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Fluorescence Resonance Energy Transfer-based Structural Analysis of the Dihydropyridine Receptor α1S Subunit Reveals Conformational Differences Induced by Binding of the β1a Subunit*

  • Mohana
    Affiliations
    From the Department of Anesthesia, Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115
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  • Claudio F. Perez
    Correspondence
    To whom correspondence may be addressed: Brigham and Women's Hospital, 75 Francis St., Boston MA 02115. Tel.: 617-525-6486; Fax: 617-732-6927
    Affiliations
    From the Department of Anesthesia, Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115
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  • James D. Fessenden
    Correspondence
    To whom correspondence may be addressed: Brigham and Women's Hospital, 75 Francis St., Boston MA 02115. Tel.: 617-525-8332; Fax: 617-732-6927
    Affiliations
    From the Department of Anesthesia, Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grants R03AR066359 (to C. F. P.), R01AR059124 (to J. D. F.), and R01AR067738 (to C. F. P. and J. D. F.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors declare that they have no conflicts of interest with the contents of this article.
Open AccessPublished:April 25, 2016DOI:https://doi.org/10.1074/jbc.M115.704049
      The skeletal muscle dihydropyridine receptor α1S subunit plays a key role in skeletal muscle excitation-contraction coupling by sensing membrane voltage changes and then triggering intracellular calcium release. The cytoplasmic loops connecting four homologous α1S structural domains have diverse functions, but their structural arrangement is poorly understood. Here, we used a novel FRET-based method to characterize the relative proximity of these intracellular loops in α1S subunits expressed in intact cells. In dysgenic myotubes, energy transfer was observed from an N-terminal-fused YFP to a FRET acceptor, ReAsH (resorufin arsenical hairpin binder), targeted to each α1S intracellular loop, with the highest FRET efficiencies measured to the α1S II-III loop and C-terminal tail. However, in HEK-293T cells, FRET efficiencies from the α1S N terminus to the II-III and III-IV loops and the C-terminal tail were significantly lower, thus suggesting that these loop structures are influenced by the cellular microenvironment. The addition of the β1a dihydropyridine receptor subunit enhanced FRET to the II-III loop, thus indicating that β1a binding directly affects II-III loop conformation. This specific structural change required the C-terminal 36 amino acids of β1a, which are essential to support EC coupling. Direct FRET measurements between α1S and β1a confirmed that both wild type and truncated β1a bind similarly to α1S. These results provide new insights into the role of muscle-specific proteins on the structural arrangement of α1S intracellular loops and point to a new conformational effect of the β1a subunit in supporting skeletal muscle excitation-contraction coupling.

      Introduction

      In skeletal muscle excitation contraction (EC)
      The abbreviations used are: EC
      excitation-contraction
      DHPR
      dihydropyridine receptor
      RyR
      ryanodine receptor
      CFP
      cyan fluorescent protein
      ReAsH
      resorufin arsenical hairpin binder
      Tc
      tetracysteine
      FRET
      fluorescence resonance energy transfer
      YFP
      yellow fluorescent protein.
      coupling, the dihydropyridine receptor (DHPR), an L-type voltage-gated Ca2+ channel, senses membrane depolarization and then initiates intracellular Ca2+ release by activating the type 1 ryanodine receptor (RyR1) embedded in the sarcoplasmic reticulum membrane. Of the five skeletal DHPR subunits, α1S and β1a are absolutely required for skeletal type EC coupling (
      • Beam K.G.
      • Knudson C.M.
      • Powell J.A.
      A lethal mutation in mice eliminates the slow calcium current in skeletal muscle cells.
      ,
      • Beurg M.
      • Sukhareva M.
      • Strube C.
      • Powers P.A.
      • Gregg R.G.
      • Coronado R.
      Recovery of Ca2+ current, charge movements, and Ca2+ transients in myotubes deficient in dihydropyridine receptor β1 subunit transfected with β1 cDNA.
      ,
      • Chaudhari N.
      • Beam K.G.
      The muscular dysgenesis mutation in mice leads to arrest of the genetic program for muscle differentiation.
      ,
      • Coronado R.
      • Ahern C.A.
      • Sheridan D.C.
      • Cheng W.
      • Carbonneau L.
      • Bhattacharya D.
      Functional equivalence of dihydropyridine receptor α1S and β1a subunits in triggering excitation-contraction coupling in skeletal muscle.
      ,
      • Gregg R.G.
      • Messing A.
      • Strube C.
      • Beurg M.
      • Moss R.
      • Behan M.
      • Sukhareva M.
      • Haynes S.
      • Powell J.A.
      • Coronado R.
      • Powers P.A.
      Absence of the β subunit (cchb1) of the skeletal muscle dihydropyridine receptor alters expression of the α1 subunit and eliminates excitation-contraction coupling.
      ,
      • Knudson C.M.
      • Chaudhari N.
      • Sharp A.H.
      • Powell J.A.
      • Beam K.G.
      • Campbell K.P.
      Specific absence of the α1 subunit of the dihydropyridine receptor in mice with muscular dysgenesis.
      ,
      • Tanabe T.
      • Beam K.G.
      • Powell J.A.
      • Numa S.
      Restoration of excitation-contraction coupling and slow calcium current in dysgenic muscle by dihydropyridine receptor complementary DNA.
      ,
      • Flucher B.E.
      • Obermair G.J.
      • Tuluc P.
      • Schredelseker J.
      • Kern G.
      • Grabner M.
      The role of auxiliary dihydropyridine receptor subunits in muscle.
      ). The 170-kDa α1S subunit contains both the voltage sensor and Ca2+ conduction pore and is composed of four homologous domains, each containing six transmembrane segments (
      • Tanabe T.
      • Takeshima H.
      • Mikami A.
      • Flockerzi V.
      • Takahashi H.
      • Kangawa K.
      • Kojima M.
      • Matsuo H.
      • Hirose T.
      • Numa S.
      Primary structure of the receptor for calcium channel blockers from skeletal muscle.
      ,
      • Catterall W.A.
      Structure and function of voltage-sensitive ion channels.
      ). These domains are connected by intracellular loops with well defined functions. For example the I-II loop has an α1S subunit interaction domain binding site for the β1a DHPR subunit (
      • Witcher D.R.
      • De Waard M.
      • Liu H.
      • Pragnell M.
      • Campbell K.P.
      Association of native Ca2+ channel β subunits with the α1 subunit interaction domain.
      ,
      • Pragnell M.
      • De Waard M.
      • Mori Y.
      • Tanabe T.
      • Snutch T.P.
      • Campbell K.P.
      Calcium channel β-subunit binds to a conserved motif in the I-II cytoplasmic linker of the α1-subunit.
      ), whereas the II-III loop contains an essential determinant (amino acids 720–765) required to activate RyR1 during skeletal-type EC coupling (
      • Grabner M.
      • Dirksen R.T.
      • Suda N.
      • Beam K.G.
      The II-III loop of the skeletal muscle dihydropyridine receptor is responsible for the Bi-directional coupling with the ryanodine receptor.
      ,
      • Nakai J.
      • Tanabe T.
      • Konno T.
      • Adams B.
      • Beam K.G.
      Localization in the II-III loop of the dihydropyridine receptor of a sequence critical for excitation-contraction coupling.
      ). Although the III-IV loop does not appear to have a direct role in RyR1 activation, a malignant hyperthermia mutation located in this loop (R1086H) has been reported to alter DHPR gating properties and EC coupling (
      • Weiss R.G.
      • O'Connell K.M.
      • Flucher B.E.
      • Allen P.D.
      • Grabner M.
      • Dirksen R.T.
      Functional analysis of the R1086H malignant hyperthermia mutation in the DHPR reveals an unexpected influence of the III-IV loop on skeletal muscle EC coupling.
      ). Finally, the C-terminal tail has been implicated as binding Ca2+/calmodulin (
      • 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.
      ) as well as mediating proper DHPR targeting to triad junctions (
      • Flucher B.E.
      • Kasielke N.
      • Grabner M.
      The triad targeting signal of the skeletal muscle calcium channel is localized in the COOH terminus of the α1S subunit.
      ). Although a recent high resolution cryo-EM reconstruction of the DHPR has resolved many of the membrane-spanning helices in the complex (
      • Wu J.
      • Yan Z.
      • Li Z.
      • Yan C.
      • Lu S.
      • Dong M.
      • Yan N.
      Structure of the voltage-gated calcium channel Cav1.1 complex.
      ), these key α1s intracellular loops are completely absent from the structure, most likely due to intrinsic flexibility. Thus, understanding the relative arrangement of these loops as well as how they change conformation due to binding of cell-specific factors remain as key questions.
      Like the α1S subunit, the β1a DHPR subunit also plays an essential role in skeletal muscle EC coupling. β1a is required to target α1S to the cell surface and to support depolarization-induced intracellular Ca2+ release (i.e. orthograde signaling) as well as to enhance DHPR inward Ca2+ current (i.e. retrograde signaling) (
      • Beurg M.
      • Sukhareva M.
      • Strube C.
      • Powers P.A.
      • Gregg R.G.
      • Coronado R.
      Recovery of Ca2+ current, charge movements, and Ca2+ transients in myotubes deficient in dihydropyridine receptor β1 subunit transfected with β1 cDNA.
      ,
      • Gregg R.G.
      • Messing A.
      • Strube C.
      • Beurg M.
      • Moss R.
      • Behan M.
      • Sukhareva M.
      • Haynes S.
      • Powell J.A.
      • Coronado R.
      • Powers P.A.
      Absence of the β subunit (cchb1) of the skeletal muscle dihydropyridine receptor alters expression of the α1 subunit and eliminates excitation-contraction coupling.
      ,
      • Karunasekara Y.
      • Dulhunty A.F.
      • Casarotto M.G.
      The voltage-gated calcium-channel β subunit: more than just an accessory.
      ). β1a is also needed to assemble the DHPR complex into tetradic arrays visualized in freeze-fracture studies (
      • Schredelseker J.
      • Di Biase V.
      • Obermair G.J.
      • Felder E.T.
      • Flucher B.E.
      • Franzini-Armstrong C.
      • Grabner M.
      The β 1a subunit is essential for the assembly of dihydropyridine-receptor arrays in skeletal muscle.
      ,
      • Schredelseker J.
      • Dayal A.
      • Schwerte T.
      • Franzini-Armstrong C.
      • Grabner M.
      Proper restoration of excitation-contraction coupling in the dihydropyridine receptor β1-null zebrafish relaxed is an exclusive function of the β1a subunit.
      ,
      • Eltit J.M.
      • Franzini-Armstrong C.
      • Perez C.F.
      Amino acid residues 489–503 of dihydropyridine receptor (DHPR) β1a subunit are critical for structural communication between the skeletal muscle DHPR complex and type 1 ryanodine receptor.
      ). An essential determinant between residues 489–503 of the β1a C-terminal tail is required for both bidirectional signaling and tetrad formation (
      • Eltit J.M.
      • Franzini-Armstrong C.
      • Perez C.F.
      Amino acid residues 489–503 of dihydropyridine receptor (DHPR) β1a subunit are critical for structural communication between the skeletal muscle DHPR complex and type 1 ryanodine receptor.
      ,
      • Sheridan D.C.
      • Cheng W.
      • Ahern C.A.
      • Mortenson L.
      • Alsammarae D.
      • Vallejo P.
      • Coronado R.
      Truncation of the carboxyl terminus of the dihydropyridine receptor β1a subunit promotes Ca2+ dependent excitation-contraction coupling in skeletal myotubes.
      ).
      Recent structural analysis of the DHPR intracellular loops has been achieved using fluorescence resonance energy transfer (FRET) measurements between cyan and yellow fluorescent proteins (CFP/YFP) fused into α1S and β1a (
      • Papadopoulos S.
      • Leuranguer V.
      • Bannister R.A.
      • Beam K.G.
      Mapping sites of potential proximity between the dihydropyridine receptor and RyR1 in muscle using a cyan fluorescent protein-yellow fluorescent protein tandem as a fluorescence resonance energy transfer probe.
      ,
      • Polster A.
      • Ohrtman J.D.
      • Beam K.G.
      • Papadopoulos S.
      Fluorescence resonance energy transfer (FRET) indicates that association with the type I ryanodine receptor (RyR1) causes reorientation of multiple cytoplasmic domains of the dihydropyridine receptor (DHPR) α1S subunit.
      ). These studies have suggested a direct role of RyR1 in the structural organization of the α1S subunit and have identified potential RyR-interacting domains in α1S and β1a. Although these studies illustrate the potential of FRET-based approaches for structural analysis of the DHPR complex in situ, they have been hampered by the relative bulkiness of the fused CFP/YFP FRET probes, as suggested by functional impairments of α1S subunits carrying a fluorescent protein fused into the III-IV loop (
      • Polster A.
      • Ohrtman J.D.
      • Beam K.G.
      • Papadopoulos S.
      Fluorescence resonance energy transfer (FRET) indicates that association with the type I ryanodine receptor (RyR1) causes reorientation of multiple cytoplasmic domains of the dihydropyridine receptor (DHPR) α1S subunit.
      ). In addition, FRET data derived exclusively from fluorescent protein insertions can be difficult to interpret due to the distance between their chromophoric centers and their insertion sites (
      • Raina S.A.
      • Tsai J.
      • Samsó M.
      • Fessenden J.D.
      FRET-based localization of fluorescent protein insertions within the ryanodine receptor type 1.
      ) as well as the bulkiness of these inserted proteins.
      In this report we used an innovative site-specific labeling method combined with FRET-based structural measurements to determine the spatial interrelationships between cytoplasmic loops of α1S subunits expressed in cultured myotubes harboring the α1s muscular dysgenesis mutation (mdg) (
      • Knudson C.M.
      • Chaudhari N.
      • Sharp A.H.
      • Powell J.A.
      • Beam K.G.
      • Campbell K.P.
      Specific absence of the α1 subunit of the dihydropyridine receptor in mice with muscular dysgenesis.
      ,
      • Tanabe T.
      • Beam K.G.
      • Powell J.A.
      • Numa S.
      Restoration of excitation-contraction coupling and slow calcium current in dysgenic muscle by dihydropyridine receptor complementary DNA.
      ), which renders these dysgenic myotubes completely deficient in native αls expression (
      • Knudson C.M.
      • Chaudhari N.
      • Sharp A.H.
      • Powell J.A.
      • Beam K.G.
      • Campbell K.P.
      Specific absence of the α1 subunit of the dihydropyridine receptor in mice with muscular dysgenesis.
      ). Energy transfer was measured from YFP fused to the N terminus of α1s to the cell-permeant resorufin arsenical hairpin binder ReAsH (
      • Adams S.R.
      • Campbell R.E.
      • Gross L.A.
      • Martin B.R.
      • Walkup G.K.
      • Yao Y.
      • Llopis J.
      • Tsien R.Y.
      New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications.
      ) targeted to short tetracysteine (Tc) motifs inserted into each of the α1S intracellular loops. Our results suggest that within the triadic environment, the α1s loops are clustered together, with the II-III loop and C terminus being closest to the α1S N terminus. However, in a non-myogenic system (HEK-293T cells), FRET to the II-III and III-IV loops and the C-terminal tail is significantly lower, thus suggesting that cellular microenvironment influences α1S structure. Finally, we found that binding of the β1a subunit affects the structure of the α1S II-III loop in situ. This study reveals the influence of muscle-specific environmental factors on the arrangement of the α1S cytosolic loops and provides the first direct evidence that the β1a subunit modulates the α1S II-III loop conformation.

      Experimental Procedures

      α1S DHPR cDNA Cloning

      A full-length rabbit α1S cDNA (amino acids 1–1873) (GenBankTM accession number M23919) was cloned in-frame downstream from the YFP-citrine gene (
      • Griesbeck O.
      • Baird G.S.
      • Campbell R.E.
      • Zacharias D.A.
      • Tsien R.Y.
      Reducing the environmental sensitivity of yellow fluorescent protein: mechanism and applications.
      ) in the bicistronic retroviral vector pCMMP-MCS-IRES-Puro, containing a puromycin resistance gene (Addgene 36952; Ref.
      • Lee D.Y.
      • Sugden B.
      The LMP1 oncogene of EBV activates PERK and the unfolded protein response to drive its own synthesis.
      ). Insertion of a Tc tag-encoding sequence (FLNCCPGCCMEP) into α1S was performed using Gibson assembly (New England BioLabs). Double-stranded DNA fragments (i.e. gBlocks, Integrated DNA Technologies) encoding the Tc tag were inserted in-frame at the following α1S locations; I-II loop (amino acid 388), II-III loop (amino acid 719 and 726), III-IV loop (amino acid 1076), and C-terminal tail (amino acid 1636). A Tc tag at the N-terminal end (amino acid 1) was inserted in-frame upstream of the YFP sequence as a positive control in FRET experiments, and all clones were confirmed by DNA sequencing.

      β1a DHPR cDNA Cloning and Purification

      Full-length mouse β1a cDNA (GenBankTM, NM_031173) was cloned into the pCold-II expression vector (TakaraTM) in-frame downstream from a streptavidin binding tag, which was used to assist with purification. The clone was expressed in the Escherichia coli BL21 strain in combination with the pG-KJE8 vector (TakaraTM) to optimize protein folding. Protein induction and purification were performed using isopropyl 1-thio-β-d-galactopyranoside/arabinose/tetracycline and Strep-trap affinity chromatography columns as described (
      • Chen Y.
      • Xue S.
      • Zou J.
      • Lopez J.R.
      • Yang J.J.
      • Perez C.F.
      Myoplasmic resting Ca2+ regulation by ryanodine receptors is under the control of a novel Ca2+-binding region of the receptor.
      ).

      Cell Culture and Immunocytochemistry

      cDNAs were expressed in dysgenic myotubes after packaging into lentivirus using a set of three packaging vectors as described (
      • Eltit J.M.
      • Franzini-Armstrong C.
      • Perez C.F.
      Amino acid residues 489–503 of dihydropyridine receptor (DHPR) β1a subunit are critical for structural communication between the skeletal muscle DHPR complex and type 1 ryanodine receptor.
      ). Dysgenic myoblasts were infected with lentiviral particles containing YFP-α1S cDNAs at a multiplicity of infection = 0.5 and then selected with 1.5 μg/ml puromycin for 2 weeks. Individual myoblast colonies stably transduced with each clone were then isolated using glass rings.
      DHPR-expressing myoblasts were grown and differentiated in 96-well plates as described (
      • Eltit J.M.
      • Franzini-Armstrong C.
      • Perez C.F.
      Amino acid residues 489–503 of dihydropyridine receptor (DHPR) β1a subunit are critical for structural communication between the skeletal muscle DHPR complex and type 1 ryanodine receptor.
      ). α1S DHPR expression was confirmed by immunocytochemistry on methanol-fixed cultured myotubes using either anti-α1S MA3–921 (Thermo Scientific) or anti-GFP G10362 (Life Technologies) monoclonal antibodies.
      HEK-293T cells were grown and polyethyleneimine-transfected with Tc-tagged YFP-α1S cDNAs as described (
      • Fessenden J.D.
      Förster resonance energy transfer measurements of ryanodine receptor type 1 structure using a novel site-specific labeling method.
      ). Two days after transfection, cells were tested in FRET-based assays.

      Calcium Imaging

      After differentiation for 4–5 days, myotubes expressing YFP-α1S cDNAs were loaded with 5 μm Fura-2 AM (Molecular Probes) in imaging buffer (125 mm NaCl, 5 mm KCl, 2 mm CaCl2, 1.2 mm MgSO4, 6 mm glucose, and 25 mm HEPES/Tris, pH 7.4). Membrane depolarization was performed by a 5-s perfusion with 5–7 volumes of imaging buffer containing increasing concentrations of KCl. Cells were imaged with an intensified 10-bit digital CCD camera (XR-Mega-10, Stanford Photonics, Stanford, CA) using a DG4 multiwavelength light source (Sutter Instruments, Novato, CA). Fluorescent emission at 510 nm was captured from regions of interest within each myotube at 33 frames per second using Piper acquisition software (Stanford Photonics) and expressed as ratio of signal collected at alternating 340/380-nm excitation wavelengths. Calcium transients quantified from the peak amplitudes were plotted as a function of added KCl and fit to a sigmoidal dose-response function (variable slope) to determine EC50 values, which were then compared via analysis of variance.

      Labeling with FRET Acceptors

      ReAsH labeling of Tc-tagged α1S- expressing intact cells was performed as described (
      • Mahalingam M.
      • Fessenden J.D.
      Methods for labeling skeletal muscle ion channels site-specifically with fluorophores suitable for FRET-based structural analysis.
      ) with some modifications. ReAsH was first complexed with ethane dithiol (EDT) for 10 min in a reaction consisting of 0.5 mm ReAsH and 12.5 mm EDT in DMSO. This reaction mixture was diluted 1:1000 in FRET buffer consisting of 125 mm NaCl, 5 mm KCl, 6 mm glucose, and 25 mm HEPES, pH 7.6, and then added to myotubes or HEK-293T cells expressing Tc-tagged YFP-α1S constructs. Cells were incubated at 37 °C for 1.5 h followed by washing with 100 μm British anti-Lewisite for 15 min to reduce nonspecific ReAsH labeling. In some experiments HEK-293T cells expressing Tc-tagged YFP-α1S constructs were permeabilized with 0.1% saponin and then incubated with 150 nm purified β1a protein for 2 h at 37C. Cells were then tested in FRET experiments (see below).
      FRET measurements between α1S and β1a subunit were conducted using the His-tag-specific FRET acceptor, Cy3NTA, as described previously (
      • Fessenden J.D.
      Förster resonance energy transfer measurements of ryanodine receptor type 1 structure using a novel site-specific labeling method.
      ). HEK-293T cells expressing YFP-α1S constructs were permeabilized using 0.1% saponin in FRET buffer containing 3 μm Cy3NTA with or without 150 nm purified β1a protein bearing 10 histidine residues (His10) at its N-terminal end. After incubation for 2 h at 37 C, cells were analyzed for FRET.

      FRET Imaging

      FRET was measured using epifluorescence microscopy as described (
      • Fessenden J.D.
      Förster resonance energy transfer measurements of ryanodine receptor type 1 structure using a novel site-specific labeling method.
      ,
      • Girgenrath T.
      • Mahalingam M.
      • Svensson B.
      • Nitu F.R.
      • Cornea R.L.
      • Fessenden J.D.
      N-terminal and central segments of the type 1 ryanodine receptor mediate its interaction with FK506-binding proteins.
      ). Briefly, YFP donor fluorescence was acquired using a 480/30-nm bandpass excitation filter and 535/40-nm bandpass emission filter as a series of 60 16-bit 672 × 516 pixel images across a z stack 60 μm in thickness. Acceptor fluorescence was photobleached for 4 min at maximum DG-4 light source intensity using a ReAsH cubeset composed of a 570/20-nm bandpass excitation filter and 620/60-nm bandpass emission filter. FRET was measured as an enhancement in donor fluorescence upon acceptor photobleaching using,
      MathJax Formula fx1
      (Eq. 1)


      where E represents FRET efficiency, and Fprebleach andFpostbleach indicate donor fluorescence intensities before and after acceptor photobleaching, respectively. Fluorescence was quantified using ImageJ version 1.43u as described (
      • Fessenden J.D.
      • Mahalingam M.
      Site-specific labeling of the type 1 ryanodine receptor using biarsenical fluorophores targeted to engineered tetracysteine motifs.
      ).

      Measurement of ReAsH Labeling Efficiency

      For each Tc-tagged YFP-α1s construct expressed in HEK-293T cells, YFP and ReAsH fluorescence was quantified both before and after ReAsH photobleaching. ReAsH labeling efficiency was then calculated as the ratio of ReAsH (pre-acceptor bleach) to YFP (post-acceptor bleach) fluorescence for each construct. Note that YFP fluorescence acquired after acceptor bleach was used for these calculations to eliminate any contribution of YFP/ReAsH FRET from these measurements.

      Molecular Visualization and Distance Measurements

      An atomic model (Protein Data Bank Accession code 3JBR) derived from a high resolution cryo-EM reconstruction of the full DHPR complex (
      • Wu J.
      • Yan Z.
      • Li Z.
      • Yan C.
      • Lu S.
      • Dong M.
      • Yan N.
      Structure of the voltage-gated calcium channel Cav1.1 complex.
      ) was used for distance measurements and preparation of Fig. 7, which was created using Chimera image processing software version 1.10.1 (build 40427) (
      • Pettersen E.F.
      • Goddard T.D.
      • Huang C.C.
      • Couch G.S.
      • Greenblatt D.M.
      • Meng E.C.
      • Ferrin T.E.
      UCSF Chimera: a visualization system for exploratory research and analysis.
      ).
      Figure thumbnail gr7
      FIGURE 7Model of the skeletal muscle DHPR complex. A, structure of the DHPR complex derived from high resolution cryo-EM reconstructions. α1s (blue) and β1a (green) subunits are shown as well as a putative location of YFP (yellow) fused to the α1s N terminus. Locations of the intradomain loops in the α1s subunit are shown as colored dots as well as the distance from the center of the II-III loop to the β1a subunit. The plasma membrane lipid bilayer is depicted as dashed lines. Structure is shown in cross-section with the extracellular face of the channel at the top of the figure. B, DHPR complex viewed from the cytoplasmic side after 900 rotation as shown. Note that because intradomain loops are not defined in the high resolution maps, arrows point to colored spheres arbitrarily placed at the center of a line connecting the nearest known sequence elements flanking each loop.

      Results

      Expression and Functional Analysis of Tc-tagged YFP-α1S DHPRs in Dysgenic Myotubes

      To characterize the relative proximity of the cytoplasmic loops in the α1S DHPR subunit using FRET, we fused citrine, a bright YFP variant (
      • Griesbeck O.
      • Baird G.S.
      • Campbell R.E.
      • Zacharias D.A.
      • Tsien R.Y.
      Reducing the environmental sensitivity of yellow fluorescent protein: mechanism and applications.
      ), to the N terminus of α1S to act as a FRET donor. Then we inserted short Tc motifs (FLNCCPGCCMEP; Ref.
      • Adams S.R.
      • Campbell R.E.
      • Gross L.A.
      • Martin B.R.
      • Walkup G.K.
      • Yao Y.
      • Llopis J.
      • Tsien R.Y.
      New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications.
      ) separately into each α1S cytoplasmic loop and domain (Fig. 1A) to act as binding sites for the FRET acceptor ReAsH. Proper targeting of these Tc-tagged YFP-α1S fusion constructs to the junctional sarcoplasmic reticulum of stably transduced dysgenic myotubes was confirmed by immunocytochemistry (Fig. 1B). After staining with an anti-GFP antibody, both YFP-α1S and Tc-tagged YFP-α1S constructs displayed discrete fluorescent foci that closely resembled the immunofluorescent pattern observed in wild type myotubes labeled with anti-α1S antibodies (Fig. 1B). This staining pattern is known to represent the peripheral couplings where the sarcoplasmic reticulum terminal cisternae interact with the surface membrane (
      • Flucher B.E.
      • Andrews S.B.
      • Daniels M.P.
      Molecular organization of transverse tubule/sarcoplasmic reticulum junctions during development of excitation-contraction coupling in skeletal muscle.
      ). These results suggest that Tc-tag insertions did not affect α1S targeting to peripheral couplings.
      Figure thumbnail gr1
      FIGURE 1Tc-tagged YFP-α1S DHPR fusion proteins are efficiently targeted to peripheral couplings in dysgenic myotubes. A, schematic representation of the α1S DHPR subunit. YFP (yellow oval) and α1S protein sequence positions of the Tc tag insertions in each intracellular loop and domain are shown. B, representative immunolocalizations of Tc-tagged YFP-α1S constructs stably expressed in dysgenic myotubes. Punctate localization pattern for these recombinant constructs probed with an anti-GFP antibody (bottom panels) is similar to immunostaining pattern for wild type myotubes labeled with an anti-α1S antibody (top panel).
      To functionally characterize these Tc-tagged α1S channels, we quantified depolarization-induced Ca2+ release (i.e. EC coupling) in Fura-2-loaded stably transduced dysgenic myotubes. Each Tc-tagged α1S construct restored robust Ca2+ release in response to K+-depolarization. This Ca2+ release was comparable with control myotubes expressing YFP-α1S lacking a Tc tag (−Tc; Fig. 2A). A small but significant reduction in average peak Ca2+ transient was observed in myotubes expressing constructs Tc-tagged at the N terminus, I-II loop, and II-III loop (Fig. 2B). Because no changes in sensitivity to K+ depolarization were observed for these constructs compared with −Tc controls (p > 0.05), it is conceivable that the smaller Ca2+ transients result from slight differences in α1S expression between individual cell clones. Myotubes expressing a Tc-tagged III-IV loop α1S construct restored robust EC coupling though with enhanced K+ sensitivity. Mutations in the III-IV loop region have been reported to affect the conductive properties of the α1S subunit (
      • Weiss R.G.
      • O'Connell K.M.
      • Flucher B.E.
      • Allen P.D.
      • Grabner M.
      • Dirksen R.T.
      Functional analysis of the R1086H malignant hyperthermia mutation in the DHPR reveals an unexpected influence of the III-IV loop on skeletal muscle EC coupling.
      ,
      • Bannister R.A.
      • Grabner M.
      • Beam K.G.
      The α1S III-IV loop influences 1,4-dihydropyridine receptor gating but is not directly involved in excitation-contraction coupling interactions with the type 1 ryanodine receptor.
      ). However, whether the insertion of the Tc tag in our study alters the voltage-sensing properties of the DHPR complex is currently unknown and will require further testing. Thus, even though small functional changes were observed in some cases, the overall targeting and function of these YFP-α1S constructs was largely unaffected by the Tc-tag insertions.
      Figure thumbnail gr2
      FIGURE 2Tc-tagged YFP-α1S DHPRs restore depolarization-induced calcium release in dysgenic myotubes. A, representative fluorescent traces of K+-induced Ca2+ release responses of dysgenic myotubes stably transduced with the indicated YFP-α1S constructs. Myotubes loaded with 5 μm Fura-2 were exposed to increasing concentrations of KCl for 5 s (black boxes). B, average peak Ca2+ transient amplitude of control (−Tc) and Tc-tagged α1S constructs expressed in dysgenic myotubes. Note that the II-III loop construct was tested in transiently transfected myotubes. The data are from 2 experiments (10–25 cells total) and are presented as the mean ± S.E.

      FRET-based Structural Analysis of Tc-tagged YFP-α1S DHPRs

      FRET-based analysis was used to characterize the structural arrangement of the α1S intracellular loops (Fig. 3). The cell-permeant biarsenical FRET acceptor, ReAsH (
      • Adams S.R.
      • Campbell R.E.
      • Gross L.A.
      • Martin B.R.
      • Walkup G.K.
      • Yao Y.
      • Llopis J.
      • Tsien R.Y.
      New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications.
      ), was targeted to the Tc-tagged YFP-α1S constructs (Fig. 3A), and energy transfer was quantified from the increase in the N-terminally fused YFP donor fluorescence after acceptor photobleaching (
      • Fessenden J.D.
      • Mahalingam M.
      Site-specific labeling of the type 1 ryanodine receptor using biarsenical fluorophores targeted to engineered tetracysteine motifs.
      ). As reported (
      • Stroffekova K.
      • Proenza C.
      • Beam K.G.
      The protein-labeling reagent FLASH-EDT2 binds not only to CCXXCC motifs but also non-specifically to endogenous cysteine-rich proteins.
      ), untransfected dysgenic myotubes were strongly labeled by ReAsH, which persisted even after washing with British anti-Lewisite (
      • Griffin B.A.
      • Adams S.R.
      • Jones J.
      • Tsien R.Y.
      Fluorescent labeling of recombinant proteins in living cells with FlAsH.
      ) to remove nonspecific ReAsH labeling (data not shown). However, this background ReAsH labeling did not impede our ability to measure FRET as only very low background energy transfer (E = 0.1) was observed in ReAsH-labeled myotubes expressing non-Tc-tagged control YFP-α1S (-−Tc). On the other hand, significant energy transfer above background was detected to ReAsH separately targeted to each intracellular domain (Fig. 3B, filled bars). As expected, a positive control construct consisting of a Tc-tag/YFP tandem fused at the α1S N terminus displayed the highest FRET efficiency (N-term, E = 0.35; Fig. 3B), similar to FRET values previously reported for this same tandem inserted into RyR1 (
      • Fessenden J.D.
      • Mahalingam M.
      Site-specific labeling of the type 1 ryanodine receptor using biarsenical fluorophores targeted to engineered tetracysteine motifs.
      ). FRET efficiencies measured to the I-II loop (E = 0.20), II-III loop (E = 0.25), and the C-terminal tail (E = 0.24) were also significantly higher compared with the −Tc negative control construct. Average FRET efficiency measured to the III-IV loop (E = 0.18) was slightly but significantly reduced compared with the II-III loop and C-terminal domain (p < 0.05), thus suggesting differences among the loops in either relative distance to the α1S N terminus and/or orientation of the Tc tags. Overall, these results indicate that FRET efficiencies measured between the α1S N terminus and each cytoplasmic domain are similar, and therefore, these loops might be tightly clustered in dysgenic myotubes.
      Figure thumbnail gr3
      FIGURE 3FRET-based structural measurements of the α1S DHPR subunit reveal key conformational differences caused by the cellular microenvironment. A, schematic representation of the FRET donor/acceptor pair in the α1S DHPR subunit. YFP (yellow oval) acts as a FRET donor, transferring energy to the FRET acceptor ReAsH, targeted specifically to tetracysteine tags (red rectangles) separately inserted into each α1S cytoplasmic loop. B, FRET efficiency values are shown from the N-terminal YFP to ReAsH targeted to the indicated α1S intracellular loops for constructs expressed in either HEK-293T cells (open bars) or dysgenic myotubes (filled bars). Data points represent mean FRET efficiency values ± S.E. for the number of cells indicated in each bar. The asterisk indicates a significant difference in energy transfer (p < 0.01 using a paired two-tailed t test) for a given construct tested in either HEK-293T cells or dysgenic myotubes. Short and Long C-term constructs contain a Tc tag at position 1636 and a stop codon inserted at either position 1664 or 1873, respectively. All other constructs are full-length (i.e. 1873 residues).
      Previous FRET studies have suggested that skeletal muscle-specific protein-protein interactions can affect the organization of the α1S cytoplasmic loops (
      • Polster A.
      • Ohrtman J.D.
      • Beam K.G.
      • Papadopoulos S.
      Fluorescence resonance energy transfer (FRET) indicates that association with the type I ryanodine receptor (RyR1) causes reorientation of multiple cytoplasmic domains of the dihydropyridine receptor (DHPR) α1S subunit.
      ). Thus, we performed a parallel series of FRET measurements on these α1S constructs expressed in HEK-293T cells (Fig. 3B, open bars), which lack skeletal muscle-specific proteins including the DHPR and RyR1. ReAsH-labeled HEK-293T cells expressing YFP-α1S (−Tc) displayed background energy transfer (E = 0.10) unchanged compared with FRET measurements for this construct expressed in dysgenic myotubes (Fig. 3B, black bars). Similarly, FRET efficiencies measured in HEK-293T cells to ReAsH targeted to the α1S N terminus and I-II loop (E = 0.39, and E = 0.21, respectively) were unchanged compared with similar measurements conducted in dysgenic myotubes. However, in intact HEK-293T cells, average FRET efficiencies to the II-III loop (E = 0.20) and III-IV loop (E = 0.14) were significantly reduced compared with measurements in dysgenic myotubes (Fig. 3B). Most surprisingly, no significant energy transfer above background was detected to the α1S C-terminal tail in HEK-293T cells (E = 0.10) despite robust FRET to this position for measurements conducted in dysgenic myotubes. This striking difference in energy transfer was unrelated to myotube-specific post-translational cleavage of the C-terminal tail at position 1664. As shown in Fig. 3B, FRET efficiencies measured from HEK-293T cells expressing α1s containing a full-length C-terminal tail were not different from those measured in cells expressing the C-terminal tail truncated at position 1668. Thus, these results suggest that the conformation of the α1S II-III and III-IV loops and particularly the C-terminal tail are influenced by the cellular microenvironment.

      ReAsH Labeling Efficiency

      To verify that differences in energy transfer observed between the various Tc-tagged α1S constructs were not due to differences in ReAsH labeling efficiency at the various Tc-tagged sites, we quantified ReAsH labeling to each construct expressed in HEK-293T cells (Fig. 4). In contrast to dysgenic myotubes, specific ReAsH labeling of each recombinant Tc-tagged α1S DHPR was readily observed after treatment with 100 μm British anti-Lewisite (Fig. 4A), as we have shown previously with Tc-tagged RyRs expressed in HEK-293T cells (
      • Fessenden J.D.
      • Mahalingam M.
      Site-specific labeling of the type 1 ryanodine receptor using biarsenical fluorophores targeted to engineered tetracysteine motifs.
      ). However, no differences in ReAsH labeling efficiency (i.e. the ReAsH/YFP fluorescence ratio) were observed between the Tc-tagged YFP-α1S constructs (Fig. 4B). Because of high nonspecific ReAsH labeling in myotubes, these control experiments were not feasible. However, in these cells FRET efficiencies measured after labeling Tc-tagged α1s YFP constructs with either 0.5 μm ReAsH (i.e. the concentration used in all FRET experiments) or a 4-fold higher ReAsH concentration (2 μm) were identical (data not shown), thus indicating that 0.5 μm ReAsH is a saturating concentration for these measurements. These results indicate that, as in HEK-293T cells, differences in FRET efficiencies measured in dysgenic myotubes does not result from trivial differences in ReAsH accessibility.
      Figure thumbnail gr4
      FIGURE 4The ReAsH FRET acceptor is targeted with equal efficiency to all α1S cytoplasmic loop positions. A, representative images of HEK-293T cells expressing ReAsH-labeled Tc-tagged YFP-α1S DHPR constructs monitored for YFP (left panels) and ReAsH fluorescence (middle panels) as well as the overlay between the two signals (right panels). Significant signal overlap was observed for all constructs except YFP-α1S, lacking a Tc tag (top panels). B, ReAsH labeling efficiency is shown for all constructs as the ratio of ReAsH/YFP fluorescence. Values represent the mean ± S.E. for the number of cells indicated in each bar. No significant differences in ReAsH labeling efficiency were noted between the Tc-tagged constructs (p > 0.05 using analysis of variance).

      Effect of β1a Binding on α1S Loop Structure

      The β1a DHPR subunit is critical for α1S membrane targeting and Ca2+ channel function as well as EC coupling (
      • Beurg M.
      • Sukhareva M.
      • Strube C.
      • Powers P.A.
      • Gregg R.G.
      • Coronado R.
      Recovery of Ca2+ current, charge movements, and Ca2+ transients in myotubes deficient in dihydropyridine receptor β1 subunit transfected with β1 cDNA.
      ,
      • Coronado R.
      • Ahern C.A.
      • Sheridan D.C.
      • Cheng W.
      • Carbonneau L.
      • Bhattacharya D.
      Functional equivalence of dihydropyridine receptor α1S and β1a subunits in triggering excitation-contraction coupling in skeletal muscle.
      ,
      • Gregg R.G.
      • Messing A.
      • Strube C.
      • Beurg M.
      • Moss R.
      • Behan M.
      • Sukhareva M.
      • Haynes S.
      • Powell J.A.
      • Coronado R.
      • Powers P.A.
      Absence of the β subunit (cchb1) of the skeletal muscle dihydropyridine receptor alters expression of the α1 subunit and eliminates excitation-contraction coupling.
      ,
      • Eltit J.M.
      • Franzini-Armstrong C.
      • Perez C.F.
      Amino acid residues 489–503 of dihydropyridine receptor (DHPR) β1a subunit are critical for structural communication between the skeletal muscle DHPR complex and type 1 ryanodine receptor.
      ). To determine whether this essential subunit was responsible for differences in energy transfer observed in the two cell expression systems, we assessed the effect of direct addition of exogenous β1a subunit on FRET efficiencies measured in HEK-293T cells. For these experiments, saponin-permeabilized cells were incubated with and without 150 nm purified recombinant β1a subunit, which saturates α1S expressed in HEK-293T cells (data not shown). In the absence of β1a, measured energy transfer to each α1S cytoplasmic loop in permeabilized HEK-293T cells (Fig. 5B, open bars) was comparable to similar measurements in intact HEK-293T cells (Fig. 3B, open bars), suggesting that cell permeabilization did not significantly affect the structure of the cytosolic loops/domains. Upon incubation with β1a (Fig. 5B, filled bars) no significant changes in energy transfer were observed to any position except the II-III loop, where β1a addition significantly increased FRET efficiency from 0.22 to 0.30 (p < 0.05). This finding was confirmed using a second II-III loop Tc-tagged construct (at position 719), where β1a enhanced FRET from 0.26 to 0.31 (Fig. 5B). Recombinant β1a lacking its C-terminal 36 amino acids (β-36), which are required to support bidirectional signaling (
      • Tanabe T.
      • Takeshima H.
      • Mikami A.
      • Flockerzi V.
      • Takahashi H.
      • Kangawa K.
      • Kojima M.
      • Matsuo H.
      • Hirose T.
      • Numa S.
      Primary structure of the receptor for calcium channel blockers from skeletal muscle.
      ), did not enhance FRET from the N-terminal YFP to the II-III loop (Fig. 5B, gray bar), thus indicating that these C-terminal tail residues are required to mediate β1a-induced conformational changes in the α1S II-III loop.
      Figure thumbnail gr5
      FIGURE 5The β1a DHPR subunit specifically alters the structure of the α1S II-III loop. A, schematic representation of the α1S DHPR subunit showing the position of YFP (yellow oval), Tc tags (red squares), and the β1a DHPR subunit (green oval) bound to its α1S subunit interacting domain determinant in the I-II loop (blue rectangle). The location of the critical Y366 residue required for β1a binding to α1S is shown. B, average FRET efficiency values are shown from YFP in the α1S N terminus to ReAsH targeted to permeabilized HEK-293T cells expressing the indicated Tc-tagged α1S DHPR constructs in either the absence (open bars) or presence (filled bars) of 150 nm recombinant β1a. The effect of β1a lacking 36 C-terminal amino acids (β-36) on FRET efficiency to the II-III loop is shown (gray bar). Data points represent mean FRET efficiencies ± S.E. for the number of cells indicated in each bar. Asterisks indicate a significant difference in energy transfer (p < 0.01 using a paired two-tailed t test) between FRET measurements for a given construct conducted in either the absence or presence of β1a. C, average FRET efficiency values are shown from YFP in the N terminus of α1S to ReAsH targeted to Tc726 in the II-III loop of α1S containing the Y366S mutation, which cannot bind β1a (
      • Witcher D.R.
      • De Waard M.
      • Liu H.
      • Pragnell M.
      • Campbell K.P.
      Association of native Ca2+ channel β subunits with the α1 subunit interaction domain.
      ). Data points represent mean FRET efficiencies ± S.E. for the number of cells indicated in each bar.
      β1a binding specificity was confirmed using a YFP-α1S Tc-tagged II-III loop construct harboring a Y366S mutation, which disrupts β1a binding to the α1S subunit interaction domain motif in the α1S I-II loop (
      • Witcher D.R.
      • De Waard M.
      • Liu H.
      • Pragnell M.
      • Campbell K.P.
      Association of native Ca2+ channel β subunits with the α1 subunit interaction domain.
      ). This Y366S α1S mutation completely prevented β1a-mediated enhancement in FRET to the II-III loop (Fig. 5C), thus confirming that β1a binding to its native α1S subunit interacting domain motif is required for its conformational effects on the II-III loop.

      FRET between α1S and β1a

      Direct binding of both wild type and truncated β1a to α1S in HEK-293T cells was confirmed by measuring FRET from the N-terminal-fused YFP of α1S to the FRET acceptor Cy3NTA (
      • Fessenden J.D.
      Förster resonance energy transfer measurements of ryanodine receptor type 1 structure using a novel site-specific labeling method.
      ) targeted to a His10 tag attached to the β1a N terminus (Fig. 6A). Significant energy transfer was observed after incubation with β1a subunit (E = 0.24) compared with background (E = 0.10). Incubation with truncated β-36 resulted in an even greater increase in energy transfer (E = 0.31), significantly higher than FRET measured with wild type β1a (Fig. 6B), thus suggesting that deletion of the β1a C terminus may affect the relative α1S1a orientation. This finding is consistent with the suggested role of the β1a C-terminal tail in supporting domain cooperativity within the subunit (
      • Dayal A.
      • Bhat V.
      • Franzini-Armstrong C.
      • Grabner M.
      Domain cooperativity in the β1a subunit is essential for dihydropyridine receptor voltage sensing in skeletal muscle.
      ). Binding specificity was confirmed using the Y366S-α1S mutation, which prevented specific energy transfer between α1S and β1a (Fig. 6C). These findings indicate that both wild type and truncated β1a bind to the I-II loop of α1S expressed in HEK-293T cells.
      Figure thumbnail gr6
      FIGURE 6Both β1a and β-36 bind to α1S DHPR. A, strategy for direct FRET measurements between α1S and β1a DHPR subunits in HEK-293T cells. FRET from the fused N-terminal YFP in α1S (yellow oval) to the His10 tag-specific FRET acceptor, Cy3NTA (red octagon), targeted to β1a (green circle) is indicative of binding between these two DHPR subunits. B and C, FRET efficiency values are shown from YFP to Cy3NTA targeted to HEK-293T cells expressing wild type YFP α1S (B) or Y366S YFP α1S (C) with and without the indicated β1a constructs. Data represent the mean FRET values ± S.E. for the number of cells indicated in each bar. The asterisk indicates a significant difference in energy transfer (p < 0.001 using a paired two-tailed t test) between FRET measurements conducted using either wild type β1a or truncated β-36.

      Discussion

      In this study using a unique FRET-based approach we have shown that the DHPR α1S subunit intracellular loops have remarkably similar structural properties when expressed either in myotubes or HEK-293T cells. However, key differences exist. To our knowledge we are the first to show that the structure of the α1s C-terminal tail is highly sensitive to muscle-specific protein-protein interactions. Our data also provide the first experimental evidence of a β1a-mediated reorientation of the α1S II-III loop domain, further supporting the idea that a synergistic α1S1a interaction could account for the conformational changes required to sustain skeletal muscle E-C coupling. The details of these findings and our unique FRET-based experimental system are outlined below.

      Labeling System

      FRET-based analysis of the DHPR using fused fluorescent proteins as FRET donors/acceptors has become a powerful tool for in situ studies of DHPR conformation and its structural interaction(s) with RyR1 (
      • Papadopoulos S.
      • Leuranguer V.
      • Bannister R.A.
      • Beam K.G.
      Mapping sites of potential proximity between the dihydropyridine receptor and RyR1 in muscle using a cyan fluorescent protein-yellow fluorescent protein tandem as a fluorescence resonance energy transfer probe.
      ,
      • Polster A.
      • Ohrtman J.D.
      • Beam K.G.
      • Papadopoulos S.
      Fluorescence resonance energy transfer (FRET) indicates that association with the type I ryanodine receptor (RyR1) causes reorientation of multiple cytoplasmic domains of the dihydropyridine receptor (DHPR) α1S subunit.
      ). Although these studies have revealed important structural aspects of DHPR/RyR interactions, they are limited by the exclusive use of fluorescent proteins, which can affect DHPR function and targeting as well as interpretation of the resulting FRET data (
      • Polster A.
      • Ohrtman J.D.
      • Beam K.G.
      • Papadopoulos S.
      Fluorescence resonance energy transfer (FRET) indicates that association with the type I ryanodine receptor (RyR1) causes reorientation of multiple cytoplasmic domains of the dihydropyridine receptor (DHPR) α1S subunit.
      ,
      • Raina S.A.
      • Tsai J.
      • Samsó M.
      • Fessenden J.D.
      FRET-based localization of fluorescent protein insertions within the ryanodine receptor type 1.
      ,
      • Bannister R.A.
      • Papadopoulos S.
      • Haarmann C.S.
      • Beam K.G.
      Effects of inserting fluorescent proteins into the α1S II-III loop: insights into excitation-contraction coupling.
      ). In the current study a small 12-residue peptide tag was used to target the FRET acceptor, ReAsH, to each of the α1S DHPR cytoplasmic loops and domains, thereby minimizing alteration of native protein conformation. Thus, we could measure FRET to the α1S III-IV loop without compromising DHPR function or proper targeting, which are both severely disrupted by insertion of larger FRET probes (i.e. fluorescent protein fusions) in this loop (
      • Polster A.
      • Ohrtman J.D.
      • Beam K.G.
      • Papadopoulos S.
      Fluorescence resonance energy transfer (FRET) indicates that association with the type I ryanodine receptor (RyR1) causes reorientation of multiple cytoplasmic domains of the dihydropyridine receptor (DHPR) α1S subunit.
      ). In addition, we could easily quantify energy transfer to a specific Tc tag via direct comparison with non-−Tc-tagged controls. And because all DHPR loop positions were equally accessible to FRET acceptor, measured FRET efficiencies could more easily be related to differences in either donor/acceptor distance or orientation. Finally, nonspecific biarsenical labeling of myotubes reported previously (
      • Stroffekova K.
      • Proenza C.
      • Beam K.G.
      The protein-labeling reagent FLASH-EDT2 binds not only to CCXXCC motifs but also non-specifically to endogenous cysteine-rich proteins.
      ) was not problematic for these studies, as ReAsH was used as FRET acceptor, and so only ReAsH fluorophores targeted to α1S Tc tags proximal to the fused YFP donor contributed to the measured FRET.

      Comparison between Loop Conformations in Heterologous and Homologous Systems

      We observed remarkable similarities and differences in the structure of α1S expressed either in HEK-293T cells or dysgenic myotubes. For example, FRET measurements to the N terminus and the I-II loop were essentially identical between the two systems, thus suggesting that cell-specific factors do not affect FRET measured to these areas. In contrast, FRET efficiencies measured from the α1S N terminus to the II-III, III-IV loops and, most significantly, to the C-terminal tail were all quite different between the two systems. We can attribute some of these differences to specific protein factors, whereas other differences will require additional experiments to investigate. A summary of these differences follows.

      II-III Loop Structure

      Compared with identical measurements in HEK-293T cells, we observed a significant elevation in energy transfer from the N terminus to the α1S II-III loop for constructs expressed in dysgenic myotubes. This increase in FRET likely reflects a specific conformational effect in the II-III loop as structural changes in the vicinity of the N-terminal YFP FRET donor common to all constructs would almost certainly have altered FRET efficiencies to all DHPR loops. This enhanced energy transfer to the II-III loop most likely results from binding of β1a to its I-II loop determinant, as we observed the same degree of elevated energy transfer to the II-III loop in HEK-293T cells incubated with recombinant β1a. Because β1a enhanced FRET to two different Tc-tagged positions in the II-III loop, this effect is reproducible and meaningful. This specific conformational change in the II-III loop also requires the C-terminal 36 amino acids of β1a, a determinant needed to support EC coupling and to organize DHPRs into tetrads (
      • Eltit J.M.
      • Franzini-Armstrong C.
      • Perez C.F.
      Amino acid residues 489–503 of dihydropyridine receptor (DHPR) β1a subunit are critical for structural communication between the skeletal muscle DHPR complex and type 1 ryanodine receptor.
      ,
      • Sheridan D.C.
      • Cheng W.
      • Ahern C.A.
      • Mortenson L.
      • Alsammarae D.
      • Vallejo P.
      • Coronado R.
      Truncation of the carboxyl terminus of the dihydropyridine receptor β1a subunit promotes Ca2+ dependent excitation-contraction coupling in skeletal myotubes.
      ). This conformational effect might reflect the natural structure of this II-III loop required to communicate with RyR1. Moreover, the II-III loop and the C-terminal tail of β1a might form a larger structural domain that then interacts with RyR1 (a notion supported by recent cryo-EM reconstructions of the DHPR; see Fig. 7A below). Although these possibilities require testing, it should be stressed that the only specific conformational effect of β1a binding we observed was within the II-III loop. Future FRET-based measurements to more defined determinants in the II-III loop may resolve more subtle structural changes occurring as a result of β1a binding or during EC coupling.

      III-IV Loop Structure

      In this study we were able to make the first direct FRET measurements to the III-IV loop. We detected very slight differences in FRET to the III-IV loop when constructs were expressed either in HEK-293T cells or dysgenic myotubes. The origin of these differences is still difficult to discern but appear to be unrelated to β1a binding. However, now that the III-IV loop can be labeled with FRET acceptors, future studies may detect structural changes in this loop that provide clues as to its function.

      C-terminal Tail Structure

      The largest difference we observed in FRET measurements conducted in the two systems was to the α1S C-terminal tail. Robust FRET was measured to this position when experiments were conducted in myotubes, whereas no significant FRET was detected in HEK-293T cells. These differences suggest changes in structural conformation of the C-terminal tail that could result from post-translational processing of the α1S subunit or differences in protein composition between the two systems as discussed below.

      Posttranslational Processing of α1S

      Differences in FRET efficiencies measured between the N terminus and the C-terminal tail may result from intrinsic differences in post-translational processing of α1S in dysgenic myotubes. For this study we used an α1S construct encoding the full 1873 amino acids of the protein. However, in myotubes, full-length α1S subunit is likely cleaved post-translationally at position 1664 (
      • Polster A.
      • Ohrtman J.D.
      • Beam K.G.
      • Papadopoulos S.
      Fluorescence resonance energy transfer (FRET) indicates that association with the type I ryanodine receptor (RyR1) causes reorientation of multiple cytoplasmic domains of the dihydropyridine receptor (DHPR) α1S subunit.
      ,
      • Hulme J.T.
      • Konoki K.
      • Lin T. W.-C.
      • Gritsenko M.A.
      • Camp 2nd, D.G.
      • Bigelow D.J.
      • Catterall W.A.
      Sites of proteolytic processing and noncovalent association of the distal C-terminal domain of CaV1.1 channels in skeletal muscle.
      ), right after our Tc-tag insertion, thus significantly shortening the C-terminal tail. To test whether post-translational processing of the C-terminal tail may have affected FRET efficiencies measured to this position, we conducted parallel FRET measurements on α1s YFP fusion constructs with either a full-length C-terminal tail or a tail shortened at position 1668. No differences in FRET for these variants were observed in either HEK-293T cells or dysgenic myotubes. Thus, it is unlikely that post-translational processing of the C-terminal tail results in the different FRET efficiency profiles to these different positions.

      Muscle-specific Proteins

      Differences in FRET efficiency to the C-terminal tail of α1S were significant and unrelated to the absence of β1a subunit in HEK-293T cells. Similarly, differences in interactions with RyR1 most likely are not responsible for changes in FRET to the α1S C terminus, as previous studies using CFP/YFP fusions showed no difference in FRET between the N and C termini for α1S constructs expressed in either dyspedic (i.e. lacking RyR1) or dysgenic systems (
      • Polster A.
      • Ohrtman J.D.
      • Beam K.G.
      • Papadopoulos S.
      Fluorescence resonance energy transfer (FRET) indicates that association with the type I ryanodine receptor (RyR1) causes reorientation of multiple cytoplasmic domains of the dihydropyridine receptor (DHPR) α1S subunit.
      ). Thus, it is possible that other proteins that make up the DHPR complex, such as the α2δ1 and γ1 subunits, might account for this difference. Similarly, interactions between the α1S C terminus tail and other muscle-specific proteins should not be ruled out. Indeed, differences in protein interactions with various functional domains of the α1S C-terminal tail, like the triad targeting signal (position 1543–1661; Refs.
      • Proenza C.
      Excitation-contraction coupling is not affected by scrambled sequence in residues 681–690 of the dihydropyridine receptor II-III loop.
      and
      • Flucher B.E.
      • Kasielke N.
      • Gerster U.
      • Neuhuber B.
      • Grabner M.
      Insertion of the full-length calcium channel α1S subunit into triads of skeletal muscle in vitro.
      ) and the AKAP/PKA binding domain (position 1724–1821; Ref.
      • Hulme J.T.
      • Konoki K.
      • Lin T. W.-C.
      • Gritsenko M.A.
      • Camp 2nd, D.G.
      • Bigelow D.J.
      • Catterall W.A.
      Sites of proteolytic processing and noncovalent association of the distal C-terminal domain of CaV1.1 channels in skeletal muscle.
      ) might lead to conformational changes in the C-terminal tail evident in myotubes but not HEK-293T cells. These possibilities await further testing using co-expression of these proteins combined with our FRET-based measurements.

      Effects of the β1a C-terminal Tail on DHPR Structure

      In this study we have made the first direct FRET measurements between α1S and β1a. These measurements confirmed that both wild type and β1a, bearing a 36-amino acid C-terminal truncation (β-36), bind to α1S in our experimental system. In addition, relative to wild type β1a, enhanced FRET was observed between the N termini of α1S and the β-36 construct. This result suggests that this truncation results in a significant reorientation of β1a, thereby bringing its N terminus closer to α1S. Thus, deletion of the β1a C terminus, which prevents bidirectional signaling (
      • Eltit J.M.
      • Franzini-Armstrong C.
      • Perez C.F.
      Amino acid residues 489–503 of dihydropyridine receptor (DHPR) β1a subunit are critical for structural communication between the skeletal muscle DHPR complex and type 1 ryanodine receptor.
      ,
      • Sheridan D.C.
      • Cheng W.
      • Ahern C.A.
      • Mortenson L.
      • Alsammarae D.
      • Vallejo P.
      • Coronado R.
      Truncation of the carboxyl terminus of the dihydropyridine receptor β1a subunit promotes Ca2+ dependent excitation-contraction coupling in skeletal myotubes.
      ), also may affect the conformation/orientation of both the α1S II-III loop and the β1a subunit. These results are consistent with the hypothesis that the C terminus of the β1a subunit is required for inducing conformational changes in the α1S subunit necessary to transmit the EC coupling signal (
      • Schredelseker J.
      • Di Biase V.
      • Obermair G.J.
      • Felder E.T.
      • Flucher B.E.
      • Franzini-Armstrong C.
      • Grabner M.
      The β 1a subunit is essential for the assembly of dihydropyridine-receptor arrays in skeletal muscle.
      ,
      • Dayal A.
      • Bhat V.
      • Franzini-Armstrong C.
      • Grabner M.
      Domain cooperativity in the β1a subunit is essential for dihydropyridine receptor voltage sensing in skeletal muscle.
      ).

      Comparison with Recent High Resolution DHPR Structures

      Recently, a near-atomic 4.2 Å resolution model of the DHPR complex has been published (
      • Wu J.
      • Yan Z.
      • Li Z.
      • Yan C.
      • Lu S.
      • Dong M.
      • Yan N.
      Structure of the voltage-gated calcium channel Cav1.1 complex.
      ). Although many parts of the DHPR complex are well defined, none of the intracellular loops tested in this study are localized in the model, most likely due to high intrinsic flexibility of the loops. However, several aspects of the model support conclusions derived from our FRET data. For example, from this new cryo-EM reconstruction, it is evident that the II-III loop is the closest of the α1s cytoplasmic loops (except the I-II loop) to the β1a subunit (Fig. 7, A and B). Thus, it is conceivable that β1a binding could modulate the structure of the II-III loop via short range allosteric interactions and that these two elements could form a structural complex. In addition, a central placement of the fused YFP would result in a relatively uniform profile of FRET efficiencies measured to the various intracellular loops (Fig. 7A). Indeed, the inherent intrinsic disorder of these loops revealed by the high resolution structures makes them excellent targets for further FRET-based structural studies to reveal subtle conformational changes underlying other important α1s functionalities.

      Author Contributions

      M. M., C. F. P., and J. D. F. designed the study, executed the experiments, analyzed the results, and wrote the paper.

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