragment modulates neuronal M-current and membrane excitability.
      ,
      • Hernandez C.C.
      • Zaika O.
      • Tolstykh G.P.
      • Shapiro M.S.
      Regulation of neural KCNQ channels: signalling pathways, structural motifs and functional implications.
      • Etzioni A.
      • Siloni S.
      • Chikvashvilli D.
      • Strulovich R.
      • Sachyani D.
      • Regev N.
      • Greitzer-Antes D.
      • Hirsch J.A.
      • Lotan I.
      Regulation of neuronal M-channel gating in an isoform-specific manner: functional interplay between calmodulin and syntaxin 1A.
      ). The case of KCNQ1-containing channels is unique in that 1) Ca2+-loaded CaM augments currents from those channels, instead of depressing them (
      • Shamgar L.
      • Ma L.
      • Schmitt N.
      • Haitin Y.
      • Peretz A.
      • Wiener R.
      • Hirsch J.
      • Pongs O.
      • Attali B.
      Calmodulin is essential for cardiac IKS channel gating and assembly: impaired function in long-QT mutations.
      ), and 2) the S2-S3 linker of KCNQ1 is wholly nonconserved with that of KCNQ2–5. Indeed, a cysteine triplet conserved among the latter is the site of reactive oxygen species, whose action dramatically increases the opening of KCNQ2, KCNQ4, and KCNQ2/3, but not KCNQ1-containing channels (
      • Gamper N.
      • Zaika O.
      • Li Y.
      • Martin P.
      • Hernandez C.C.
      • Perez M.R.
      • Wang A.Y.
      • Jaffe D.B.
      • Shapiro M.S.
      Oxidative modification of M-type K+ channels as a mechanism of cytoprotective neuronal silencing.
      ,
      • Linley J.E.
      • Ooi L.
      • Pettinger L.
      • Kirton H.
      • Boyle J.P.
      • Peers C.
      • Gamper N.
      Reactive oxygen species are second messengers of neurokinin signaling in peripheral sensory neurons.
      • Ooi L.
      • Gigout S.
      • Pettinger L.
      • Gamper N.
      Triple cysteine module within M-type K+ channels mediates reciprocal channel modulation by nitric oxide and reactive oxygen species.
      ), whereas the S2-S3 linker of KCNQ1 has been recently suggested to gently engage bound CaM, resulting in shifts in voltage dependence of those channels also by ∼10 mV (
      • Sun J.
      • MacKinnon R.
      Cryo-EM Structure of a KCNQ1/CaM complex reveals insights into congenital long-QT syndrome.
      ). A recent crystal structure of apoCaM with the joined A and B domains of KCNQ4 suggested a very large lobe-specific action of CaM on the voltage dependence of activation of KCNQ4, by >35 mV, and a strong effect on activation kinetics ∼3.4-fold in magnitude (
      • Chang A.
      • Abderemane-Ali F.
      • Hura G.L.
      • Rossen N.D.
      • Gate R.E.
      • Minor Jr., D.L.
      A calmodulin C-lobe Ca2+-dependent switch governs Kv7 channel function.
      ). Because that group coupled such dramatic effects on voltage dependence and kinetics with their suggested structural model and were so divergent from our previous studies (
      • Gamper N.
      • Shapiro M.S.
      Calmodulin mediates Ca2+-dependent modulation of M-type K+ channels.
      ,
      • Gamper N.
      • Li Y.
      • Shapiro M.S.
      Structural requirements for differential sensitivity of KCNQ K+ channels to modulation by Ca2+/calmodulin.
      ), we decided to revisit this issue.
      Chinese hamster ovary (CHO) cells were co-transfected with KCNQ4 and either WT CaM, CaM(1,2), CaM(3,4), or CaM(1,2,3,4), where CaM(1,2) and CaM(3,4) refer to N- or C-lobe mutants that cannot bind Ca2+ ions in that lobe, respectively, and CaM(1,2,3,4) (i.e. D20A/D56A/D93A/D129A) cannot bind Ca2+ in either lobe. All of the constructs, which were the same cDNA as used previously by us (
      • Gamper N.
      • Li Y.
      • Shapiro M.S.
      Structural requirements for differential sensitivity of KCNQ K+ channels to modulation by Ca2+/calmodulin.
      ), were resequenced in their entirety and found to be correct. As before, we performed experiments in the perforated patch variant of whole-cell voltage clamp and, moreover, ensured that the resulting currents were not so large as to either induce series resistance errors or “soak up” all of the PIP2 molecules in the plasma membrane, both of which might cause artifacts in our data. Cell lysates from each group were also immunoblotted with anti-KCNQ4 or anti-CaM antibodies to ensure a reasonable balance of expression of the two proteins. We found that such cells expressed very similar currents from each group, with very similar properties (Fig. 9). For cells co-transfected with KCNQ4 and WT CaM, CaM(1,2), CaM(3,4), or CaM(1,2,3,4), the midpoint voltage of activation (V½) values were −20.3 ± 0.7, −18.1 ± 5.0, −18.2 ± 5.3, and −19.1 ± 1.1 mV, respectively (n = 6, 5, 4, and 5). The activation kinetics at 10 mV were likewise not significantly divergent for cells in each group, having the weighted time constants (see “Experimental procedures”) of 420 ± 145, 395 ± 96, 355 ± 68, and 291 ± 37 ms, respectively. Likewise, there were no significant differences in the deactivation kinetics at −60 mV, which were 70 ± 5, 50 ± 6, 90 ± 15, and 85 ± 6 ms, respectively. Whereas the modest number of cells studied does not rule out minor changes in activation kinetics in the presence of different forms of CaM, any such differences could only be small.
      Figure thumbnail gr9
      Figure 9WT or mutant CaM-lobe mutants do not affect the voltage dependence of KCNQ4 currents. A, representative perforated patch voltage-clamp recordings from CHO cells expressing KCNQ4 channels together with either WT or the indicated CaM mutants. The kinetics of activation at 10 mV and deactivation (inset) at −60 mV after the prepulse were quantified by fits to a double and single exponential, respectively. Fits are shown in gray. B, superimposed are the voltage-dependent activation curves for the KCNQ4 + CaM combinations shown in A, assayed as the amplitude of the tail current at −60 mV after the 500-ms prepulse to the indicated voltages. C, comparison of the activation and deactivation time constant values. D, a table summarizes the data shown in A–C. Overexpression of CaM WT or the indicated CaM mutants did not induce significant changes in the voltage dependence of activation or in the kinetics of activation or deactivation. Error bars, S.E., as these are group data.

      Discussion

      The results from this study highlight several key elements of the dynamics of CaM interaction with the A and B domains of KCNQ2–4. First, we showed that the A and B domains of the channels are likely to be intrinsically disordered in the absence of CaM and that interactions with CaM Ca2+-loaded in at least its N-lobe imposes α-helical secondary structure on those domains. Next, we demonstrated that apoCaM does indeed bind KCNQ4 subunits with moderate affinity with only the C-lobe of apoCaM interacting with the B domain of KCNQ4. Finally, the major advance of this work is our finding that a dramatic lobe switching of CaM interaction with the KCNQ4 A and B domain occurs from low to high [Ca2+].
      Despite our findings that the A and B domains are likely to be disordered in the absence of CaM, the above experiments do not allow us to conclude whether apoCaM induces the helical structure on the B domain, a question planned for future studies. However, the co-crystal structures of KCNQ A-B with CaM all clearly show the A and B domains helical, whether in the presence of divalent cations, such as Mg2+ or Ca2+, or after removal of either divalent from the preformed structure (
      • Chang A.
      • Abderemane-Ali F.
      • Hura G.L.
      • Rossen N.D.
      • Gate R.E.
      • Minor Jr., D.L.
      A calmodulin C-lobe Ca2+-dependent switch governs Kv7 channel function.
      ). This induced conformational change in CaM target proteins has been widely observed, such as for Ca2+/CaM-dependent kinases (
      • Wang Q.
      • Zhang P.
      • Hoffman L.
      • Tripathi S.
      • Homouz D.
      • Liu Y.
      • Waxham M.N.
      • Cheung M.S.
      Protein recognition and selection through conformational and mutually induced fit.
      ) and other ion channels (
      • Liu Z.
      • Vogel H.J.
      Structural basis for the regulation of L-type voltage-gated calcium channels: interactions between the N-terminal cytoplasmic domain and Ca2+-calmodulin.
      ,
      • Wang Q.
      • Zhang P.
      • Hoffman L.
      • Tripathi S.
      • Homouz D.
      • Liu Y.
      • Waxham M.N.
      • Cheung M.S.
      Protein recognition and selection through conformational and mutually induced fit.
      ,
      • Wang C.
      • Chung B.C.
      • Yan H.
      • Wang H.G.
      • Lee S.Y.
      • Pitt G.S.
      Structural analyses of Ca2+/CaM interaction with NaV channel C-termini reveal mechanisms of calcium-dependent regulation.
      • Gabelli S.B.
      • Boto A.
      • Kuhns V.H.
      • Bianchet M.A.
      • Farinelli F.
      • Aripirala S.
      • Yoder J.
      • Jakoncic J.
      • Tomaselli G.F.
      • Amzel L.M.
      Regulation of the NaV1.5 cytoplasmic domain by calmodulin.
      ). Our finding that apoCaM binds the B domain might account for the obligatory need for the presence of CaM reported by several laboratories either for functional expression of KCNQ channels or their assembly in the plasma membrane of either tissue culture cells or neurons (
      • Etxeberria A.
      • Aivar P.
      • Rodriguez-Alfaro J.A.
      • Alaimo A.
      • Villacé P.
      • Gómez-Posada J.C.
      • Areso P.
      • Villarroel A.
      Calmodulin regulates the trafficking of KCNQ2 potassium channels.
      ,
      • Shamgar L.
      • Ma L.
      • Schmitt N.
      • Haitin Y.
      • Peretz A.
      • Wiener R.
      • Hirsch J.
      • Pongs O.
      • Attali B.
      Calmodulin is essential for cardiac IKS channel gating and assembly: impaired function in long-QT mutations.
      ,
      • Alaimo A.
      • Gómez-Posada J.C.
      • Aivar P.
      • Etxeberría A.
      • Rodriguez-Alfaro J.A.
      • Areso P.
      • Villarroel A.
      Calmodulin activation limits the rate of KCNQ2 K+ channel exit from the endoplasmic reticulum.
      ,
      • Wen H.
      • Levitan I.B.
      Calmodulin is an auxiliary subunit of KCNQ2/3 potassium channels.
      ,
      • Shahidullah M.
      • Santarelli L.C.
      • Wen H.
      • Levitan I.B.
      Expression of a calmodulin-binding KCNQ2 potassium channel fragment modulates neuronal M-current and membrane excitability.
      ). The reason likely has to do with the obligatory interactions of all KCNQ channels for interactions with PIP2 in the plasma membrane for function (
      • Falkenburger B.H.
      • Jensen J.B.
      • Dickson E.J.
      • Suh B.C.
      • Hille B.
      Phosphoinositides: lipid regulators of membrane proteins.
      ,
      • Suh B.C.
      • Hille B.
      Regulation of KCNQ channels by manipulation of phosphoinositides.
      ). Given that the two most important domains of the channels for PIP2 interactions are in the proximal C terminus (Fig. 10) (
      • Hernandez C.C.
      • Zaika O.
      • Shapiro M.S.
      A carboxy-terminal inter-helix linker as the site of phosphatidylinositol 4,5-bisphosphate action on Kv7 (M-type) K+ channels.
      ,
      • Sachyani D.
      • Dvir M.
      • Strulovich R.
      • Tria G.
      • Tobelaim W.
      • Peretz A.
      • Pongs O.
      • Svergun D.
      • Attali B.
      • Hirsch J.A.
      Structural basis of a Kv7.1 potassium channel gating module: studies of the intracellular C-terminal domain in complex with calmodulin.
      ,
      • Zaydman M.A.
      • Silva J.R.
      • Delaloye K.
      • Li Y.
      • Liang H.
      • Larsson H.P.
      • Shi J.
      • Cui J.
      Kv7.1 ion channels require a lipid to couple voltage sensing to pore opening.
      ), such a change in structure may prevent the RD from stable interactions with PIP2 (
      • Kosenko A.
      • Hoshi N.
      A change in configuration of the calmodulin-KCNQ channel complex underlies Ca2+-dependent modulation of KCNQ channel activity.
      ), thus hindering opening. Another group has also shown an intricate relationship between channel interactions with PIP2 and CaM (
      • Kosenko A.
      • Kang S.
      • Smith I.M.
      • Greene D.L.
      • Langeberg L.K.
      • Scott J.D.
      • Hoshi N.
      Coordinated signal integration at the M-type potassium channel upon muscarinic stimulation.
      ). However, as opposed to test tube experiments, all cells, especially excitable cells, express CaM at high levels in cytoplasm. As CaM is increasingly Ca2+-loaded in response to increasing [Ca2+]i, we propose that the tight embrace of the A and B domains (now certainly helices) condenses the RD to a springlike structure, pulling it away from the plasma membrane and interfering with those KCNQ-PIP2 interactions, thus causing suppression of M current. We present our model in more detail in Fig. 10.
      To properly interpret our data, we must consider the physiology of CaM molecules and Ca2+ ions in the cytoplasm. Because the affinity of CaM for almost all of its cellular targets greatly increases upon Ca2+ loading (neurogranin being a notable converse example (
      • Zhang P.
      • Tripathi S.
      • Trinh H.
      • Cheung M.S.
      Opposing intermolecular tuning of Ca2+ affinity for calmodulin by neurogranin and CaMKII peptides.
      ,
      • Cui Y.
      • Wen J.
      • Hung Sze K.
      • Man D.
      • Lin D.
      • Liu M.
      • Zhu G.
      Interaction between calcium-free calmodulin and IQ motif of neurogranin studied by nuclear magnetic resonance spectroscopy.
      ,
      • Kaleka K.S.
      • Petersen A.N.
      • Florence M.A.
      • Gerges N.Z.
      Pull-down of calmodulin-binding proteins.
      • Kubota Y.
      • Putkey J.A.
      • Waxham M.N.
      Neurogranin controls the spatiotemporal pattern of postsynaptic Ca2+/CaM signaling.
      )), free [CaM], either Ca2+-bound or not, is exquisitely dependent upon [Ca2+]i, with available CaM ranging as high as perhaps 40 μm at the lowest conceivable values of free [Ca2+] in the cytoplasm, down to <10 nm upon high elevations in global [Ca2+] (
      • Black D.J.
      • Persechini A.
      In calmodulin-IQ domain complexes, the Ca2+-free and Ca2+-bound forms of the calmodulin C-lobe direct the N-lobe to different binding sites.
      ,
      • Black D.J.
      • Tran Q.K.
      • Persechini A.
      Monitoring the total available calmodulin concentration in intact cells over the physiological range in free Ca2+.
      • Tran Q.K.
      • Black D.J.
      • Persechini A.
      Intracellular coupling via limiting calmodulin.
      ), such as in response to strong neuronal stimulation and rapid firing. It is important to note that, unlike the case of CaM actions on VGCCs, for which highly local free [Ca2+] at the inner mouth of the pore may approach millimolar concentrations (
      • Ben-Johny M.
      • Yue D.T.
      Calmodulin regulation (calmodulation) of voltage-gated calcium channels.
      ,
      • Klingauf J.
      • Neher E.
      Modeling buffered Ca2+ diffusion near the membrane: implications for secretion in neuroendocrine cells.
      ,
      • Mori M.X.
      • Erickson M.G.
      • Yue D.T.
      Functional stoichiometry and local enrichment of calmodulin interacting with Ca2+ channels.
      ), no Ca2+ ions are flowing through open K+ channels, and so it is likely that, as for the analysis of CaM actions on SK K+ channels, it is global [Ca2+] that should be most relevant for our thinking. This supposition is tempered, however, by the discovery of KCNQ channels and Ca2+-permeable channels clustered together in microdomains in sensory neurons (
      • Zhang J.
      • Carver C.M.
      • Choveau F.S.
      • Shapiro M.S.
      Clustering and functional coupling of diverse ion channels and signaling proteins revealed by super-resolution STORM microscopy in neurons.
      ), and similar multichannel complexes likely exist in brain as well (
      • Li B.
      • Tadross M.R.
      • Tsien R.W.
      Sequential ionic and conformational signaling by calcium channels drives neuronal gene expression.
      ,
      • Dixon R.E.
      • Yuan C.
      • Cheng E.P.
      • Navedo M.F.
      • Santana L.F.
      Ca2+ signaling amplification by oligomerization of L-type CaV1.2 channels.
      • Vivas O.
      • Moreno C.M.
      • Santana L.F.
      • Hille B.
      Proximal clustering between BK and CaV1.3 channels promotes functional coupling and BK channel activation at low voltage.
      ). Thus, we cannot say with certainty the precise [Ca2+] in the local micro-environment of KCNQ channels in nerve, heart, and muscle that corresponds to CaM being maximally “switched on.”
      Studied in isolation via ITC, we found Ca2+/CaM to have a much higher affinity for the B domains than for the A domains of KCNQ2–4, with KCNQ3 and KCNQ4 most notably so. However, the affinity increased by an order of magnitude when the A domain was present as well. Consistent with those measurements is the high-resolution crystal structure we obtained of Ca2+-loaded CaM wrapped around the A and B domains, very similar to that reported earlier for a variety of KCNQ subtypes (
      • Strulovich R.
      • Tobelaim W.S.
      • Attali B.
      • Hirsch J.A.
      Structural insights into the M channel proximal C-terminus/calmodulin complex.
      ,
      • Sachyani D.
      • Dvir M.
      • Strulovich R.
      • Tria G.
      • Tobelaim W.
      • Peretz A.
      • Pongs O.
      • Svergun D.
      • Attali B.
      • Hirsch J.A.
      Structural basis of a Kv7.1 potassium channel gating module: studies of the intracellular C-terminal domain in complex with calmodulin.
      • Sun J.
      • MacKinnon R.
      Cryo-EM Structure of a KCNQ1/CaM complex reveals insights into congenital long-QT syndrome.
      ,
      • Bernardo-Seisdedos G.
      • Nuñez E.
      • Gomis-Perez C.
      • Malo C.
      • Villarroel Á.
      • Millet O.
      Structural basis and energy landscape for the Ca2+ gating and calmodulation of the Kv7.2 K+ channel.
      ). None of that work suggests a role of CaM in cross-linking the C termini of the subunits in the tetramer, but rather suggests that CaM interacts within individual subunits. A caveat to our conclusions is our ITC results, in which the addition of the A domain to a preformed Ca2+/CaM-B helix complex did not induce a thermodynamic signal; thus, we cannot rule out a configuration of Ca2+/CaM wrapped around the B domain alone under certain cytoplasmic conditions, as suggested by another group (
      • Chang A.
      • Abderemane-Ali F.
      • Hura G.L.
      • Rossen N.D.
      • Gate R.E.
      • Minor Jr., D.L.
      A calmodulin C-lobe Ca2+-dependent switch governs Kv7 channel function.
      ,
      • Xu Q.
      • Chang A.
      • Tolia A.
      • Minor Jr., D.L.
      Structure of a Ca2+/CaM:Kv7.4 (KCNQ4) B-helix complex provides insight into M-current modulation.
      ). It is important to remember that absolute affinities assayed in test tube experiments with isolated A and B domains and CaM molecules are likely much different from those when the RD is attached to the rest of the channel, namely coupled to the gating machinery, as well as the changes in affinities of the EF-hands of CaM for Ca2+ when pre-associated with the relevant domains of the channels (
      • Erickson M.G.
      • Alseikhan B.A.
      • Peterson B.Z.
      • Yue D.T.
      Preassociation of calmodulin with voltage-gated Ca2+ channels revealed by FRET in single living cells.
      ,
      • Zamponi G.W.
      Calmodulin lobotomized: novel insights into calcium regulation of voltage-gated calcium channels.
      ,
      • Evans T.I.
      • Hell J.W.
      • Shea M.A.
      Thermodynamic linkage between calmodulin domains binding calcium and contiguous sites in the C-terminal tail of CaV1.2.
      ). However, we assert that the relative affinities and thermodynamic parameters are likely to parallel our findings in intact cell experiments, giving us insight into conformational changes over ranges of physiological [Ca2+] and [CaM].
      Our observations of the Ca2+ titrations shifting from emission changes in C-lobe residues to those including the N-lobe of CaM prebound to Q4B lead us to propose a lobe-switching model. The issue of lobe dependence and/or specificity for CaM actions (both CDI and CDF) on VGCCs is represented by a vast literature that reveals surprisingly stark differences between L-type (CaV1) and N and P/Q-type (CaV2) Ca2+ channels. For the former, the C-lobe is recognized as the “Ca2+ sensor” provoking CDI, whereas for the latter, the N-lobe has been proposed to fulfill that role (
      • Ben-Johny M.
      • Yue D.T.
      Calmodulin regulation (calmodulation) of voltage-gated calcium channels.
      ). Moreover, for both CaV1 and CaV2 channels, lobe switching has been proposed as a key structural mechanism, involving an N-terminal region of the channels (
      • Dick I.E.
      • Tadross M.R.
      • Liang H.
      • Tay L.H.
      • Yang W.
      • Yue D.T.
      A modular switch for spatial Ca2+ selectivity in the calmodulin regulation of CaV channels.
      ,
      • Ben Johny M.
      • Yang P.S.
      • Bazzazi H.
      • Yue D.T.
      Dynamic switching of calmodulin interactions underlies Ca2+ regulation of CaV1.3 channels.
      ), and it is tempting to think that this CaM-mediated regulatory mechanism is conserved among channel types (
      • Ben-Johny M.
      • Yang P.S.
      • Niu J.
      • Yang W.
      • Joshi-Mukherjee R.
      • Yue D.T.
      Conservation of Ca2+/calmodulin regulation across Na and Ca2+ channels.
      ). Notably, there have not been any structural analyses of CaM/KCNQ channel interactions that include the N terminus of KCNQ channels, which have been suggested to play key roles in gating by interacting with the C terminus, involving syntaxin and CaM (
      • Etzioni A.
      • Siloni S.
      • Chikvashvilli D.
      • Strulovich R.
      • Sachyani D.
      • Regev N.
      • Greitzer-Antes D.
      • Hirsch J.A.
      • Lotan I.
      Regulation of neuronal M-channel gating in an isoform-specific manner: functional interplay between calmodulin and syntaxin 1A.
      ,
      • Siloni S.
      • Singer-Lahat D.
      • Esa M.
      • Tsemakhovich V.
      • Chikvashvili D.
      • Lotan I.
      Regulation of the neuronal KCNQ2 channel by Src–a dual rearrangement of the cytosolic termini underlies bidirectional regulation of gating.
      ). Our thinking also takes into account our earlier work indicating Ca2+ loading of the N-lobe of CaM to be critical for Ca2+/CaM-mediated suppression of KCNQ2, KCNQ4, and KCNQ2/3 channels, with Ca2+ loading of the C-lobe unimportant. That work was performed on living cells expressing full-length, functional channels, studied under perforated patch whole-cell recording (
      • Gamper N.
      • Li Y.
      • Shapiro M.S.
      Structural requirements for differential sensitivity of KCNQ K+ channels to modulation by Ca2+/calmodulin.
      ) rather than only analyses of C-terminal fragments.
      We were unable to obtain a crystal of this or any other “apo” state under metal-free conditions using our crystallization screen of 1,728 conditions by assembling the discrete proteins in the absence of Ca2+. Our apoCaM data contrast with a recent report showing co-expressed (pre-assembled) apoCaM co-crystallized with the AB fragment of KCNQ4 (PDB entry 6B8L) in which Ca2+ had been removed from the preformed complex (
      • Chang A.
      • Abderemane-Ali F.
      • Hura G.L.
      • Rossen N.D.
      • Gate R.E.
      • Minor Jr., D.L.
      A calmodulin C-lobe Ca2+-dependent switch governs Kv7 channel function.
      ) and another showing the solution NMR structure of apoCaM:KCNQ2-AB (PDB code 6FEG) that had been similarly co-expressed and purified (
      • Bernardo-Seisdedos G.
      • Nuñez E.
      • Gomis-Perez C.
      • Malo C.
      • Villarroel Á.
      • Millet O.
      Structural basis and energy landscape for the Ca2+ gating and calmodulation of the Kv7.2 K+ channel.
      ). Aside from their absence of Ca2+ ions, these “apo” structures are still quite similar to that found for the Ca2+/CaM:KCNQ-AB complexes (
      • Strulovich R.
      • Tobelaim W.S.
      • Attali B.
      • Hirsch J.A.
      Structural insights into the M channel proximal C-terminus/calmodulin complex.
      ,
      • Sachyani D.
      • Dvir M.
      • Strulovich R.
      • Tria G.
      • Tobelaim W.
      • Peretz A.
      • Pongs O.
      • Svergun D.
      • Attali B.
      • Hirsch J.A.
      Structural basis of a Kv7.1 potassium channel gating module: studies of the intracellular C-terminal domain in complex with calmodulin.
      • Sun J.
      • MacKinnon R.
      Cryo-EM Structure of a KCNQ1/CaM complex reveals insights into congenital long-QT syndrome.
      ). We note that the primary difference between these apo structures and the apo model we propose is in how the complex was assembled; in this study, we combined each Ca2+-free protein/peptide separately with the crystallization buffer, whereas all others had been co-expressed and preassembled in a divalent cation-rich medium (LB/2YT) prior to removal of the Ca2+ ions with EGTA. We think this suggests that the complex itself is more stable than the transient Ca2+ state of the CaM-EF-hands.
      Millet and colleagues (
      • Bernardo-Seisdedos G.
      • Nuñez E.
      • Gomis-Perez C.
      • Malo C.
      • Villarroel Á.
      • Millet O.
      Structural basis and energy landscape for the Ca2+ gating and calmodulation of the Kv7.2 K+ channel.
      ) called their structure “intermediate,” with regard to metal, consistent with this complex not being wholly Ca2+-free, and reported that complete unloading of all Ca2+ from the preformed complexes was difficult. Under those circumstances, any further loading of Ca2+ ions to the complex displayed a Kd value using FRET assays of ∼1 μm (
      • Bernardo-Seisdedos G.
      • Nuñez E.
      • Gomis-Perez C.
      • Malo C.
      • Villarroel Á.
      • Millet O.
      Structural basis and energy landscape for the Ca2+ gating and calmodulation of the Kv7.2 K+ channel.
      ), a value similar to that globally in the cytoplasm of neurons in response to a stimulus. Our solution NMR and MST experiments suggest that the C-lobe of apoCaM weakly interacts with the KCNQ4 B domain under Ca2+ conditions at which at most 1% of the CaM EF-hands could be Ca2+-loaded, and at extremely low [Ca2+]i, free CaM may be within that range in the cytoplasm (
      • Black D.J.
      • Tran Q.K.
      • Persechini A.
      Monitoring the total available calmodulin concentration in intact cells over the physiological range in free Ca2+.
      ,
      • Tran Q.K.
      • Black D.J.
      • Persechini A.
      Intracellular coupling via limiting calmodulin.
      ). It is unclear what physiological condition would correspond to such a scarcity of Ca2+ ions relative to CaM molecules, but under those conditions, we found the C-lobe of CaM to interact solely with the B domain of KCNQ4. Titration of Ca2+ into the apoCaM+Q4B complex measured by NMR suggests that Ca2+ must reach a stoichiometry of 1:4 (EF-hands) to displace the B domain from the C-lobe. Taken together, our results can only be reconciled by a profound change in the configuration of the KCNQ4 RD when CaM is half-loaded by Ca2+ (two of the four EF-hands bound by Ca2+), under which the Ca2+-loaded N-lobe now strongly interacts with the B domain with very high affinity. This configuration seems to be particularly stable, as consistently found by all investigators.
      Like many studies, we here ignore the significant concentration of free Mg2+ ions, estimated to be ∼0.5–1 mm in neurons, and given that the known affinities of Mg2+ ions for the CaM EF-hands are within that range, predict significant occupancy of CaM EF-hands at tonic [Ca2+]i (
      • Grabarek Z.
      Insights into modulation of calcium signaling by magnesium in calmodulin, troponin C and related EF-hand proteins.
      ,
      • Senguen F.T.
      • Grabarek Z.
      X-ray structures of magnesium and manganese complexes with the N-terminal domain of calmodulin: insights into the mechanism and specificity of metal ion binding to an EF-hand.
      • Malmendal A.
      • Linse S.
      • Evenäs J.
      • Forsén S.
      • Drakenberg T.
      Battle for the EF-hands: magnesium-calcium interference in calmodulin.
      ). Thus, it is likely that in resting cells at which free [Ca2+] is low, some or all of the EF-hands of CaM are not empty but rather are occupied by Mg2+ ions. The high-resolution crystal structure of the Mg2+-loaded N-lobe of CaM reveals Mg2+ ions in both EF-hands, but in contrast with Ca2+ occupancy, 2–4 H2O molecules are also included in each EF-hand (
      • Senguen F.T.
      • Grabarek Z.
      X-ray structures of magnesium and manganese complexes with the N-terminal domain of calmodulin: insights into the mechanism and specificity of metal ion binding to an EF-hand.
      ), which could electrostatically shield much of the charge of divalent ions and perhaps alter interactions of the RD with the membrane. The occupancy by CaM by Mg2+ ions when CaM is “off” was explicitly assumed for the solved CaM/NaV1.2/FGFHF complex (
      • Gabelli S.B.
      • Yoder J.B.
      • Tomaselli G.F.
      • Amzel L.M.
      Calmodulin and Ca2+ control of voltage gated Na+ channels.
      ) and recently examined for CaM/KCNQ4 RD interactions (
      • Chang A.
      • Abderemane-Ali F.
      • Hura G.L.
      • Rossen N.D.
      • Gate R.E.
      • Minor Jr., D.L.
      A calmodulin C-lobe Ca2+-dependent switch governs Kv7 channel function.
      ). This raises the likelihood of “alkali earth-metal exchange” being part of the switching of CaM “on” or “off” in its functional interactions with the channels. This topic has also been explored for the case of synaptotagmin, whose structure when loaded with Mg2+ (under which exocytosis is inhibited) has also been determined (
      • Sutton R.B.
      • Ernst J.A.
      • Brunger A.T.
      Crystal structure of the cytosolic C2A-C2B domains of synaptotagmin III: implications for Ca2+-independent snare complex interaction.
      ,
      • Zhou Q.
      • Lai Y.
      • Bacaj T.
      • Zhao M.
      • Lyubimov A.Y.
      • Uervirojnangkoorn M.
      • Zeldin O.B.
      • Brewster A.S.
      • Sauter N.K.
      • Cohen A.E.
      • Soltis S.M.
      • Alonso-Mori R.
      • Chollet M.
      • Lemke H.T.
      • Pfuetzner R.A.
      • et al.
      Architecture of the synaptotagmin-SNARE machinery for neuronal exocytosis.
      ). In all of these cases, the structure of the CaM/target complex was suggested to be only subtly distinct between Mg2+-loaded and Ca2+-loaded forms. How these results can be in accord with the indisputable role of Ca2+ as the “switch” for CaM actions on these proteins remains to be elucidated.
      Based on our results, we propose a “lobe-switching model” in which Ca2+ ions compete with the B domain for binding the C-lobe under low [Ca2+], causing the B domain to dissociate from CaM in a stepwise CaM action on KCNQ4 channels (Fig. 10). The true “apo” configuration (Fig. 10, 1) can only occur during extremely low (<10 nm) [Ca2+], and we do not know whether and when such a low value occurs in the cytoplasm of living cells, which contain up to 1 mm free [Mg2+]. Nonetheless, in this state, the C-lobe of apoCaM binds the B domain with modest affinity, and the N-lobe of CaM and the A domain of the channel are not involved. Because the RD is in a flexible, “open” configuration, we suppose it could be anchored to the plasma membrane via the two PIP2-binding sites located on both ends of the A domain (the “S6Jx” or “pre-A helix” site and the linker between the A and B domain). In this state, both the A and B domains are likely still disordered and nonhelical. It is unclear whether the channel can function in this fully “apoCaM” state. At local free [Ca2+] near resting levels in excitable cells (50–100 nm), the complex rapidly adopts a CaM displacement configuration, in which the B domain dissociates from the C-lobe and CaM is reconfigured to a “target-ready” state that can recognize the A domain (Fig. 10, 2). This brief state then likely quickly transitions to the CaM rebinding configuration (Fig. 10, 3), as a result of Ca2+-loaded C-lobe (half-loaded CaM) binding to the A domain, imposing an α-helical structure on the A domain and condensing the proximal C terminus. In the first three of these configurations, the RD is relatively relaxed, allowing the C terminus to extend the two proposed PIP2-binding sites, the “S6Jx” or “pre-A helix,” and the “A-B linker,” to interact with PIP2 at the plasma membrane–cytoplasm interface.
      We take note, however, that our earlier work found exogenous expression of mutant C-lobe CaM that cannot bind Ca2+ ions to not perturb Ca2+-mediated suppression of KCNQ2, KCNQ4, and KCNQ2/3, channels expressed in CHO cells (
      • Gamper N.
      • Li Y.
      • Shapiro M.S.
      Structural requirements for differential sensitivity of KCNQ K+ channels to modulation by Ca2+/calmodulin.
      ). How can that be reconciled with our data and model here, which require Ca2+ ions in the C-lobe in steps 1 and 3 in Fig. 10? We think the answer lies in the extremely high affinity of Ca2+ ions for the C-lobe, and the presence of endogenous CaM in nearly all mammalian cells, with even more so in neurons. Thus, KCNQ channels already assembled in the Golgi (with the C termini in the cytoplasm) are pre-bound by endogenous CaM with Ca2+ ions in the C-lobe and do not lose those ions. Overexpression of C-lobe mutant CaM is very much higher than expression of endogenous CaM. However, no inhibition of neuronal KCNQ channels can occur without the Ca2+/N lobe-dependent embrace of the A and B helices, which is what pulls the RD away from the plasma membrane, disrupting the obligatory interactions between PIP2 and the two PIP2 interaction sites in the proximal C terminus of the channels that are required for channel opening.
      We hypothesize that upon a physiological signal that causes a substantial rise in free [Ca2+]i in the vicinity of the “primed” CaM/RD complex, the triplex configuration occurs (Fig. 10, 4), as Ca2+ binds the N-lobe of CaM, causing it to latch around the B domain. This induces a pulling/torsional Ca2+-induced motion, as suggested recently (
      • Bernardo-Seisdedos G.
      • Nuñez E.
      • Gomis-Perez C.
      • Malo C.
      • Villarroel Á.
      • Millet O.
      Structural basis and energy landscape for the Ca2+ gating and calmodulation of the Kv7.2 K+ channel.
      ), likely disrupting the critical interactions between the RD and PIP2, causing inhibition of channel gating for neuronal M channels. We note that this “condensed” trimolecular configuration, as opposed to the “open” structure of free Ca2+-loaded CaM, is due to its intimate interaction with the A and B helices, with which its many bonds and sites of interaction provide the energetics for the resultant tight configuration of CaM. Thus, we suggest here that both the A and B helices and CaM rearrange their secondary and tertiary structures in response to each other, similar to the “mutually induced fit” already noted for CaM interactions with Ca2+/CaM kinases (
      • Wang Q.
      • Zhang P.
      • Hoffman L.
      • Tripathi S.
      • Homouz D.
      • Liu Y.
      • Waxham M.N.
      • Cheung M.S.
      Protein recognition and selection through conformational and mutually induced fit.
      ). This insight we believe to represent another advance of this paper.
      Finally, with a rise in [Ca2+] that is more than transient, this configuration is “locked,” and the double anti-parallel helical conformation of the complex is stabilized, creating a compact geometry that severely disrupts PIP2 interactions with KCNQ C termini that likely takes some minutes to reverse, as seen physiologically. Although Ca2+ must be bound to the C-lobe first to displace CaM from the B domain, it is Ca2+ loading of the N-lobe that creates this highly stable trimeric complex, which allows freedom within EF-hands III and IV of the C-lobe, rendering their ligation of Ca2+ optional at this final step. Our lobe-switching model could provide the explanation for the discrepancies reported in the literature of how Ca2+ directs CaM in its binding to, and regulation of, KCNQ channels, as it does incorporate some role of metal loading of the C-lobe as (an early) part of the mechanism (
      • Chang A.
      • Abderemane-Ali F.
      • Hura G.L.
      • Rossen N.D.
      • Gate R.E.
      • Minor Jr., D.L.
      A calmodulin C-lobe Ca2+-dependent switch governs Kv7 channel function.
      ) but retains Ca2+ binding to the N-lobe as the modulatory switch, in accord with our physiological experiments in cells (
      • Gamper N.
      • Li Y.
      • Shapiro M.S.
      Structural requirements for differential sensitivity of KCNQ K+ channels to modulation by Ca2+/calmodulin.
      ). It also is in accord with the need for CaM for functional expression (
      • Etxeberria A.
      • Aivar P.
      • Rodriguez-Alfaro J.A.
      • Alaimo A.
      • Villacé P.
      • Gómez-Posada J.C.
      • Areso P.
      • Villarroel A.
      Calmodulin regulates the trafficking of KCNQ2 potassium channels.
      ,
      • Wen H.
      • Levitan I.B.
      Calmodulin is an auxiliary subunit of KCNQ2/3 potassium channels.
      ,
      • Alaimo A.
      • Alberdi A.
      • Gomis-Perez C.
      • Fernández-Orth J.
      • Gómez-Posada J.C.
      • Areso P.
      • Villarroel A.
      Cooperativity between calmodulin-binding sites in Kv7.2 channels.
      ). However, we do not believe that CaM interacts with the voltage-sensor domain of KCNQ2–5 channels and that as for PIP2 actions on those channels (
      • Choveau F.S.
      • De la Rosa V.
      • Bierbower S.M.
      • Hernandez C.C.
      • Shapiro M.S.
      Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates KCNQ3 K+ channels through multiple sites of action.
      ), effects on voltage dependence are minimal. Future studies to test the affinities of the peptides with inactive N- or C-lobe CaM mutants and these domains will further probe whether this lobe-switching mechanism is indeed correct.

      Experimental procedures

      Buffers and protein preparation

      Peptides corresponding to the A and B domains of KCNQ2–4 were synthesized to 95% purity (Peptide 2.0, Chantilly, VA). The lyophilized peptides were reconstituted in HBS buffer, which consists of 20 mm HEPES and 150 mm NaCl at pH 7.4, made with deionized water. We name each peptide according to the subunit isoform and the CaM binding domain: Q2A, Q2B, Q3A, Q3B, Q4A and Q4B, referring to the A or B domains of KCNQ2–4, respectively. A plasmid containing WT, untagged vertebrate CaM (plasmid pETGQ.HCaM) was a gift from William N. Zagotta (University of Washington). Vertebrate CaM was expressed in BL21 competent cells at 37 °C for 6 h in LB or minimal essential medium containing [15N]ammonium chloride, depending on the experimental design, and then purified using phenyl-Sepharose matrix (GE Healthcare). For experiments requiring higher resolution of NMR spectra we expressed in D2O (Millipore-Sigma). The eluent was further purified through a Superdex 75 column on an AKTA FPLC system (GE Healthcare). For divalent metal-free CaM (apoCaM) studies, the protein was exchanged to HBS buffer that had been soaked with Chelex reagent (Bio-Rad). We refer to this chelexed buffer as “ChHBS.” Glassware and other containers used for apoCaM measurements were prerinsed with 10 mm EGTA, followed by 2× ChHBS rinses, prior to the addition of proteins. The total [Ca2+] in the ChHBS buffer was determined by inductively coupled plasma MS to be ∼500 nm (Northwestern University, Evanston, IL). The peptides used for apoCaM interactions were reconstituted in ChHBS. The concentrations of the peptides and CaM were determined by amino acid analysis at the Texas A&M University Protein Chemistry Laboratory core (College Station, TX).

      CD

      Proteins were diluted to ∼30 μm in potassium phosphate buffer (KH2PO4), pH 7.4, containing 5 mm NaCl and ∼5–30 μm Ca2+ (determined by inductively coupled plasma MS and fluorescence spectroscopy using Ca2+ indicator dyes) and placed in a 0.5-cm path length quartz cuvette. The molar ellipticity of each peptide was reported using a Jasco J-810 CD spectrometer at 4 °C. CD spectra were recorded from 270 to 190 nm in 0.1-nm steps. The CD signals were corrected by subtracting the spectra from buffer only. The CD analysis plotting tool, CAPITO, was used to determine the predicted content of α-helices, β-sheets, and random coils of the indicated peptides and proteins (
      • Wiedemann C.
      • Bellstedt P.
      • Görlach M.
      CAPITO–a web server-based analysis and plotting tool for circular dichroism data.
      ).

      ITC

      ITC titrations were performed at 25 or 37 °C using a VP-ITC microcalorimeter (MicroCal/Malvern Instruments). Titrations were conducted in HBS buffer supplemented with 1 mm EGTA or 0.5 mm CaCl2. Samples were degassed for at least 15 min. 5–10 μm CaM was placed in the ITC cell, and 50–200 μm peptide was added to the titration syringe. Each ITC experiment consisted of at least 24 injections of 10 μl of titrant, preceded by one 2-μl injection, which is traditionally used to “prime” the system for ligand diffusion during the temperature equilibration between the syringe and cell contents (
      • Dutta A.K.
      • Rösgen J.
      • Rajarathnam K.
      Erratum: using isothermal titration calorimetry to determine thermodynamic parameters of protein-glycosaminoglycan interactions.
      ). Data were analyzed with MicroCal Origin version 7.0, using the built-in curve-fitting models.

      Crystallization, structure determination, and refinement

      WT CaM purified in-house (see above) was mixed with Q4A in an equimolar ratio at room temperature, in HBS buffer supplemented with 2 mm CaCl2 or 2 mm EGTA, and Q4B was added at an equimolar ratio 30 min later. The final concentration of each protein was 222 μm. Automated screening for crystallization was carried out using the sitting drop vapor-diffusion method with an Art Robbins Instruments Phoenix system in the X-ray Crystallography Core Laboratory at UT Health San Antonio. Crystals of Ca2+-loaded complexes were initially obtained from Microlytic MCSG-III screen condition 60; optimized in 1.3 m sodium citrate, 0.1 m HEPES, pH 7.0; and flash-cooled in liquid nitrogen prior to data collection. Data for two crystals were collected at the Advanced Photon Source NE-CAT beamline 24-ID-E and integrated and scaled together using XDS (
      • Kabsch W.
      XDS.
      ). The structure of Ca2+/CaM:Q4A:Q4B was determined by the molecular replacement method implemented in PHASER (
      • McCoy A.J.
      • Grosse-Kunstleve R.W.
      • Adams P.D.
      • Winn M.D.
      • Storoni L.C.
      • Read R.J.
      Phaser crystallographic software.
      ) using a truncated version of PDB entry 4UMO as the search model. Coordinates were refined using PHENIX (
      • Adams P.D.
      • Afonine P.V.
      • Bunkóczi G.
      • Chen V.B.
      • Davis I.W.
      • Echols N.
      • Headd J.J.
      • Hung L.W.
      • Kapral G.J.
      • Grosse-Kunstleve R.W.
      • McCoy A.J.
      • Moriarty N.W.
      • Oeffner R.
      • Read R.J.
      • Richardson D.C.
      • et al.
      PHENIX: a comprehensive Python-based system for macromolecular structure solution.
      ), including simulated annealing with torsion angle dynamics and TLS refinement, alternated with manual rebuilding using COOT (
      • Emsley P.
      • Lohkamp B.
      • Scott W.G.
      • Cowtan K.
      Features and development of Coot.
      ). The model was verified using composite omit map analysis (
      • Afonine P.V.
      • Grosse-Kunstleve R.W.
      • Echols N.
      • Headd J.J.
      • Moriarty N.W.
      • Mustyakimov M.
      • Terwilliger T.C.
      • Urzhumtsev A.
      • Zwart P.H.
      • Adams P.D.
      Towards automated crystallographic structure refinement with phenix.refine.
      ,
      • Terwilliger T.C.
      • Grosse-Kunstleve R.W.
      • Afonine P.V.
      • Moriarty N.W.
      • Adams P.D.
      • Read R.J.
      • Zwart P.H.
      • Hung L.W.
      Iterative-build OMIT maps: map improvement by iterative model building and refinement without model bias.
      ) to minimize model bias. Data collection and refinement statistics are shown in Table S1. Renderings of the structures were performed using PyMOL software (PyMOL Molecular Graphics System, version 2.0, Schrödinger, LLC).

      MST

      apoCaM in ChHBS was conjugated with Alexa Fluor 594 (Alexa-CaM) using a protein-labeling kit (Invitrogen). Q4B or Q4A peptides were serially diluted 1:2 in ChHBS containing an additional 0.5 mm EGTA. The final concentration of Alexa-CaM in each well of the dilution series was 200 nm, and each sample was centrifuged prior to loading to standard capillaries. After capturing the proteins in 16 different capillaries, fluorescence was measured in a Nanotemper Monolith 1.115, using an excitation power of 60 and MST power of 80. Microscale thermophoresis was recorded using the MO control software, and the binding affinities were analyzed using PALMIST and GUSSI (Chad Brautigam, UT Southwestern, Dallas, TX) (
      • Brautigam C.A.
      Calculations and publication-quality illustrations for analytical ultracentrifugation data.
      ,
      • Scheuermann T.H.
      • Padrick S.B.
      • Gardner K.H.
      • Brautigam C.A.
      On the acquisition and analysis of microscale thermophoresis data.
      ).

      HSQC-NMR

      NMR titrations of [15N]CaM with unlabeled Q4A and Q4B peptides were performed using HSQC (non-TROSY) experiments. All non-TROSY experiments were conducted in HBS, 1 mm EGTA, and 10% (v/v) D2O at 298 K on a Bruker Avance 700 NMR spectrometer. NMR titrations of deuterated [2H-15N]CaM with Ca2+ were performed in ChHBS using TROSY-HSQC to reduce line broadening by cancelling dipole–dipole coupling and chemical shift anisotropy, thus producing well-resolved spectra. The methods were performed similarly to those described elsewhere (
      • Pervushin K.
      • Riek R.
      • Wider G.
      • Wüthrich K.
      Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution.
      ,
      • Biris N.
      • Tomashevski A.
      • Bhattacharya A.
      • Diaz-Griffero F.
      • Ivanov D.N.
      Rhesus monkey TRIM5α SPRY domain recognizes multiple epitopes that span several capsid monomers on the surface of the HIV-1 mature viral core.
      ).
      The assignments for mammalian apoCaM were shared with us by John Putkey (UT Health, Houston, TX), and the assignments for Ca2+/CaM were kindly provided by Walter Chazin (Vanderbilt University, Nashville, TN) and Adriaan Bax (National Institutes of Health, Bethesdsa, MD). The raw spectrometer format data were processed using nmrPipe and nmrDraw (
      • Delaglio F.
      • Grzesiek S.
      • Vuister G.W.
      • Zhu G.
      • Pfeifer J.
      • Bax A.
      NMRPipe: a multidimensional spectral processing system based on UNIX pipes.
      ). The peaks were calculated and visualized using SPARKY version 3.115 software (T. D. Goddard and D. G. Kneller, University of California, San Francisco), and in some cases, the overlays were formatted using Adobe Illustrator.
      Estimation of equilibrium constants of apoCaM for the KCNQ4 B domain were evaluated by NMR titration data as follows. The peak heights of the titration spectra were normalized against the reference spectrum without titrant, followed by calculation of the mean and S.D. of the normalized peak heights after titration. Only those residues showing normalized peak heights at least 2 × S.D. above the mean were plotted. Using these peak data, the Kd values were determined using GraphPad Prism version 7, by fitting those data to the binding equation, y = Bmax·x/(Kd + x) + NS·x + background, in the one-site–total nonlinear model. Although plotting peak height (line broadening) is not a widely accepted or accurate method for determining affinities, we used this method as an approximation to guide us in further experimental approaches for testing binding affinities.

      Perforated patch-clamp electrophysiology

      CHO cells were grown in 100-mm tissue culture dishes (Falcon, Franklin Lakes, NJ) in Dulbecco's modified Eagle's medium with 10% heat-inactivated fetal bovine serum plus 0.1% penicillin/streptomycin in a humidified incubator at 37 °C (5% CO2) and passaged every 4 days. Cells were discarded after ∼30 passages. CHO cells were first passaged onto 35-mm plastic tissue culture dishes and transfected 24 h later with FuGENE HD reagent (Promega), according to the manufacturer's instructions. The total amount of cDNA used was 0.55 μg, which is less than that typical but required for such high-expressing channels like KCNQ4 (
      • Hernandez C.C.
      • Zaika O.
      • Shapiro M.S.
      A carboxy-terminal inter-helix linker as the site of phosphatidylinositol 4,5-bisphosphate action on Kv7 (M-type) K+ channels.
      ), which otherwise might “soak-up” all of the PIP2 in the membrane, allowing artifactual interactions between CaM and the channels that are unphysiological. The next day, cells were plated onto coverglass chips, and experiments were performed over the following 1–2 days. Pipettes were pulled from borosilicate glass capillaries (BF150-86-10HP; Sutter Instruments) using a Flaming/Brown micropipette puller P-97 (Sutter Instruments) and had resistances of 2–3 MΩ when filled with internal solution and measured in standard bath solution. The external Ringer's solution contained 160 mm NaCl, 5 mm KCl, 2 mm CaCl2, 1 mm MgCl2, and 10 mm HEPES, pH 7.4, with NaOH. The pipette solution contained 160 mm KCl, 5 mm MgCl2, and 10 mm HEPES, pH 7.4, with KOH with added amphotericin B.
      Membrane current was measured with pipette and membrane capacitance cancellation, sampled at 5 ms, and filtered at 1 kHz using an EPC10 amplifier and Patchmaster software (HEKA). In all experiments, the perforated patch method of recording was used with amphotericin B (600 ng/ml) in the pipette solution (
      • Rae J.
      • Cooper K.
      • Gates P.
      • Watsky M.
      Low access resistance perforated patch recordings using amphotericin B.
      ). Amphotericin was prepared as a stock solution as 60 mg/ml in DMSO. In these experiments, the access resistance was typically 7–15 MΩ, 5–10 min after seal formation. Series resistance compensation was routinely compensated ∼60%, and liquid junction potential corrections (<2 mV in this case) were not applied. Cells were placed in a 500-μl perfusion chamber through which solution flowed at 1–2 ml/min. Inflow to the chamber was by gravity from several reservoirs, selectable by activation of solenoid valves (Warner Scientific). Bath solution exchange was essentially complete by <30 s. Experiments were performed at room temperature. Cells that displayed KCNQ4 currents over 1 nA at 0 mV were not studied, nor were experiments accepted if the uncorrected series resistance was >5 MΩ, due to undue sequestering of free [PIP2] or to residual series resistance voltage errors. Currents were studied by holding the membrane potential at −80 mV and applying 500-ms depolarizing pulses from −80 to 40 mV, followed by a 400-ms step to −60 mV, every 3 s. To estimate voltage dependence, tail current amplitudes at −60 mV were fit to a single exponential starting at a time ∼5–10 ms after the repolarization (when the residual capacity transient has subsided), and the amplitudes were normalized and plotted as a function of test potential. The data were fit with Boltzmann relations of the form, I/Imax = Imax/(1 + exp((V½V)/k)), where Imax is the maximum tail current, V½ is the voltage that produces half-maximal activation of the conductance, and k is the slope factor. Values from cell populations were compared using a two-tailed t test. In such group comparisons, the use of S.E. values is most statistically correct. The activation kinetics were estimated using a weighted double-exponential fit (A·exp(−τ/t1) + B·exp(−τ/t2)) of the first 400 ms of the currents, where τ1 and τ2 are the fast and slow time constants and A and B are the “weights.” Thus, the weighted deactivation time constants reported are given by (A·τ1 + B·τ2)/(A + B). The deactivation time constants were measured by fitting the deactivating current to a single exponential at −60 mV, as described above.

      Author contributions

      C. R. A., A. B. T., and M. S. S. conceptualization; C. R. A., A. B. T., V. D. l. R., A. B., and M. S. S. formal analysis; C. R. A. and M. S. S. funding acquisition; C. R. A., B. T. E., A. B. T., A. B., and M. S. S. validation; C. R. A., B. T. E., A. B. T., V. D. l. R., A. B., and M. S. S. investigation; C. R. A., A. B. T., V. D. l. R., and A. B. visualization; C. R. A., B. T. E., A. B. T., and V. D. l. R. writing-original draft; C. R. A., A. B., and M. S. S. writing-review and editing; B. T. E., A. B. T., A. B., and M. S. S. data curation; B. T. E., A. B. T., A. B., and M. S. S. methodology; A. B. T., A. B., and M. S. S. resources; A. B. T. and A. B. software; M. S. S. supervision and project administration.

      Acknowledgments

      We thank Yinghua Chen (Case Western Reserve University, Cleveland, OH) for preparing the Alexa Fluor 594 proteins and providing expertise in MST analysis and Pamela Reed, MaryAnn Hobbs, and Isamar Sanchez for expert technical support. We are indebted to the following facilities of the Institutional Research Cores at UT Health San Antonio in the Department of Biochemistry: the Center for Macromolecular Interactions, directed by Drs. Eileen Lafer and Bo Demeler; the X-ray Crystallography Core Laboratory, directed by Drs. P. John Hart and Alex Taylor; and the NMR spectroscopy core, directed by Dr. Dmitri Ivanov. Institutional Research Cores at UT Health San Antonio are supported by the Office of the Vice President for Research and the Mays Cancer Center, the center home to the UT Health San Antonio MD Anderson Cancer Center (National Institutes of Health Grant P30 CA054174). We also gratefully thank Drs. Eileen Lafer, Bo Demeler, and Dmitri Ivanov (Department of Biochemistry and Structural Biology), Robert Brenner (Department of Cell and Integrative Physiology), Brad Rothberg (Temple University), Chad Brautigam (UT Southwestern), Sudha Chakrapani (Case Western Reserve University), and William N. Zagotta (University of Washington) for many helpful discussions regarding this project. We are further indebted to James D. Stockand for providing the necessary time for C. R. A. to finish the manuscript.

      Supplementary Material

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