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CaV channels reject signaling from a second CaM in eliciting Ca2+-dependent feedback regulation

Open AccessPublished:August 20, 2020DOI:https://doi.org/10.1074/jbc.RA120.013777
      Calmodulin (CaM) regulation of voltage-gated calcium (CaV1-2) channels is a powerful Ca2+-feedback mechanism to adjust channel activity in response to Ca2+ influx. Despite progress in resolving mechanisms of CaM-CaV feedback, the stoichiometry of CaM interaction with CaV channels remains ambiguous. Functional studies that tethered CaM to CaV1.2 suggested that a single CaM sufficed for Ca2+ feedback, yet biochemical, FRET, and structural studies showed that multiple CaM molecules interact with distinct interfaces within channel cytosolic segments, suggesting that functional Ca2+ regulation may be more nuanced. Resolving this ambiguity is critical as CaM is enriched in subcellular domains where CaV channels reside, such as the cardiac dyad. We here localized multiple CaMs to the CaV nanodomain by tethering either WT or mutant CaM that lack Ca2+-binding capacity to the pore-forming α-subunit of CaV1.2, CaV1.3, and CaV2.1 and/or the auxiliary β2A subunit. We observed that a single CaM tethered to either the α or β2A subunit tunes Ca2+ regulation of CaV channels. However, when multiple CaMs are localized concurrently, CaV channels preferentially respond to signaling from the α-subunit–tethered CaM. Mechanistically, the introduction of a second IQ domain to the CaV1.3 carboxyl tail switched the apparent functional stoichiometry, permitting two CaMs to mediate functional regulation. In all, Ca2+ feedback of CaV channels depends exquisitely on a single CaM preassociated with the α-subunit carboxyl tail. Additional CaMs that colocalize with the channel complex are unable to trigger Ca2+-dependent feedback of channel gating but may support alternate regulatory functions.
      Calmodulin (CaM) regulation of high-voltage activated calcium channels (CaV1-2) is a dynamic feedback modulation that sculpts calcium entry into neurons and cardiac myocytes (
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      ) channels, hinting that insights from CaV modulation may shed light on common regulatory mechanisms.
      Indeed, progress over the past three decades has revealed core mechanistic details regarding CaV calmodulation (
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      ). First, CaM tunes multiple aspects of CaV1 and CaV2 function, including channel gating (
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      γCaMKII shuttles Ca2+/CaM to the nucleus to trigger CREB phosphorylation and gene expression.
      ). Second, to elicit gating changes, Ca2+-free CaM pore-forming α1 subunit with the pore-forming channel subunit (
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      ). Following channel activation, permeant Ca2+ ions bind CaM, and ensuing conformational rearrangements (
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      Identification of the components controlling inactivation of voltage-gated Ca2+ channels.
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      ) trigger Ca2+-dependent feedback modulation. For many CaV1 and CaV2 channels, this conformational change diminishes channel activity, a process termed Ca2+-dependent inactivation (CDI) (
      • Zühlke R.D.
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      Calmodulin supports both inactivation and facilitation of L-type calcium channels.
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      • Lin T.
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      ,
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      Calmodulin bifurcates the local Ca2+ signal that modulates P/Q-type Ca2+ channels.
      ). For CaV2.1, however, this modulatory process can also enhance channel activity, a positive feedback known as Ca2+-dependent facilitation (CDF) (
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      Ca2+/calmodulin-dependent facilitation and inactivation of P/Q-type Ca2+ channels.
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      ,
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      ). Third, multiple channel domains that interact with CaM have been identified. Specifically, Ca2+-free CaM preassociates with a canonical CaM-binding IQ motif localized to the channel carboxyl tail (CT) and the closely juxtaposed EF-hand segment (
      • Black D.J.
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      Calmodulin interactions with IQ peptides from voltage-dependent calcium channels.
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      ,
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      FRET two-hybrid mapping reveals function and location of L-type Ca2+ channel CaM preassociation.
      ). Ca2+/CaM, on the other hand, interacts with multiple domains, including the IQ domain (
      • Zühlke R.D.
      • Pitt G.S.
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      • Reuter H.
      Calmodulin supports both inactivation and facilitation of L-type calcium channels.
      ,
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      • Minor Jr., D.L.
      Insights into voltage-gated calcium channel regulation from the structure of the CaV1.2 IQ domain-Ca2+/calmodulin complex.
      ,
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      ,
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      ,
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      A modular switch for spatial Ca2+ selectivity in the calmodulin regulation of CaV channels.
      ,
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      ,
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      Modulation of L-type Ca2+ channels by Gβγ and calmodulin via interactions with N and C termini of α1C.
      ), and a CaM-binding domain (CBD) distal to the CaV2.1 IQ domain (
      • Lee A.
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      • Catterall W.A.
      Molecular determinants of Ca2+/calmodulin-dependent regulation of Cav2.1 channels.
      ). Fourth, CaM signaling to the CaV channel is ultimately conveyed to a selectivity filter gate (
      • Abderemane-Ali F.
      • Findeisen F.
      • Rossen N.D.
      • Minor Jr., D.L.
      A selectivity filter gate controls voltage-gated calcium channel calcium-dependent inactivation.
      ).
      Despite these advances, one uncertainty pertains to the stoichiometry of CaM interaction with the CaV complex. Early functional studies that tethered CaM onto the CT of the pore-forming α1 subunit suggested that a single CaM is both necessary and sufficient for CaV1 regulation (
      • Mori M.X.
      • Erickson M.G.
      • Yue D.T.
      Functional stoichiometry and local enrichment of calmodulin interacting with Ca2+ channels.
      ). However, biochemical and structural analysis point to the binding of multiple CaM molecules within the CaV complex. Briefly, atomic structures of the CaV1.2 and CaV2.1 CT show Ca2+/CaM interaction with the IQ domain (
      • Van Petegem F.
      • Chatelain F.C.
      • Minor Jr., D.L.
      Insights into voltage-gated calcium channel regulation from the structure of the CaV1.2 IQ domain-Ca2+/calmodulin complex.
      ,
      • Fallon J.L.
      • Halling D.B.
      • Hamilton S.L.
      • Quiocho F.A.
      Structure of calmodulin bound to the hydrophobic IQ domain of the cardiac Cav1.2 calcium channel.
      ,
      • Mori M.X.
      • Vander Kooi C.W.
      • Leahy D.J.
      • Yue D.T.
      Crystal structure of the CaV2 IQ domain in complex with Ca2+/calmodulin: high-resolution mechanistic implications for channel regulation by Ca2+.
      ), as well as two pre-IQ domains cross-bridged by two additional Ca2+/CaM molecules (
      • Fallon J.L.
      • Baker M.R.
      • Xiong L.
      • Loy R.E.
      • Yang G.
      • Dirksen R.T.
      • Hamilton S.L.
      • Quiocho F.A.
      Crystal structure of dimeric cardiac L-type calcium channel regulatory domains bridged by Ca2+ calmodulins.
      ,
      • Mori M.X.
      • Vander Kooi C.W.
      • Leahy D.J.
      • Yue D.T.
      Crystal structure of the CaV2 IQ domain in complex with Ca2+/calmodulin: high-resolution mechanistic implications for channel regulation by Ca2+.
      ). For CaV1.2, NMR structures show the binding conformation of Ca2+/CaM to the CaV1.2 NSCaTE domain (
      • 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.
      ). It remains unknown whether a single CaM molecule switches between conformations (
      • Taiakina V.
      • Boone A.N.
      • Fux J.
      • Senatore A.
      • Weber-Adrian D.
      • Guillemette J.G.
      • Spafford J.D.
      The calmodulin-binding, short linear motif, NSCaTE is conserved in L-type channel ancestors of vertebrate Cav1.2 and Cav1.3 channels.
      ) or whether multiple CaM molecules engage distinct sites (
      • Benmocha Guggenheimer A.
      • Almagor L.
      • Tsemakhovich V.
      • Tripathy D.R.
      • Hirsch J.A.
      • Dascal N.
      Interactions between N and C termini of α1C subunit regulate inactivation of CaV1.2 L-type Ca2+ channel.
      ) to orchestrate channel regulation. This mechanistic ambiguity is biologically important as CaM is enriched in subcellular regions, such as the cardiac dyad where CaV1 channels also reside (
      • Yang Y.
      • Guo T.
      • Oda T.
      • Chakraborty A.
      • Chen L.
      • Uchinoumi H.
      • Knowlton A.A.
      • Fruen B.R.
      • Cornea R.L.
      • Meissner G.
      • Bers D.M.
      Cardiac myocyte Z-line calmodulin is mainly RyR2-bound, and reduction is arrhythmogenic and occurs in heart failure.
      ). In vitro analysis suggests that Ca2+/CaM is not capable of bridging the aforementioned channel domains (
      • Benmocha Guggenheimer A.
      • Almagor L.
      • Tsemakhovich V.
      • Tripathy D.R.
      • Hirsch J.A.
      • Dascal N.
      Interactions between N and C termini of α1C subunit regulate inactivation of CaV1.2 L-type Ca2+ channel.
      ,
      • Benmocha A.
      • Almagor L.
      • Oz S.
      • Hirsch J.A.
      • Dascal N.
      Characterization of the calmodulin-binding site in the N terminus of CaV1.2.
      ). Furthermore, previous FRET-based analysis of CaM stoichiometry showed that whereas a single apo-CaM preassociates with the holo-CaV1.2 channel, in the presence of Ca2+, up to two CaMs can bind to the holo-channel complex (
      • Ben-Johny M.
      • Yue D.N.
      • Yue D.T.
      Detecting stoichiometry of macromolecular complexes in live cells using FRET.
      ). Given this ambiguity, we here sought to dissect the potential role of multiple CaMs in eliciting Ca2+-dependent modulation of CaV channel gating by tethering mutant or WT CaM to distinct locations within the channel complex. We found that CaM linked to the channel CT is privileged in eliciting Ca2+ regulation of CaV channels. Furthermore, when additional Ca2+/CaMs are present in the channel complex, signaling by these molecules is rejected by the channel pore domain with regard to dynamic Ca2+-feedback modulation.

      Results

      Strategy for probing effects of multiple CaM in tuning CaV1 CDI

      To dissect the potential functional contribution of multiple CaM in evoking CDI of CaV1.2, we localized either WT (CaMWT) or mutant CaM that lacks Ca2+ binding (CaM1234) with known stoichiometries through genetic fusion to either the pore-forming α1C subunit
      α1C, α1D, and α1A denote CaV1.2, CaV1.3, and CaV2.1, respectively.
      or the auxiliary β2A subunit (
      • Mori M.X.
      • Erickson M.G.
      • Yue D.T.
      Functional stoichiometry and local enrichment of calmodulin interacting with Ca2+ channels.
      ,
      • Bazzazi H.
      • Ben Johny M.
      • Adams P.J.
      • Soong T.W.
      • Yue D.T.
      Continuously tunable Ca2+ regulation of RNA-edited CaV1.3 channels.
      ) (Fig. 1A). Here, CaM1234 mutant is generated by alanine substitution of key Ca2+-coordinating aspartate residues in all four EF-hand domains of CaM. This overall strategy allows us to localize either one or two CaM molecules to the channel complex and assess changes in CDI. Fig. 1B shows baseline extent of CDI for full-length CaV1.2 in the absence of CaM fusion to either the α1C or β2A subunits. In response to a step-voltage depolarization to +10 mV, Ca2+ current decay (red) is accelerated compared with Ba2+ current (black). Population data shows the fraction of peak current remaining after a 300-ms depolarization (r300) with either Ca2+ (red) or Ba2+ (black) as the charge carrier. As Ba2+ binds poorly to CaM (
      • Chao S.H.
      • Suzuki Y.
      • Zysk J.R.
      • Cheung W.Y.
      Activation of calmodulin by various metal cations as a function of ionic radius.
      ), the Ba2+ relation provides a baseline measure of voltage-dependent inactivation (VDI). The magnitude of CDI is quantified as the fractional difference between r300 relations obtained with Ca2+ and Ba2+ as permeant ions (i.e. CDI300 = 1 – r300,Ca/r300,Ba) (Table 1). Further representative currents and current-voltage relationships are provided in Fig. S1A. Previous studies have demonstrated that fusion of CaMWT to the carboxyl terminus of the truncated α1CΔ1671 subunit preserves strong CDI, whereas tethering CaM1234 to the same location abolishes CDI, suggesting that CaM fusion to the channel carboxyl terminus preserves modulatory function and permits interaction with key effector interface on the channel (
      • Mori M.X.
      • Erickson M.G.
      • Yue D.T.
      Functional stoichiometry and local enrichment of calmodulin interacting with Ca2+ channels.
      ,
      • Yang P.S.
      • Johny M.B.
      • Yue D.T.
      Allostery in Ca2+ channel modulation by calcium-binding proteins.
      ). As such, we confirmed that truncation of the distal carboxyl tail does not appreciably alter CDI (Fig. 1C, Fig. S1B, and Table 1). We further validated that α1CΔ1671-CaMWT supported strong CDI (Fig. 1D and Fig. S1C), whereas α1CΔ1671-CaM1234 abolished CDI (Fig. 1E and Fig. S1D). To determine whether genetic fusion of CaM to the β2A subunit similarly supports CaV1.2 regulation, we tethered CaMWT and CaM1234 onto the β2A subunit, yielding β2A-CaMWT or β2A-CaM1234, respectively. Notably, the β2A subunit binds to the α1 subunit with a high affinity and 1:1 stoichiometry (
      • Dalton S.
      • Takahashi S.X.
      • Miriyala J.
      • Colecraft H.M.
      A single CaVβ can reconstitute both trafficking and macroscopic conductance of voltage-dependent calcium channels.
      ,
      • Wu J.
      • Yan Z.
      • Li Z.
      • Qian X.
      • Lu S.
      • Dong M.
      • Zhou Q.
      • Yan N.
      Structure of the voltage-gated calcium channel Cav1.1 at 3.6 Å resolution.
      ), and this subunit is obligatory for channel function in HEK293 cells. As such, we co-expressed α1C with either β2A-CaMWT or β2A-CaM1234. β2A-CaMWT co-expression with α1C subunit preserved strong CDI (Fig. 1F, Fig. S1E, and Table 1) similar to control conditions. By contrast, co-expression of β2A-CaM1234 abolished CDI (Fig. 1G, Fig. S1F, and Table 1). Thus, CaM tethered to the β2A subunit is also capable of binding to critical channel effector motifs and eliciting functional regulation.
      Figure thumbnail gr1
      Figure 1CaM tethered to either CaV1.2 α- or β-subunit is capable of eliciting Ca2+-dependent regulation. A, schematic illustrates strategy for dissecting the effect of multiple CaMs in tuning channel regulation. Either CaMWT or mutant CaM1234 is localized to the CaV1.2 complex by genetic fusion to either α- or β-subunits using a polyglycine linker. A 2:1 stoichiometry can be attained by co-expressing both α- and β-subunits with CaM tethered. B, left, cartoon illustrates WT CaV1.2 L-type channels presumably bound to endogenous CaM with its preferred stoichiometry. Middle, representative current traces evoked in response to the +10 mV voltage step show enhanced decay of Ca2+ (red) versus Ba2+ currents (black), confirming robust CDI. Throughout, Ba2+ traces for CDI are scaled to about one-third actual magnitude to match peak Ca2+ traces (at scale with bar). Right, population data show the extent of baseline CDI. r300 values report the fraction of peak current remaining following 300-ms depolarization. Black dots and error bars represent mean ± S.E. C, truncation of the distal carboxyl tail of CaV1.2 L-typechannel does not alter CDI (α1CΔ1671 + β2A-CaM). D, fusion of CaMWT to the carboxyl terminus of α1C subunit (α1CΔ1671-CaMWT) supports normal CDI. E, fusion of CaM1234 to the α1C subunit (α1CΔ1671-CaM1234) abolishes CDI. F, CaM fusion to the β2A subunit (i.e. β2A-CaMWT) supports strong CDI. G, CDI is absent when the β2A subunit is fused to CaM12342A-CaM1234). The format is as described for B.
      Table 1Comparison of CaV1.2 CDI300 values when one or two CaM molecules are localized to the channel complex
      ConstructCa2+ r300 at +10 mVBa2+ r300 at +10 mVCDI300 at +10 mVp value
      α1C + β2A0.46 ± 0.071.08 ± 0.090.57 ± 0.05 (n = 5)NA
      α1CΔ1671 + β2A0.42 ± 0.040.81 ± 0.080.48 ± 0.02 (n = 5)0.910
      Tukey's multiple-comparison test shows p values in comparison with α1C + β2A.
      α1CΔ1671-CaMWT + β2A0.42 ± 0.030.85 ± 0.070.49 ± 0.04 (n = 5)>0.9777
      Tukey's multiple-comparison test shows p values in comparison with α1C + β2A.
      ; >0.9999
      Tukey's multiple-comparison test shows p values in comparison with α1CΔ1671 + β2A.
      α1CΔ1671-CaM1234 + β2A0.86 ± 0.050.91 ± 0.010.05 ± 0.05 (n = 6)<0.0001
      Tukey's multiple-comparison test shows p values in comparison with α1C + β2A.
      ; <0.0001
      Tukey's multiple-comparison test shows p values in comparison with α1CΔ1671 + β2A.
      α1C + β2A-CaMWT0.35 ± 0.020.96 ± 0.020.64 ± 0.02 (n = 8)0.9688
      Tukey's multiple-comparison test shows p values in comparison with α1C + β2A.
      ; 0.1326
      Tukey's multiple-comparison test shows p values in comparison with α1CΔ1671 + β2A.
      ; 0.2591
      Tukey's multiple-comparison test shows p values in comparison with α1C-CaMWT + β2A.
      α1C + β2A-CaM12340.89 ± 0.050.98 ± 0.040.09 ± 0.05 (n = 7)<0.0001
      Tukey's multiple-comparison test shows p values in comparison with α1C + β2A.
      ; <0.0001
      Tukey's multiple-comparison test shows p values in comparison with α1CΔ1671 + β2A.
      α1CΔ1671-CaMWT + β2A-CaMWT0.63 ± 0.031.00 ± 0.020.36 ± 0.03 (n = 7)0.0271
      Tukey's multiple-comparison test shows p values in comparison with α1C + β2A.
      ; 0.6832
      Tukey's multiple-comparison test shows p values in comparison with α1CΔ1671 + β2A.
      α1CΔ1671-CaMWT + β2A-CaM12340.47 ± 0.040.92 ± 0.050.49 ± 0.05 (n = 8)0.9095
      Tukey's multiple-comparison test shows p values in comparison with α1C + β2A.
      ; >0.9999
      Tukey's multiple-comparison test shows p values in comparison with α1CΔ1671 + β2A.
      α1CΔ1671-CaM1234 + β2A-CaMWT0.80 ± 0.030.86 ± 0.030.063 ± 0.04 (n = 6)<0.0001
      Tukey's multiple-comparison test shows p values in comparison with α1C + β2A.
      ; <0.0001
      Tukey's multiple-comparison test shows p values in comparison with α1CΔ1671 + β2A.
      α1CΔ1671-CaM1234 + β2A + CaM0.86 ± 0.040.89 ± 0.020.003 ± 0.03 (n = 5)<0.0001
      Tukey's multiple-comparison test shows p values in comparison with α1C + β2A.
      ; <0.0001
      Tukey's multiple-comparison test shows p values in comparison with α1CΔ1671 + β2A.
      α1C + β2A-CaM1234 + CaM0.79 ± 0.070.90 ± 0.070.13 ± 0.03 (n = 5)<0.0001
      Tukey's multiple-comparison test shows p values in comparison with α1C + β2A.
      ; <0.0001
      Tukey's multiple-comparison test shows p values in comparison with α1CΔ1671 + β2A.
      a Tukey's multiple-comparison test shows p values in comparison with α1C + β2A.
      b Tukey's multiple-comparison test shows p values in comparison with α1CΔ1671 + β2A.
      c Tukey's multiple-comparison test shows p values in comparison with α1C-CaMWT + β2A.

      CaV1 preferentially responds to CaM tethered to the channel carboxyl terminus

      Having verified the functionality of tethered CaM, we sought to determine channel regulation when multiple CaM molecules are localized within the channel nanodomain. As both the α1C CT and the β2A subunit are within close proximity of the channel pore (<10 nm) based on cryo-EM structure (
      • Wu J.
      • Yan Z.
      • Li Z.
      • Qian X.
      • Lu S.
      • Dong M.
      • Zhou Q.
      • Yan N.
      Structure of the voltage-gated calcium channel Cav1.1 at 3.6 Å resolution.
      ), CaM tethered to either domain is exposed to similar local Ca2+ fluctuations (
      • Tadross M.R.
      • Tsien R.W.
      • Yue D.T.
      Ca2+ channel nanodomains boost local Ca2+ amplitude.
      ,
      • Tay L.H.
      • Dick I.E.
      • Yang W.
      • Mank M.
      • Griesbeck O.
      • Yue D.T.
      Nanodomain Ca2+ of Ca2+ channels detected by a tethered genetically encoded Ca2+ sensor.
      ). Thus, if two WT CaM molecules are attached to the channel complex, we anticipate strong CDI akin to channels that lack tethered CaM, because either one or both CaM molecules can interact with respective channel effector interfaces. Indeed, co-expression of β2A-CaMWT with α1CΔ1671-CaMWT resulted in appreciable CDI (Fig. 2A, Fig. S1G, and Table 1), albeit modestly reduced compared with channels lacking tethered CaM (∼25% reduction). However, if the channel complex comprises of one mutant and one WT CaM, then four distinct functional outcomes emerge, depending on the underlying mechanism of channel modulation: If two Ca2+/CaM independently orchestrate channel modulation, then this maneuver would result in a partial disruption of CDI regardless of whether CaM1234 is tethered to α1C CT or β2A (Scenario I); if two Ca2+/CaM cooperatively modulate CaV1.2 regulation, then the presence of one CaM1234 in the channel complex tethered to either α1C or β2A would exert a dominant negative effect and fully inhibit CDI (Scenario II); if instead, functional channel modulation relied on a single Ca2+-bound CaM, then the presence of one CaMWT tethered to either α1C CT or β2A would elicit full CDI (Scenario III); and a final nuanced possibility is that CDI depends only on a single CaM, but one that is prebound to a particular interface (Scenario IV). In this last scenario, the modulatory effect will be binary, depending on whether CaMWT or CaM1234 occupies the interface responsible for triggering CDI. To dissect between these possibilities, we first co-expressed α1CΔ1671-CaM1234 with β2A-CaMWT. Comparison of Ca2+ versus Ba2+ current decay demonstrates a strong reduction of CDI (Fig. 2B, Fig. S1H, and Table 1). This result eliminates both Scenarios I and III. To distinguish between Scenarios II and IV, we co-expressed α1CΔ1671-CaMWT with β2A-CaM1234 (Fig. 2C and Fig. S1I). This maneuver resulted in strong CDI indistinguishable from that observed upon co-expression of either α1C or α1CΔ1671 with β2A (Table 1). This result confirmed Scenario IV with a single CaM prebound to the channel carboxyl tail being privileged in triggering CDI. To further ensure that the glycine linkage of CaM to either the α1CΔ1671 or the β2A subunit did not occlude accessibility of CaM to effector interfaces, we measured CDI of α1CΔ1671-CaM1234 with β2A and α1C in the presence of freely diffusible CaM. As with α1CΔ1671-CaM1234, we observed no CDI even upon CaM overexpression (Fig. S2 (A and B) and Table 1). As a further control, we also co-expressed α1C with β2A-CaM1234 and freely diffusible CaM. In this case, we again found no CDI, consistent with β2A-localized CaM occupying the carboxyl tail site (Fig. S2 (C and D) and Table 1). These findings further confirm that glycine linkage does not prevent CaM from reaching critical sites. In all, these results demonstrate that CaV1.2 is preferentially regulated by a single CaM associated with the channel CT, in effect rejecting Ca2+/CaM signaling from the β2A-tethered CaM.
      Figure thumbnail gr2
      Figure 2CaV1.2 is preferentially regulated by CaM tethered to the channel carboxyl terminus. A, localizing two WT CaM to the channel complex via fusion to both the α1C and β2A subunits (α1CΔ1671-CaMWT + β2A-CaMWT) supports CDI, albeit the extent of CDI is modestly diminished compared with channels with a single tethered CaMWT. B, co-expression of α1CΔ1671-CaM1234 with β2A-CaMWT sufficed to abolish CDI, suggesting that CT-tethered CaM is critical for CDI. C, co-expression of α1CΔ1671-CaMWT with β2A-CaM1234 resulted in strong CDI, suggesting that CaV1.2 is preferentially regulated by a single CaM associated with the channel carboxyl tail. Data are presented as mean ± S.E. obtained from a specified number of cells (n).
      To assess generality, we considered the stoichiometric basis for CaM regulation of CaV1.3. Accordingly, CaV1.3 exhibits strong CDI at baseline as shown in Fig. 3A (Fig. S3A and Table 2), consistent with previous studies. As with CaV1.2, we have previously demonstrated that fusion of CaMWT to the CaV1.3 CT (α1D-CaMWT) supports strong CDI, whereas attaching CaM1234 at the same locus (α1D-CaM1234) abolishes CDI (
      • Yang P.S.
      • Johny M.B.
      • Yue D.T.
      Allostery in Ca2+ channel modulation by calcium-binding proteins.
      ,
      • Banerjee R.
      • Yoder J.B.
      • Yue D.T.
      • Amzel L.M.
      • Tomaselli G.F.
      • Gabelli S.B.
      • Ben-Johny M.
      Bilobal architecture is a requirement for calmodulin signaling to CaV1.3 channels.
      ). To confirm functionality of CaM linkage to the β2A subunit, we co-expressed β2A-CaMWT or β2A-CaM1234 with the α1D pore-forming subunit. We observed robust CDI for CaV1.3 in the presence of β2A-CaMWT similar to that observed with the β2A subunit alone (Fig. 3B, Fig. S3B, and Table 2). By contrast, co-expression of β2A-CaM1234 abolished CDI (Fig. 3C, Fig. S3C, and Table 2), suggesting that CaM linked to the β2A subunit is capable of eliciting functional regulation. Thus assured, we sought to deduce the effect of localizing two CaMWT to the CaV1.3 complex. As anticipated, strong CDI was observed when α1D-CaMWT was co-expressed with β2A-CaMWT (Fig. 3D, Fig. S3D, and Table 2). Subsequently, we co-expressed α1D-CaM1234 with β2A-CaMWT and measured CDI (Fig. 3E, Fig. S3E, and Table 2). As with CaV1.2, this combination sufficed to strongly attenuate CDI. In contrast, co-expression of α1D-CaMWT with β2A-CaM1234 fully spared CDI (Fig. 3F, Fig. S3F, Table 2). To ensure that these findings did not result from a steric limitation imposed by tethered CaM, we considered whether overexpression of freely diffusible recombinant CaMWT could reverse CDI deficits of either α1D-CaM1234 with β2A (Fig. S4 (A and B) and Table 2) or α1D with β2A-CaM1234 (Fig. S4 (C and D) and Table 2). In both cases, we observed no CDI, confirming that CaM localized to the channel preferentially regulated channel function (Fig. S4). Taken together, the binary switching of channel regulatory behavior observed with localizing one CaMWT and one CaM1234 suggests that functional CaV1.3 regulation is preferentially triggered by CaM in close vicinity of the channel CT.
      Figure thumbnail gr3
      Figure 3CaV1.3 preferentially responds to CaM tethered to the channel carboxyl terminus. A, at baseline, CaV1.3 exhibits strong CDI. The format is as in B. Dots and error bars, mean ± S.E. obtained from the specified number of cells (n). B, as with CaV1.2, co-expression of β2A-CaMWT supports strong CDI of CaV1.3. C, co-expression of β2A-CaM1234 abolishes CDI of CaV1.3. D, localizing two CaMWT to the CaV1.3 complex by co-expressing α1D-CaMWT with β2A-CaMWT supports CDI, although the extent of CDI is modestly reduced compared with control conditions. E, co-expression of α1D-CaM1234 with β2A-CaMWT results in a strong reduction of CDI. F, co-expression of α1D-CaMWT with β2A-CaM1234 supports strong CDI, suggesting that CaM bound to the channel carboxyl terminus is privileged in signaling CDI.
      Table 2Comparison of CaV1.3 CDI300 values when one or two CaM molecules are localized to the channel complex
      ConstructCa22+ r300Ba2+ r300CDI300 at +10 mV (mean ± S.E.)p value
      α1D + β2A0.23 ± 0.020.97 ± 0.010.765 ± 0.025 (n = 8)NA
      α1D + β2A-CaMWT0.17 ± 0.060.94 ± 0.030.820 ± 0.057 (n = 6)0.9231
      α1D + β2A-CaM12340.96 ± 0.021.00 ± 0.010.033 ± 0.029 (n = 7)<0.0001
      α1d-CaMWT + β2A-CaMWT0.32 ± 0.030.98 ± 0.010.673 ± 0.036 (n = 8)0.3858
      α1d-CaMWT + β2A-CaM12340.23 ± 0.030.90 ± 0.030.746 ± 0.025 (n = 6)0.9996
      α1d-CaM1234 + β2A-CaMWT0.97 ± 0.090.99 ± 0.010.025 ± 0.084 (n = 5)<0.0001
      α1d-CaM1234 + β2A + CaM0.91 ± 0.050.95 ± 0.050.041 ± 0.047 (n = 5)<0.0001
      α1D + β2A-CaM1234 + CaM0.83 ± 0.050.86 ± 0.060.035 ± 0.025 (n = 5)<0.0001

      Functional CaM stoichiometry for CaV1 is limited by the number of CaV IQ domains

      To delineate mechanisms that govern CaM stoichiometry for channel regulation, we considered whether CaV1 could be engineered to be responsive to multiple CaM molecules. Accordingly, we constructed CaV1.3 channels containing two IQ domains in tandem in the carboxyl tail (CaV1.32×IQ), fused to either CaMWT (termed α1D/2×IQ-CaMWT to denote CaMWT fusion to the pore-forming α-subunit) or CaM12341D/2×IQ-CaM1234) and co-expressed with β2A, β2A-CaMWT, or β2A-CaM1234. We observed strong CDI for α1D/2×IQ-CaMWT similar to CaV1.3 (Fig. 4A, Fig. S5A, and Table 3). By comparison, CDI of α1D/2×IQ-CaM1234 was sharply diminished similarly to α1D-CaM1234, although not fully eliminated (Fig. 4B, Fig. S5B, and Table 3). These findings suggest that CT-linked CaM remains vital for CDI of CaV1.3 containing tandem IQ domains. Furthermore, co-expression of α1D/2×IQ-CaMWT with β2A-CaMWT also revealed strong CDI similarly to α1D/2×IQ-CaMWT co-expressed with β2A alone (Fig. 4C, Fig. S5C, and Table 3). However, when α1D/2×IQ-CaMWT is co-expressed with β2A-CaM1234, we observed a partial reduction in CDI (Fig. 4D, Fig. S5D, and Table 3). In like manner, co-expression of α1D/2×IQ-CaM1234 with β2A-CaMWT also showed a partial reduction in CDI (Fig. 4E, Fig. S5E, and Table 3). By contrast, localizing two-mutant CaM1234 to the channel complex by co-expressing α1D/2×IQ-CaM1234 with β2A-CaM1234 revealed a complete disruption of CDI (Fig. 4F, Fig. S5F, and Table 3). This behavior is distinctly different from a single IQ domain–containing CaV1.3 (Fig. 3), where a binary change in CDI is observed, depending on the Ca2+-binding ability of carboxyl-terminally linked CaM. Instead, the stepwise change in CDI with one- versus two-mutant CaM1234 is consistent with Scenario I considered above. This outcome suggests a 2:1 functional CaM stoichiometry for mutant CaV1.32×IQ.
      Figure thumbnail gr4
      Figure 4Engineering CaV1.3 with tandem IQ domains switches functional CaM stoichiometry. A, α1D/2×IQ-CaMWT with β2A exhibits strong CDI. The format is as in B. B, α1D/2×IQ-CaM1234 with β2A exhibit strongly reduced CDI. C, co-expression of α1D/2×IQ-CaMWT with β2A-CaMWT also demonstrates strong CDI. D, localizing one CaMWT and one CaM1234 to the CaV1.32×IQ channel complex by co-expressing α1D/2×IQ-CaMWT with β2A-CaM1234 results in a partial reduction in CDI. E, similarly, co-expression of α1D/2×IQ-CaM1234 with β2A-CaMWT also revealed a partial reduction in CDI. F, by contrast, localizing two CaM1234 molecules by expressing α1D/2×IQ-CaM1234 with β2A-CaM1234 results in a complete absence of CDI.
      Table 3Comparison of CaV1.3 tandem IQ CDI300 values when one or two CaM molecules are localized to the channel complex
      ConstructCa22+ r300Ba2+ r300CDI300 at +10 mV (mean ± S.E.)p value
      α1D/2×IQ-CaMWT + β2A0.13 ± 0.020.81 ± 0.070.836 ± 0.020 (n = 5)0.3953
      α1D/2×IQ-CaM1234 + β2A0.89 ± 0.041.01 ± 0.040.124 ± 0.017 (n = 6)<0.0001
      α1D/2×IQ-CaMWT + β2A-CaMWT0.26 ± 0.010.97 ± 0.030.724 ± 0.017 (n = 6)0.7614
      α1D/2×IQ-CaMWT + β2A-CaM12340.60 ± 0.050.94 ± 0.030.354 ± 0.070 (n = 5)<0.0001
      α1D/2×IQ-CaM1234 + β2A-CaMWT0.48 ± 0.050.98 ± 0.010.478 ± 0.061 (n = 5)<0.0001
      α1D/2×IQ-CaM1234 + β2A-CaM12340.99 ± 0.011.00 ± 0.010.011 ± 0.006 (n = 5)<0.0001
      Two mechanistic possibilities may engender this switch in functional CaM stoichiometry. First, the number of apo-CaM molecules within the CaV1 complex may be the determining parameter for functional CaM stoichiometry. Our previous work using a holo-channel FRET two-hybrid assay showed that although two Ca2+/CaM molecules bind the holo-CaV1 channels, only a single apo-CaM preassociates with the full-length channel (
      • Ben-Johny M.
      • Yue D.N.
      • Yue D.T.
      Detecting stoichiometry of macromolecular complexes in live cells using FRET.
      ). Furthermore, with two IQ domains, up to two apo-CaM molecules may interact with the channel complex. Second, functional CaM stoichiometry may be fundamentally limited by the number of IQ domains, a critical segment for initiating CDI. We previously showed that Ca2+-binding to prebound CaM triggers a conformational rearrangement of the channel CT, resulting in the formation of a tripartite complex involving CaM, the channel dual vestigial EF-hand domains, and the IQ domain (
      • Ben-Johny M.
      • Yang P.S.
      • Bazzazi H.
      • Yue D.T.
      Dynamic switching of calmodulin interactions underlies Ca2+ regulation of CaV1.3 channels.
      ). If so, the functional CaM stoichiometry may be limited by the number of IQ domains available to initiate formation of the tripartite EF/CaM/IQ complex. To test these possibilities, we replaced the CaV1.3 IQ domain with three different CaM-binding segments (
      • Rhoads A.R.
      • Friedberg F.
      Sequence motifs for calmodulin recognition.
      ): 1) M13 peptide from the myosin light-chain kinase (CaV1.3-M13), 2) the IQ domain 1 of unconventional myosin Va (CaV1.3-MyoIQ), and 3) the IQ domain of related NaV1.4 channels (CaV1.3-NaV14IQ). Thus probed, the CaV1.3-M13 channels revealed minimal CDI (Fig. 5A, Fig. S6A, and Table S1) compared with WT CaV1.3. As M13 interacts only with the Ca2+-bound form of CaM, this result suggests that apo-CaM preassociation is obligatory for CDI. Notably, CaV1.3-M13 channels also exhibited increased VDI, reminiscent of previous observations of increased VDI upon disrupting apo-CaM binding. Unlike the M13 peptide, the IQ domain of unconventional myosin Va interacts with both apo-CaM and Ca2+/CaM with a high affinity comparable with CaV channel IQ domain. If the number of apo-CaM molecules in the channel complex sufficed to determine functional regulation and stoichiometry, then substitution of the CaV1.3 IQ domain with the IQ domain from the unconventional myosin Va would preserve CDI triggered by a single CaM. However, CaV1.3-MyoIQ channels failed to trigger appreciable CDI, suggesting that high-affinity apo-CaM and Ca2+/CaM interaction with the channel alone are insufficient for CDI (Fig. 5B, Fig. S6B, and Table S1). As a further test, we considered whether substitution of the CaV1.3 IQ domain with the IQ domain from the related NaV1.4 channels might support functional channel regulation. Of note, NaV1.4 undergoes CDI with similar underlying mechanisms as CaV1.3 (
      • 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.
      ). Intriguingly, whole-cell recordings of CaV1.3-NaV1.4IQ revealed recognizable CDI although with reduced magnitude compared with WT CaV1.3 (Fig. 5C, Fig. S6C, and Table S1). Taken together, these findings suggest that the CaV and NaV IQ domains are privileged in CaV/NaV channel modulation, and this domain likely plays an important for orchestrating downstream structural rearrangements of channel cytosolic domains. Overall, these results are consistent with the possibility that the functional CaM stoichiometry for CaV1 is dictated by the number of IQ domains in the channel carboxyl terminus.
      Figure thumbnail gr5
      Figure 5The IQ domain is essential for functional CaM regulation of CaV1.3. A, replacing the CaV1.3 IQ domain with M13 peptide from myosin light-chain kinase that only interacts with Ca2+/CaM strongly reduces CDI. The format is as in B. Data are presented as mean ± S.E. obtained from a specified number of cells (n). B, substitution of CaV1.3 IQ domain with IQ domain 1 from unconventional myosin Va also abolished CDI. C, by contrast, replacement of CaV1.3 IQ domain with IQ domain of NaV1.4 supports CDI. The magnitude of CDI is diminished compared with WT CaV1.3.

      Distinct modes of CaV2.1 regulation are preferentially evoked by carboxyl-terminally linked CaM

      CaM regulation of CaV2.1 is bifurcated resulting in two mechanistically distinct forms of regulation: 1) rapid CDF that evolves over ∼1–10 ms and is sensitive to local Ca2+ fluctuations, and 2) kinetically slower CDI that evolves over ∼300-800 ms and is sensitive to global Ca2+ elevations (
      • 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.
      ,
      • Lee A.
      • Wong S.T.
      • Gallagher D.
      • Li B.
      • Storm D.R.
      • Scheuer T.
      • Catterall W.A.
      Ca2+/calmodulin binds to and modulates P/Q-type calcium channels.
      ). The two modes of channel regulation rely on Ca2+/CaM interaction with distinct channel domains (
      • Kim E.Y.
      • Rumpf C.H.
      • Fujiwara Y.
      • Cooley E.S.
      • Van Petegem F.
      • Minor Jr., D.L.
      Structures of CaV2 Ca2+/CaM-IQ domain complexes reveal binding modes that underlie calcium-dependent inactivation and facilitation.
      ,
      • Mori M.X.
      • Vander Kooi C.W.
      • Leahy D.J.
      • Yue D.T.
      Crystal structure of the CaV2 IQ domain in complex with Ca2+/calmodulin: high-resolution mechanistic implications for channel regulation by Ca2+.
      ). CDF is triggered primarily by CaM C-lobe interaction with the canonical IQ domain (
      • Kim E.Y.
      • Rumpf C.H.
      • Fujiwara Y.
      • Cooley E.S.
      • Van Petegem F.
      • Minor Jr., D.L.
      Structures of CaV2 Ca2+/CaM-IQ domain complexes reveal binding modes that underlie calcium-dependent inactivation and facilitation.
      ), whereas CDI relies on Ca2+/CaM N-lobe interacting with binding sites elsewhere on the channel (
      • Lee A.
      • Wong S.T.
      • Gallagher D.
      • Li B.
      • Storm D.R.
      • Scheuer T.
      • Catterall W.A.
      Ca2+/calmodulin binds to and modulates P/Q-type calcium channels.
      ). To determine whether both modes of channel regulation are triggered by a single CaM, we again applied our strategy of localizing multiple CaM molecules to the CaV2 complex through linkage to the pore-forming α1A and the β2A subunits. For these experiments, the whole-cell dialyzate contained low Ca2+ buffering (1 mm EGTA) to permit global Ca2+ elevations necessary to trigger CDI. A family of depolarizing voltage pulses of 800-ms duration were utilized to elicit Ca2+ and Ba2+ currents for CDI measurements. Thus probed, Fig. 6A shows baseline CDI of CaV2.1 (Fig. S7A and Table S2). Tethering CaMWT to the CT of α1A subunit (α1A-CaMWT) and co-expression with the β2A subunit yields CDI comparable with baseline conditions (Fig. 6B, Fig. S7B, and Table S2). By contrast, fusion of CaM1234 to the CT of α1A subunit results in a reduction in CDI (Fig. 6C, Fig. S7C, and Table S2). Subsequently, we tested whether CaM fusion to the β2A subunit also evoked similar regulatory effects. Accordingly, co-expression of α1A subunit with β2A-CaMWT showed robust CDI comparable with baseline conditions (Fig. 6D, Fig. S7D, and Table S2), whereas β2A-CaM1234 strongly diminished CDI (Fig. 6E, Fig. S7E, and Table S2). Therefore, fusion of CaM to either the α1A or the β2A subunit permits interaction with key effector interfaces and preserves functional modulation.
      Figure thumbnail gr6
      Figure 6CaM to CaV2.1 via fusion to either the α or β subunits preserves functional channel regulation. A, baseline CDI of CaV2.1 is assessed at low Ca2+ buffering. Representative traces correspond to Ca2+ and Ba2+ currents evoked in response to a +20 mV voltage step. Ba2+ traces are scaled to about one-third actual magnitude to match Ca2+ traces (at scale with bar). Right, levels of inactivation at different voltages are assessed as the fraction of peak current remaining following 800-ms depolarization (r800) and averaged from n cells. Symbols and error bars, mean ± S.E. B, fusion of CaMWT to the carboxyl terminus of α1A subunit (α1A-CaMWT) supports normal CDI. C, fusion of mutant CaM12341A-CaM1234) abolishes CDI. D, localizing a single CaM to the CaV2.1 complex by co-expression of β2A-CaMWT supports robust CDI. E, CDI of CaV2.1 is strongly diminished when β2A-CaM1234 is co-expressed.
      Thus informed, we considered changes in channel regulatory behavior in the presence of multiple CaM molecules. Accordingly, we co-expressed α1A-CaMWT with β2A-CaMWT. As expected, this maneuver elicited strong CDI (Fig. 7A, Fig. S7F, and Table S2). To determine stoichiometric requirements, we measured CDI of α1A-CaM1234 in the presence of β2A-CaMWT. Comparison of Ca2+ versus Ba2+ currents revealed markedly blunted CDI (Fig. 7B, Fig. S7G, and Table S2). In comparison, co-expression of α1A-CaMWT with β2A-CaM1234 revealed strong CDI (Fig. 7C, Fig. S7H, and Table S2) similar to untagged channels. As a control, we co-expressed of α1A-CaM1234 with β2A-CaM1234 and found nearly complete inhibition of CDI (Fig. 7D, Fig. S7I, and Table S2). Once again, to further corroborate these findings, we considered whether overexpression of freely diffusible recombinant CaMWT could reverse CDI deficits of α1A-CaM1234 in the presence of β2A (Fig. S8 (A, B, and D) and Table S2). Indeed, CDI remained blunted for α1A-CaM1234 despite CaM overexpression. Similarly, overexpression of CaMWT failed to reverse the reduction in CDI of α1A in the presence of β2A-CaM1234 (Fig. S8 (E and F), Fig. 8H, and Table S2). These findings suggest that a single CaM bound to the CaV2.1 CT is primarily responsible for signaling CDI.
      Figure thumbnail gr7
      Figure 7CaM fused to the CaV2.1 carboxyl terminus is privileged in triggering CDI. A, co-expression of α1A-CaMWT with β2A-CaMWT results in robust CDI. The format is as in B. B, by contrast, α1A-CaM1234 co-expressed with β2A-CaMWT results in strong reduction in CDI, suggesting that CT-fused CaM is privileged in modulating CDI. C, strong CDI is evident for α1A-CaMWT in the presence of β2A-CaM1234. D, CDI is strongly diminished when mutant CaM1234 is tethered to both the α1A and β2A subunits.
      Figure thumbnail gr8
      Figure 8CaM tethered to both the CaV2.1 α and β subunits is able to trigger CDF. A, top left, schematic of WT CaV2.1 co-expressed with α2δ and β2A subunits. Top right, a two-pulse protocol is used to quantify the extent of CDF. Ba2+ current kinetics are similar in the presence (black) or absence (gray) of a depolarizing prepulse. Bottom right, without prepulse, a 0 mV step depolarization elicits a biphasic inward Ca2+ current with an initial rapid phase followed by a slow phase corresponding to Ca2+-dependent facilitation. With a +20-mV prepulse, the channels are already facilitated, and as such, the ensuing test pulse elicits currents that exhibit enhanced channel activation. The area between the two current traces (ΔQ) approximates CDF triggered by the prepulse. Bottom left, population data (mean ± S.E.) shows RF (relative facilitation) at different prepulse potentials averaged form n cells and assessed as ΔQ divided by the time constant (t) of facilitation. CDF is determined as the difference in RF with Ba2+ versus Ca2+ as charge carrier. B, fusion of CaMWT to the carboxyl terminus of α1A subunit (α1A-CaMWT) supports strong CDF. C, fusion of CaM1234 to the α1A carboxyl terminus (α1A-CaM1234) abolishes CDF. D, co-expression of β2A-CaMWT with α1A elicits strong CDF. E, CDF is abolished in the presence of β2A-CaM1234.
      To determine whether CaM localized to the α1A CT is also responsible for eliciting CDF, we first established baseline CDF of CaV2.1 using a paired-pulse facilitation protocol (Fig. 8A and Table S3). Briefly, in the absence of a prepulse, CaV2.1 current displays biphasic kinetics corresponding to rapid activation of the channel and a subsequent slower interconversion into a facilitated gating configuration following Ca2+ binding to CaM. With a prepulse, CaV2.1 current is monophasic with enhanced activation, Ca2+ entry during the prepulse having already triggered facilitation. RF is quantified as the excess charge entry following prepulse, and CDF is quantified as the difference in RF with Ca2+ versus Ba2+ as charge carriers. Once again, we validated that CaM fusion to the α1A and β2A supports CDF. Briefly, co-expression of α1A-CaMWT with the β2A subunit elicits strong CDF, whereas α1A-CaM1234 exhibits strongly diminished CDF (Fig. 8 (B and C) and Table S3). In like manner, expressing β2A-CaMWT with α1A subunit supports strong CDF, whereas β2A-CaM1234 diminishes CDF (Fig. 8 (D and E) and Table S3), thus confirming functionality of tethered CaM. Furthermore, robust CDF was also observed when both α1A and β2A subunits were both fused with CaMWT (Fig. 9A and Table S3), albeit the magnitude of CDF was modestly reduced compared with WT channels. To determine whether CDF of CaV2.1 is also dependent on carboxyl-terminally linked CaM, we probed CDF of α1A-CaM1234 in the presence of β2A-CaMWT. CDF was nearly absent for this pair (Fig. 9B and Table S3). By contrast, α1A-CaMWT in the presence of β2A-CaM1234 revealed no appreciable change in CDF (Fig. 9C and Table S3). Taken together, these findings suggest that CaM linked to the α1A CT is privileged in triggering CDF. Of note, co-expression of α1A-CaM1234 with β2A-CaM1234 exhibited minimal CDF (Fig. 9D and Table S3). As a further test, we probed whether freely diffusible recombinant CaMWT could reverse reduced CDF observed for α1A-CaM1234 in the presence β2A or for α1A in the presence of β2A-CaM1234. Indeed, CDF was strongly diminished in both cases (Fig. S8, C and G). Thus, localized CaM is privileged in initiating CDF. Taken together, these results also indicate that the same CaM molecule prebound to the channel CT is responsible for mediating both CDI and CDF.
      Figure thumbnail gr9
      Figure 9CDF of CaV2.1 channel is preferentially evoked by carboxyl terminus linked CaM. A, localization of two CaMWT molecules via co-expression of α1A-CaMWT with β2A-CaMWT supports CDF, although its magnitude is blunted compared with WT channels that lack tethered CaM. B, localizing a mutant CaM1234 via fusion to the α1A subunit CT (α1A-CaM1234) and a CaMWT via linkage to the β2a subunit (β2A-CaMWT) resulted in a partial reduction of CDF. C, fusion of α1A-CaMWT with β2A-CaM1234 demonstrates strong CDF comparable with channels that lack a tethered CaM. D, co-expression of α1A-CaM1234 with β2A-CaM1234 completely abolishes CDF.

      Discussion

      The stoichiometry of CaM interaction with the CaV channel complex and the functional requirements for channel regulation have long been debated (
      • Ben-Johny M.
      • Yue D.T.
      Calmodulin regulation (calmodulation) of voltage-gated calcium channels.
      ,
      • Minor Jr., D.L.
      • Findeisen F.
      Progress in the structural understanding of voltage-gated calcium channel (CaV) function and modulation.
      ). Biochemical and structural studies demonstrate the interaction of multiple CaM with distinct channel peptide segments (
      • Tang W.
      • Halling D.B.
      • Black D.J.
      • Pate P.
      • Zhang J.Z.
      • Pedersen S.
      • Altschuld R.A.
      • Hamilton S.L.
      Apocalmodulin and Ca2+ calmodulin-binding sites on the CaV1.2 channel.
      ,
      • Fallon J.L.
      • Baker M.R.
      • Xiong L.
      • Loy R.E.
      • Yang G.
      • Dirksen R.T.
      • Hamilton S.L.
      • Quiocho F.A.
      Crystal structure of dimeric cardiac L-type calcium channel regulatory domains bridged by Ca2+ calmodulins.
      ,
      • Kim E.Y.
      • Rumpf C.H.
      • Fujiwara Y.
      • Cooley E.S.
      • Van Petegem F.
      • Minor Jr., D.L.
      Structures of CaV2 Ca2+/CaM-IQ domain complexes reveal binding modes that underlie calcium-dependent inactivation and facilitation.
      ,
      • Kim E.Y.
      • Rumpf C.H.
      • Van Petegem F.
      • Arant R.J.
      • Findeisen F.
      • Cooley E.S.
      • Isacoff E.Y.
      • Minor Jr., D.L.
      Multiple C-terminal tail Ca2+/CaMs regulate CaV1.2 function but do not mediate channel dimerization.
      ,
      • 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.
      ,
      • Ivanina T.
      • Blumenstein Y.
      • Shistik E.
      • Barzilai R.
      • Dascal N.
      Modulation of L-type Ca2+ channels by Gβγ and calmodulin via interactions with N and C termini of α1C.
      ,
      • Fallon J.L.
      • Halling D.B.
      • Hamilton S.L.
      • Quiocho F.A.
      Structure of calmodulin bound to the hydrophobic IQ domain of the cardiac Cav1.2 calcium channel.
      ,
      • Benmocha A.
      • Almagor L.
      • Oz S.
      • Hirsch J.A.
      • Dascal N.
      Characterization of the calmodulin-binding site in the N terminus of CaV1.2.
      ). FRET analysis suggests a stoichiometry of up to two Ca2+/CaM molecules associating with the holo-CaV channel complex (
      • Ben-Johny M.
      • Yue D.N.
      • Yue D.T.
      Detecting stoichiometry of macromolecular complexes in live cells using FRET.
      ). Functional studies, however, indicate that a single CaM suffices for Ca2+-dependent feedback regulation (
      • Mori M.X.
      • Erickson M.G.
      • Yue D.T.
      Functional stoichiometry and local enrichment of calmodulin interacting with Ca2+ channels.
      ). To reconcile these differences, we dissected the potential role of multiple CaM in orchestrating CaV feedback modulation. We localized up to two WT CaM or mutant CaM1234 to the CaV1-2 channel complex through linkage to the α and β subunits. Consistent with prior studies, we found that a single CaM tethered to the CaV complex through either the α or β subunits is fully capable of replacing endogenous CaM (
      • Mori M.X.
      • Erickson M.G.
      • Yue D.T.
      Functional stoichiometry and local enrichment of calmodulin interacting with Ca2+ channels.
      ,
      • Bazzazi H.
      • Ben Johny M.
      • Adams P.J.
      • Soong T.W.
      • Yue D.T.
      Continuously tunable Ca2+ regulation of RNA-edited CaV1.3 channels.
      ,
      • Banerjee R.
      • Yoder J.B.
      • Yue D.T.
      • Amzel L.M.
      • Tomaselli G.F.
      • Gabelli S.B.
      • Ben-Johny M.
      Bilobal architecture is a requirement for calmodulin signaling to CaV1.3 channels.
      ). Nonetheless, when multiple CaMs are localized to the CaV1-2 channel complex, functional Ca2+-regulation of channel gating depends primarily on CaM tethered to the CT of the α-subunit. More specifically, when CaMWT is attached to the CaV1/2 CT locus, both CDI (for CaV1.2/1.3/2.1) and CDF (for CaV2.1) are fully intact; however, when CaM1234 is attached at this locus, Ca2+ regulation is absent. Furthermore, we found that introduction of a second IQ domain in the channel carboxyl tail switches the functional CaM stoichiometry for CaV1.3 channels such that channel regulation is responsive to two CaM molecules. These results are consistent with a model whereby a single CaM preassociated with the channel CT serves as a dedicated sensor for Ca2+-dependent modulation of CaV1/2 gating.
      A few mechanistic implications merit further attention. First, in vitro measurements of CaM affinity have demonstrated that apo-CaM interaction with the channel CT peptides is much weaker (∼1 μm) compared with Ca2+/CaM interaction (<1 nM) (
      • 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.
      ,
      • Findeisen F.
      • Rumpf C.H.
      • Minor Jr., D.L.
      Apo states of calmodulin and CaBP1 control CaV1 voltage-gated calcium channel function through direct competition for the IQ domain.
      ). Thus, if both a mutant CaM incapable of binding Ca2+ and a WT CaM are within the same channel complex, one would expect the WT CaM to competitively displace the mutant CaM on the CT, owing to the 3-order of magnitude affinity advantage. However, we found that this was not the case for CaV1/2 channels; β-subunit–tethered CaMWT was unable to displace CaM1234 to trigger CDI. One possibility is that the apo-CaM affinity for the CaV channel complex may be stronger than estimated in vitro, presumably reflecting unconventional interactions with the channel complex, as has been observed in cryo-EM structures of holo-KV7 channels (
      • Sun J.
      • MacKinnon R.
      Cryo-EM structure of a KCNQ1/CaM complex reveals insights into congenital long QT syndrome.
      ) and ryanodine receptors (
      • Gong D.
      • Chi X.
      • Wei J.
      • Zhou G.
      • Huang G.
      • Zhang L.
      • Wang R.
      • Lei J.
      • Chen S.R.W.
      • Yan N.
      Modulation of cardiac ryanodine receptor 2 by calmodulin.
      ). Indeed, previous studies have shown that reducing free apo-CaM levels to nanomolar concentrations was insufficient to appreciably deplete apo-CaM preassociation from the CaV1.3 channel, suggesting a higher apo-CaM affinity (
      • Liu X.
      • Yang P.S.
      • Yang W.
      • Yue D.T.
      Enzyme-inhibitor-like tuning of Ca2+ channel connectivity with calmodulin.
      ). An alternative possibility is that additional channel regulatory proteins, such as α-actinin, may fine-tune CaM interactions with the CaV channel, thereby imparting distinct effects on channel gating (
      • Turner M.
      • Anderson D.E.
      • Bartels P.
      • Nieves-Cintron M.
      • Coleman A.M.
      • Henderson P.B.
      • Man K.N.M.
      • Tseng P.Y.
      • Yarov-Yarovoy V.
      • Bers D.M.
      • Navedo M.F.
      • Horne M.C.
      • Ames J.B.
      • Hell J.W.
      α-Actinin-1 promotes activity of the L-type Ca2+ channel Cav1.2.
      ). Second, our findings also point toward potential mechanisms that underlie the singular CaM stoichiometry observed for Ca2+ regulation of CaV gating. For CaV1.3 channels, we previously found that Ca2+-binding to CaM elicits a conformational rearrangement of the channel CT, resulting in the formation of a tripartite complex involving the channel IQ domain, Ca2+/CaM, and the channel dual vestigial EF-hand segments (
      • Ben-Johny M.
      • Yang P.S.
      • Bazzazi H.
      • Yue D.T.
      Dynamic switching of calmodulin interactions underlies Ca2+ regulation of CaV1.3 channels.
      ). Thus, one possibility is that functional stoichiometry for CaM regulation of channel gating may be ultimately limited by the number of IQ domains available to initiate formation of the tripartite complex. Consistent with this possibility, when CaV1.3 channels contained two IQ domains, functional Ca2+ regulation appeared to depend on both the channel CT-tethered CaM and the β-subunit–tethered CaM. Furthermore, we found that replacement of the CaV1.3 IQ domain with either M13 peptide or an IQ domain from the unconventional myosin Va resulted in a nearly complete inhibition of CDI. By contrast, substitution of the NaV1.4 IQ domain still permits functional Ca2+ regulation. Importantly, NaV1.4 channels are homologous to CaV channels, and they undergo CDI in a similar manner as CaV1 channels. In this scenario, it is possible that specific residues unique to the CaV/NaV IQ domain and not in myosin Va IQ may be critical in triggering tripartite complex formation. It is also possible that the precise orientation or arrangement of CaM may also be relevant in this process (
      • Kim E.Y.
      • Rumpf C.H.
      • Fujiwara Y.
      • Cooley E.S.
      • Van Petegem F.
      • Minor Jr., D.L.
      Structures of CaV2 Ca2+/CaM-IQ domain complexes reveal binding modes that underlie calcium-dependent inactivation and facilitation.
      ). Furthermore, although M13 and myosin Va IQ domain are widely recognized as CaM-binding peptides, it is possible that attachment to the CaV channel carboxyl tail may perturb the ability of these peptides to interact with CaM. Third, the traditional model of Ca2+-dependent regulation is that Ca2+/CaM interaction with effector sites is sufficient to signal to the pore domain. For most CaV channels, the carboxyl tail IQ domain is thought to harbor key effector sites for triggering Ca2+/CaM regulation (
      • Minor Jr., D.L.
      • Findeisen F.
      Progress in the structural understanding of voltage-gated calcium channel (CaV) function and modulation.
      ); however, for CaV1.2 and CaV1.3, one critical interface for N-lobe–mediated CDI is the NSCaTE motif located on the channel N terminus (
      • 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.
      ). Importantly, NSCaTE only interacts with CaM in the presence of Ca2+ (
      • 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.
      ,
      • Ivanina T.
      • Blumenstein Y.
      • Shistik E.
      • Barzilai R.
      • Dascal N.
      Modulation of L-type Ca2+ channels by Gβγ and calmodulin via interactions with N and C termini of α1C.
      ). As such, if mutant CaM1234 were prebound to the channel CT, then the NSCaTE motif remains unoccupied at basal conditions (
      • 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.
      ,
      • Ivanina T.
      • Blumenstein Y.
      • Shistik E.
      • Barzilai R.
      • Dascal N.
      Modulation of L-type Ca2+ channels by Gβγ and calmodulin via interactions with N and C termini of α1C.
      ,
      • Taiakina V.
      • Boone A.N.
      • Fux J.
      • Senatore A.
      • Weber-Adrian D.
      • Guillemette J.G.
      • Spafford J.D.
      The calmodulin-binding, short linear motif, NSCaTE is conserved in L-type channel ancestors of vertebrate Cav1.2 and Cav1.3 channels.
      ). Following Ca2+ influx, the second CaMWT within the channel complex localized via the β subunit would be able to interact with NSCaTE segment and trigger CDI. However, the complete absence of Ca2+/CaM regulation when CaV1.2 and CaV1.3 α subunit is linked to mutant CaM1234 and β2A-CaM suggests that the simple interaction of the N-lobe of Ca2+/CaM with the NSCaTE domain is insufficient. Instead, these results suggest that a Ca2+-dependent conformational rearrangement of the CT-bound CaM is obligatory for CaM regulation. Fourth, for CaV2 channels, CaM regulation manifests as both CDF and CDI with distinct spatial Ca2+ selectivity and kinetics (
      • 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.
      ,
      • Lee S.R.
      • Adams P.J.
      • Yue D.T.
      Large Ca2+-dependent facilitation of CaV2.1 channels revealed by Ca2+ photo-uncaging.
      ). These findings support the possibility that both modes of channel regulation are mediated by the same CaM that is initially preassociated with the channel CT.
      Our findings also bear important biological implications. In cardiac myocytes, a vast majority of Ca2+-free CaM is enriched in the dyad with a large fraction bound to the RyR (
      • Yang Y.
      • Guo T.
      • Oda T.
      • Chakraborty A.
      • Chen L.
      • Uchinoumi H.
      • Knowlton A.A.
      • Fruen B.R.
      • Cornea R.L.
      • Meissner G.
      • Bers D.M.
      Cardiac myocyte Z-line calmodulin is mainly RyR2-bound, and reduction is arrhythmogenic and occurs in heart failure.
      ,
      • Wu X.
      • Bers D.M.
      Free and bound intracellular calmodulin measurements in cardiac myocytes.
      ). Following Ca2+ binding, however, CaM is mobilized and is available to interact with targets including Ca2+/CaM-dependent kinases and phosphatases that are also localized at the dyad (
      • Wu X.
      • Bers D.M.
      Free and bound intracellular calmodulin measurements in cardiac myocytes.
      ). Our findings suggest that Ca2+-mobilized CaM would be unable to inhibit the L-type Ca2+-channels. Instead, only CaM initially preassociated with the channel CT would be able to trigger Ca2+ regulation. Physiologically, this scheme is advantageous in cardiomyocytes as additional CaMs in the dyad are free to signal to other regulatory processes, including activation of kinases and phosphatases (
      • Oliveria S.F.
      • Dell'Acqua M.L.
      • Sather W.A.
      AKAP79/150 anchoring of calcineurin controls neuronal L-type Ca2+ channel activity and nuclear signaling.
      ,
      • Hudmon A.
      • Schulman H.
      • Kim J.
      • Maltez J.M.
      • Tsien R.W.
      • Pitt G.S.
      CaMKII tethers to L-type Ca2+ channels, establishing a local and dedicated integrator of Ca2+ signals for facilitation.
      ,
      • Tandan S.
      • Wang Y.
      • Wang T.T.
      • Jiang N.
      • Hall D.D.
      • Hell J.W.
      • Luo X.
      • Rothermel B.A.
      • Hill J.A.
      Physical and functional interaction between calcineurin and the cardiac L-type Ca2+ channel.
      ,
      • Gao H.
      • Wang F.
      • Wang W.
      • Makarewich C.A.
      • Zhang H.
      • Kubo H.
      • Berretta R.M.
      • Barr L.A.
      • Molkentin J.D.
      • Houser S.R.
      Ca2+ influx through L-type Ca2+ channels and transient receptor potential channels activates pathological hypertrophy signaling.
      ), channel coupling (
      • 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.
      ), or translocation to nucleus (
      • Bossuyt J.
      • Bers D.M.
      Visualizing CaMKII and CaM activity: a paradigm of compartmentalized signaling.
      ), all without disrupting CaV channel inactivation, an essential factor for normal cardiac repolarization (
      • Alseikhan B.A.
      • DeMaria C.D.
      • Colecraft H.M.
      • Yue D.T.
      Engineered calmodulins reveal the unexpected eminence of Ca2+ channel inactivation in controlling heart excitation.
      ). In the disease context, this arrangement, however, makes L-type channels particularly vulnerable to misregulation in cardiac arrhythmias associated with calmodulinopathies (
      • Limpitikul W.B.
      • Dick I.E.
      • Joshi-Mukherjee R.
      • Overgaard M.T.
      • George Jr., A.L.
      • Yue D.T.
      Calmodulin mutations associated with long QT syndrome prevent inactivation of cardiac L-type Ca2+ currents and promote proarrhythmic behavior in ventricular myocytes.
      ). The singular functional stoichiometry implies that the preassociation of even a small fraction of mutant CaM with weakened Ca2+ binding could appreciably disrupt L-type channel inactivation and increase risk of arrhythmogenesis. In like manner, in neurons, CaM localized to Ca2+ channels serve multiple functions (
      • Simms B.A.
      • Zamponi G.W.
      Neuronal voltage-gated calcium channels: structure, function, and dysfunction.
      ), including modulation of channel gating, trafficking (
      • Wang H.G.
      • George M.S.
      • Kim J.
      • Wang C.
      • Pitt G.S.
      Ca2+/calmodulin regulates trafficking of CaV1.2 Ca2+ channels in cultured hippocampal neurons.
      ,
      • Hall D.D.
      • Dai S.
      • Tseng P.Y.
      • Malik Z.
      • Nguyen M.
      • Matt L.
      • Schnizler K.
      • Shephard A.
      • Mohapatra D.P.
      • Tsuruta F.
      • Dolmetsch R.E.
      • Christel C.J.
      • Lee A.
      • Burette A.
      • Weinberg R.J.
      • et al.
      Competition between α-actinin and Ca2+-calmodulin controls surface retention of the L-type Ca2+ channel CaV1.2.
      ), and a key role in excitation-transcription coupling, where local Ca2+ signaling near L-type channels results in rapid shuttling of Ca2+/CaM to the nucleus through γCaMKII (
      • Ma H.
      • Groth R.D.
      • Cohen S.M.
      • Emery J.F.
      • Li B.
      • Hoedt E.
      • Zhang G.
      • Neubert T.A.
      • Tsien R.W.
      γCaMKII shuttles Ca2+/CaM to the nucleus to trigger CREB phosphorylation and gene expression.
      ,
      • Cohen S.M.
      • Suutari B.
      • He X.
      • Wang Y.
      • Sanchez S.
      • Tirko N.N.
      • Mandelberg N.J.
      • Mullins C.
      • Zhou G.
      • Wang S.
      • Kats I.
      • Salah A.
      • Tsien R.W.
      • Ma H.
      Calmodulin shuttling mediates cytonuclear signaling to trigger experience-dependent transcription and memory.
      ,
      • Wang X.
      • Marks C.R.
      • Perfitt T.L.
      • Nakagawa T.
      • Lee A.
      • Jacobson D.A.
      • Colbran R.J.
      A novel mechanism for Ca2+/calmodulin-dependent protein kinase II targeting to L-type Ca2+ channels that initiates long-range signaling to the nucleus.
      ). Having a resident CaM dedicated for CaV channel feedback modulation ensures that local Ca2+/CaM signaling can be multiplexed without detrimental effects on cellular electrical excitability.

      Experimental procedures

      Molecular biology

      CaV1.2, CaV1.3, and CaV2.1 variant were unmodified from previously published constructs: rabbit CaV1.2 (NM001136522) (
      • Wei X.Y.
      • Perez-Reyes E.
      • Lacerda A.E.
      • Schuster G.
      • Brown A.M.
      • Birnbaumer L.
      Heterologous regulation of the cardiac Ca2+ channel α1 subunit by skeletal muscle β and γ subunits: implications for the structure of cardiac L-type Ca2+ channels.
      ), CaV1.2-glycine-(12)-CaMWT and CaV1.2-glycine-(12)-CaM1234 (
      • Mori M.X.
      • Erickson M.G.
      • Yue D.T.
      Functional stoichiometry and local enrichment of calmodulin interacting with Ca2+ channels.
      ); rat CaV1.3 (AF370009.1) (
      • Liu X.
      • Yang P.S.
      • Yang W.
      • Yue D.T.
      Enzyme-inhibitor-like tuning of Ca2+ channel connectivity with calmodulin.
      ), CaV1.3-glycine-(12)-CaMWT and CaV1.3-glycine-(12)-CaM1234 (
      • Yang P.S.
      • Johny M.B.
      • Yue D.T.
      Allostery in Ca2+ channel modulation by calcium-binding proteins.
      ). Human CaV2.1 clones (NP_001120693.1) were as previously published (
      • Lee S.R.
      • Adams P.J.
      • Yue D.T.
      Large Ca2+-dependent facilitation of CaV2.1 channels revealed by Ca2+ photo-uncaging.
      ) with the exact splice background being Δ10A (+G); 16+/17+; Δ17A (−VEA); −31* (−NP); 37a (EFa); 43+/44−; 47− (
      • Soong T.W.
      • DeMaria C.D.
      • Alvania R.S.
      • Zweifel L.S.
      • Liang M.C.
      • Mittman S.
      • Agnew W.S.
      • Yue D.T.
      Systematic identification of splice variants in human P/Q-type channel α12.1 subunits: implications for current density and Ca2+-dependent inactivation.
      ). CaV2.1-glycine-(12)-CaMWT and CaV2.1-glycine-(12)-CaM1234 were obtained by attaching a polyglycine linker of length 8 fused to CaMWT/CaM1234 immediately downstream of the channel IQ domain (following the protein sequence MREEQ, residue 1981) using PCR amplification and ligation using restriction enzyme sites XbaI and SalI. β2A (M80545) (
      • Perez-Reyes E.
      • Kim H.S.
      • Lacerda A.E.
      • Horne W.
      • Wei X.Y.
      • Rampe D.
      • Campbell K.P.
      • Brown A.M.
      • Birnbaumer L.
      Induction of calcium currents by the expression of the α1-subunit of the dihydropyridine receptor from skeletal muscle.
      ), β2A-glycine-(8)-CaMWT and β2A-glycine-(8)-CaM1234 were unchanged from previously published rat β2A modifications (
      • Yang P.S.
      • Johny M.B.
      • Yue D.T.
      Allostery in Ca2+ channel modulation by calcium-binding proteins.
      ). Rat CaM (M80545) was used for fusion to α- and β-subunits. The tandem IQ domain–containing CaV1.3 were constructed using gene synthesis. To do so, we utilized a CaV1.3 channel with a silent mutation at 1538GT1539 that introduces a KpnI restriction site (by replacing GGGACA with GGTACC). We synthesized (GENEWIZ) a tandem IQ domain–containing gene fragment linked to either CaMWT or CaM1234 with a 12-glycine linker flanked by KpnI and XbaI restriction sites. The amino acid sequence for the tandem IQ domain segment is as follows: EVTVGKFYATFLIQDYFRKFKKRKEQGLVTVGKFYATFLIQDYFRKFKKRKEQGLV. The gene fragments were ligated into the aforementioned engineered CaV1.3 plasmid following restriction digest using KpnI/XbaI. To replace the CaV1.3 IQ domain with M13, IQ domain 1 from unconventional myosin Va, and IQ domain of NaV1.4, we followed an identical strategy of synthesis of gene fragments (GENEWIZ) flanked by KpnI and XbaI sites ligated into the engineered channels following restriction digest. For CaV1.3-M13, the gene fragment encoded the following amino acid sequence GTVMFNATLFALVRTALKIKTEGNLEQANEELRAVIKKIWKKTSMKLLDQVVPPAGDDEVTKRRWKKNFIAVSAANRFKKISSSGAL*. The M13 segment is italicized. For CaV1.3-MyoIQ, the gene fragment encoded GTVMFNATLFALVRTALKIKTEGNLEQANEELRAVIKKIWKKTSMKLLDQVVPPAGDDEV-TKLRAACIRIQKTIRGWLLRKRYL* with the IQ domain italicized. For CaV1.3-NaV1.4IQ, the gene fragment encoded GTVMFNATLFALVRTALKIKTEGNLEQANEELRAVIKKIWKKTSMKLLDQVVPPAGDDEVTLKRKHEEVCAIKIQRAYRRHLLQRSMKQAS* with the NaV1.4 segment italicized.

      Cell culture and transfection of HEK293 cells

      For whole-cell electrophysiology, HEK293 cells were cultured on glass coverslips in 60-mm dishes and transfected using a calcium phosphate method (
      • 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.
      ). We applied 2–4 μg of cDNA encoding the desired channel α1 subunit (WT or CaM-linked variant), along with 4 μg of rat brain β2A (WT or CaM-linked) and 4 μg of rat brain α2δ subunits (NM012919.2) (
      • Tomlinson W.J.
      • Stea A.
      • Bourinet E.
      • Charnet P.
      • Nargeot J.
      • Snutch T.P.
      Functional properties of a neuronal class C L-type calcium channel.
      ). To enhance expression, cDNA for simian virus 40 T antigen (1 μg) was co-transfected. Electrophysiology recordings were done at room temperature 1–2 days after transfection.

      Whole-cell electrophysiology recordings

      Whole-cell voltage-clamp recordings for HEK293 were collected at room temperature using an Axopatch 200A amplifier (Axon Instruments). Glass pipettes (World Precision Instruments, MTW 150-F4) were pulled with a horizontal puller (P-97; Sutter Instruments Co.) and fire-polished (Microforge, Narishige, Tokyo, Japan), resulting in 1–3-megaohm resistances, before series resistance compensation of 70%. For CaV1.2 and CaV1.3 recordings, the internal solutions contained 135 mm CsMeSO3, 5 mm CsCl2, 1 mm MgCl2, 4 mm MgATP, 10 mm HEPES, 10 mm BAPTA, adjusted to 295 mosm with CsMeSO3 and pH 7.4 with CsOH. The external solution contained 140 mm TEA-MeSO3, 10 mm HEPES, 40 mm CaCl2 or BaCl2, adjusted to 300 mosm with TEA-MeSO3 and pH 7.4 with TEA-OH. This external solution composition was chosen based on previous studies to ensure that local Ca2+ signals are saturating to drive maximal local CDI (
      • Tadross M.R.
      • Tsien R.W.
      • Yue D.T.
      Ca2+ channel nanodomains boost local Ca2+ amplitude.
      ,
      • Tadross M.R.
      • Dick I.E.
      • Yue D.T.
      Mechanism of local and global Ca2+ sensing by calmodulin in complex with a Ca2+ channel.
      ). For CaV2.1 recording, the internal solutions contained 135 mm CsMeSO3, 5 mm CsCl2, 1 mm MgCl2, 4 mm MgATP, 10 mm HEPES, 1 mm EGTA, adjusted to 295 mosm with CsMeSO3 and pH 7.4 with CsOH. The external solution contained 140 mm TEA-MeSO3, 10 mm HEPES, 5 mm CaCl2 or BaCl2, adjusted to 300 mosm with TEA-MeSO3 and pH 7.4 with TEA-OH. For CDI measurements, we used a family of test pulses from −50 to +50 mV with repetition intervals of 20 s, at a holding potential of −80 mV. Custom MATLAB (Mathworks) software was used to determine peak current and fraction of peak current remaining after either 300 ms (r300) of depolarization for CaV1 or 800 ms (r800) of depolarization for CaV2. Ca2+-dependent facilitation was quantified using the normalized charge difference ΔQ, obtained by integrating the difference between normalized traces ± prepulse as reported previously (
      • 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.
      ). The fraction of channels facilitated by prepulse is directly proportional to ΔQ divided by the slow time constant (t) of facilitation, yielding relative facilitation (RF = ΔQ/t). For knockouts of Ca2+-dependent facilitation, t was set to 12 ms (matching the average time constant for facilitation in channels lacking tethered CaM). RFCa corresponds to relative facilitation with Ca2+ as charge carrier, whereas RFBa corresponds to that obtained with Ba2+ as charge carrier representing voltage-dependent facilitation. CDF is measured as the difference RFCaRFBa (
      • 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.
      ).

      Data availability

      All original data are fully available upon request from Manu Ben-Johny.

      Acknowledgments

      We thank members of the Ben-Johny laboratory for insightful comments and Dr. Jacqueline Niu for helpful comments on experimental design and for assistance with data analysis. We thank Dr. Shin Rong Lee for construction of CaV2.1 fused to CaM. We thank the late Dr. David Yue, who was instrumental in the initial conceptualization and experiment design for this project. Dr. Yue passed away on December 23, 2014. He was an extraordinary mentor and teacher, and his unbridled passion for science continues to inspire us.

      Supplementary Material

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