The II-III loop of the skeletal muscle dihydropyridine receptor is responsible for the Bi-directional coupling with the ryanodine receptor.

The dihydropyridine receptor (DHPR) in the skeletal muscle plasmalemma functions as both voltage-gated Ca(2+) channel and voltage sensor for excitation-contraction (EC) coupling. As voltage sensor, the DHPR regulates intracellular Ca(2+) release via the skeletal isoform of the ryanodine receptor (RyR-1). Interaction with RyR-1 also feeds back to increase the Ca(2+) current mediated by the DHPR. To identify regions of the DHPR important for receiving this signal from RyR-1, we expressed in dysgenic myotubes a chimera (SkLC) having skeletal (Sk) DHPR sequence except for a cardiac (C) II-III loop (L). Tagging with green fluorescent protein (GFP) enabled identification of expressing myotubes. Dysgenic myotubes expressing GFP-SkLC or SkLC lacked EC coupling and had very small Ca(2+) currents. Introducing a short skeletal segment (alpha(1S) residues 720-765) into the cardiac II-III loop (replacing alpha(1C) residues 851-896) of GFP-SkLC restored both EC coupling and Ca(2+) current densities like those of the wild type skeletal DHPR. This 46-amino acid stretch of skeletal sequence was recently shown to be capable of transferring strong, skeletal-type EC coupling to an otherwise cardiac DHPR (Nakai, J., Tanabe, T., Konno, T., Adams, B., and Beam, K.G. (1998) J. Biol. Chem. 273, 24983-24986). Thus, this segment of the skeletal II-III loop contains a motif required for both skeletal-type EC coupling and RyR-1-mediated enhancement of Ca(2+) current.     

The monoclonal antibody mAB 1A binds to the excitation--contraction coupling domain in the II-III loop of the skeletal muscle calcium channel alpha(1S) subunit

Interactions of the II-III loop of the voltage-gated Ca(2+) channel alpha(1S) subunit with the Ca(2+) release channel (RyR1) are essential for skeletal-type excitation-contraction (EC) coupling. Here, we characterized the binding site of the monoclonal alpha(1S) antibody mAB 1A and used it to probe the structure of the II-III loop in chimeras with different EC coupling properties. Phage-display epitope mapping of mAB 1A revealed a minimal consensus binding sequence X-P-X-X-D-X-P. Immunofluorescence labeling of (1S), alpha(1C), alpha(1D), and of II-III loop chimeras expressed in dysgenic myotubes established that mAB 1A reacted specifically with amino acids 737-744 in the II-III loop of alpha(1S), which is within the domain (D734-L764) critical for bidirectional coupling with RyR1. Comparing mAB 1A immunoreactivity with known structural and functional properties of II-III loop chimeras in which the non-conserved skeletal residues were systematically mutated to their cardiac counterparts indicated a correlation of mAB 1A immunoreactivity and skeletal-type EC coupling.     

Excitation-contraction coupling is not affected by scrambled sequence in residues 681-690 of the dihydropyridine receptor II-III loop

A peptide corresponding to residues 681-690 of the II-III loop of the skeletal muscle dihydropyridine receptor alpha(1) subunit (DHPR, alpha(1S)) has been reported to activate the skeletal muscle ryanodine receptor (RyR1) in vitro. Within this region of alpha(1S), a cluster of basic residues, Arg(681)-Lys(685), was previously reported to be indispensable for the activation of RyR1 in microsomal preparations and lipid bilayers. We have used an intact alpha(1S) subunit with scrambled sequence in this region of the II-III loop (alpha(1S)-scr) to test the importance of residues 681-690 and the basic motif for skeletal-type excitation-contraction (EC) coupling and retrograde signaling in vivo. When expressed in dysgenic myotubes (which lack endogenous alpha(1S)), alpha(1S)-scr restored calcium currents that were indistinguishable, in current density and voltage dependence, from those restored by wild-type alpha(1S). The scrambled DHPR also rescued skeletal-type EC coupling, as indicated by electrically evoked contractions in the presence of 0.5 mm Cd(2+) and 0.1 mm La(3+). Furthermore, the release of intracellular Ca(2+), as assayed by the indicator dye, Fluo-3, had similar kinetics and voltage dependence for alpha(1S) and alpha(1S)-scr. These data suggest that residues 681-690 of the alpha(1S) II-III loop are not essential in muscle cells for normal functioning of the DHPR, including skeletal-type EC coupling and retrograde signaling.     

The alpha1S N-terminus is not essential for bi-directional coupling with RyR1

The dihydropyridine receptor (DHPR) alpha(1S) II-III loop has been shown to be critical for excitation-contraction (EC) coupling in skeletal muscle, but the importance of other cytoplasmic regions, especially the N-terminus (residues 1-51), remains unclear. In this study, we found that deletion of alpha(1S) residues 2-37 (weakly conserved with N-termini of other L-type Ca(2+) channels) had little effect on the ability of alpha(1S) to serve as a Ca(2+) channel or voltage sensor for EC coupling. Strikingly, deletion of 10 additional residues, which are conserved in L-type channels, resulted in ablation of DHPR function. Specifically, confocal microscopy and measurement of charge movement showed that removal of residues 2-47 resulted in a failure of sarcolemmal insertion. Our results indicate that the weakly conserved, distal alpha(1S) N-terminus is not critical for EC coupling or function as a Ca(2+) channel. However, integrity of the proximal alpha(1S) N-terminus is necessary for sarcolemmal expression of the DHPR.     

Differential contribution of skeletal and cardiac II-III loop sequences to the assembly of dihydropyridine-receptor arrays in skeletal muscle

The plasmalemmal dihydropyridine receptor (DHPR) is the voltage sensor in skeletal muscle excitation-contraction (e-c) coupling. It activates calcium release from the sarcoplasmic reticulum via protein-protein interactions with the ryanodine receptor (RyR). To enable this interaction, DHPRs are arranged in arrays of tetrads opposite RyRs. In the DHPR alpha(1S) subunit, the cytoplasmic loop connecting repeats II and III is a major determinant of skeletal-type e-c coupling. Whether the essential II-III loop sequence (L720-L764) also determines the skeletal-specific arrangement of DHPRs was examined in dysgenic (alpha(1S)-null) myotubes reconstituted with distinct alpha(1) subunit isoforms and II-III loop chimeras. Parallel immunofluorescence and freeze-fracture analysis showed that alpha(1S) and chimeras containing L720-L764, all of which restored skeletal-type e-c coupling, displayed the skeletal arrangement of DHPRs in arrays of tetrads. Conversely, alpha(1C) and those chimeras with a cardiac II-III loop and cardiac e-c coupling properties were targeted into junctional membranes but failed to form tetrads. However, an alpha(1S)-based chimera with the heterologous Musca II-III loop produced tetrads but did not reconstitute skeletal muscle e-c coupling. These findings suggest an inhibitory role in tetrad formation of the cardiac II-III loop and that the organization of DHPRs in tetrads vis-a-vis the RyR is necessary but not sufficient for skeletal-type e-c coupling.     

Accessibility of targeted DHPR sites to streptavidin and functional effects of binding on EC coupling

In skeletal muscle, the dihydropyridine receptor (DHPR) in the plasma membrane (PM) serves as a Ca(2+) channel and as the voltage sensor for excitation-contraction (EC coupling), triggering Ca(2+) release via the type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR) membrane. In addition to being functionally linked, these two proteins are also structurally linked to one another, but the identity of these links remains unknown. As an approach to address this issue, we have expressed DHPR alpha(1S) or beta(1a) subunits, with a biotin acceptor domain fused to targeted sites, in myotubes null for the corresponding, endogenous DHPR subunit. After saponin permeabilization, the approximately 60-kD streptavidin molecule had access to the beta(1a) N and C termini and to the alpha(1S) N terminus and proximal II-III loop (residues 671-686). Steptavidin also had access to these sites after injection into living myotubes. However, sites of the alpha(1S) C terminus were either inaccessible or conditionally accessible in saponin- permeabilized myotubes, suggesting that these C-terminal regions may exist in conformations that are occluded by other proteins in PM/SR junction (e.g., RyR1). The binding of injected streptavidin to the beta(1a) N or C terminus, or to the alpha(1S) N terminus, had no effect on electrically evoked contractions. By contrast, binding of streptavidin to the proximal alpha(1S) II-III loop abolished such contractions, without affecting agonist-induced Ca(2+) release via RyR1. Moreover, the block of EC coupling did not appear to result from global distortion of the DHPR and supports the hypothesis that conformational changes of the alpha(1S) II-III loop are necessary for EC coupling in skeletal muscle.     

Multiple loops of the dihydropyridine receptor pore subunit are required for full-scale excitation-contraction coupling in skeletal muscle

Understanding which cytosolic domains of the dihydropyridine receptor participate in excitation-contraction (EC) coupling is critical to validate current structural models. Here we quantified the contribution to skeletal-type EC coupling of the alpha1S (CaV1.1) II-III loop when alone or in combination with the rest of the cytosolic domains of alpha1S. Chimeras consisting of alpha1C (CaV1.2) with alpha1S substitutions at each of the interrepeat loops (I-II, II-III, and III-IV loops) and N- and C-terminal domains were evaluated in dysgenic (alpha1S-null) myotubes for phenotypic expression of skeletal-type EC coupling. Myotubes were voltage-clamped, and Ca2+ transients were measured by confocal line-scan imaging of fluo-4 fluorescence. In agreement with previous results, the alpha1C/alpha1S II-III loop chimera, but none of the other single-loop chimeras, recovered a sigmoidal fluorescence-voltage curve indicative of skeletal-type EC coupling. To quantify Ca2+ transients in the absence of inward Ca2+ current, but without changing the external solution, a mutation, E736K, was introduced into the P-loop of repeat II of alpha1C. The Ca2+ transients expressed by the alpha1C(E736K)/alpha1S II-III loop chimera were approximately 70% smaller than those expressed by the Ca2+-conducting alpha1C/alpha1S II-III variant. The low skeletal-type EC coupling expressed by the alpha1C/alpha1S II-III loop chimera was confirmed in the Ca2+-conducting alpha1C/alpha1S II-III loop variant using Cd2+ (10(-4) M) as the Ca2+ current blocker. In contrast to the behavior of the II-III loop chimera, Ca2+ transients expressed by an alpha1C/alpha1S chimera carrying all tested skeletal alpha1S domains (all alpha1S interrepeat loops, N- and C-terminus) were similar in shape and amplitude to wild-type alpha1S, and did not change in the presence of the E736K mutation or in the presence of 10(-4) M Cd2+. Controls indicated that similar dihydropyridine receptor charge movements were expressed by the non-Ca2+ permeant alpha1S(E1014K) variant, the alpha1C(E736K)/alpha1S II-III loop chimera, and the alpha1C(E736K)/alpha1S chimera carrying all tested alpha1S domains. The data indicate that the functional recovery produced by the alpha1S II-III loop is incomplete and that multiple cytosolic domains of alpha1S are necessary for a quantitative recovery of the EC-coupling phenotype of skeletal myotubes. Thus, despite the importance of the II-III loop there may be other critical determinants in alpha1S that influence the efficiency of EC coupling.     

Effects of conformational peptide probe DP4 on bidirectional signaling between DHPR and RyR1 calcium channels in voltage-clamped skeletal muscle fibers

In skeletal muscle, excitation-contraction coupling involves the activation of dihydropyridine receptors (DHPR) and type-1 ryanodine receptors (RyR1) to produce depolarization-dependent sarcoplasmic reticulum Ca²⁺ release via orthograde signaling. Another form of DHPR-RyR1 communication is retrograde signaling, in which RyRs modulate the gating of DHPR. DP4 (domain peptide 4), is a peptide corresponding to residues Leu²⁴⁴²-Pro²⁴⁷⁷ of the central domain of the RyR1 that produces RyR1 channel destabilization. Here we explore the effects of DP4 on orthograde excitation-contraction coupling and retrograde RyR1-DHPR signaling in isolated murine muscle fibers. Intracellular dialysis of DP4 increased the peak amplitude of Ca²⁺ release during step depolarizations by 64% without affecting its voltage-dependence or kinetics, and also caused a similar increase in Ca²⁺ release during an action potential waveform. DP4 did not modify either the amplitude or the voltage-dependence of the intramembrane charge movement. However, DP4 augmented DHPR Ca²⁺ current density without affecting its voltage-dependence. Our results demonstrate that the conformational changes induced by DP4 regulate both orthograde E-C coupling and retrograde RyR1-DHPR signaling.     

Two domains in dihydropyridine receptor activate the skeletal muscle Ca(2+) release channel

The II-III cytoplasmic loop of the skeletal muscle dihydropyridine receptor (DHPR) alpha(1)-subunit is essential for skeletal-type excitation-contraction coupling. Single channel and [(3)H]ryanodine binding studies with a full-length recombinant peptide (p(666-791)) confirmed that this region specifically activates skeletal muscle Ca2+ release channels (CRCs). However, attempts to identify shorter domains of the II-III loop specific for skeletal CRC activation have yielded contradictory results. We assessed the specificity of the interaction of five truncated II-III loop peptides by comparing their effects on skeletal and cardiac CRCs in lipid bilayer experiments; p(671-680) and p(720-765) specifically activated the submaximally Ca2+-activated skeletal CRC in experiments using both mono and divalent ions as current carriers. A third peptide, p(671-690), showed a bimodal activation/inactivation behavior indicating a high-affinity activating and low-affinity inactivating binding site. Two other peptides (p(681-690) and p(681-685)) that contained an RKRRK-motif and have previously been suggested in in vitro studies to be important for skeletal-type E-C coupling, failed to specifically stimulate skeletal CRCs. Noteworthy, p(671-690), p(681-690), and p(681-685) induced similar subconductances and long-lasting channel closings in skeletal and cardiac CRCs, indicating that these peptides interact in an isoform-independent manner with the CRCs.     

Effects of inserting fluorescent proteins into the α1S II–III loop: insights into excitation–contraction coupling

In skeletal muscle, intermolecular communication between the 1,4-dihydropyridine receptor (DHPR) and RYR1 is bidirectional: orthograde coupling (skeletal excitation–contraction coupling) is observed as depolarization-induced Ca²⁺ release via RYR1, and retrograde coupling is manifested by increased L-type Ca²⁺ current via DHPR. A critical domain (residues 720–765) of the DHPR α_1S II–III loop plays an important but poorly understood role in bidirectional coupling with RYR1. In this study, we examine the consequences of fluorescent protein insertion into different positions within the α_1S II–III loop. In four constructs, a cyan fluorescent protein (CFP)–yellow fluorescent protein (YFP) tandem was introduced in place of residues 672–685 (the peptide A region). All four constructs supported efficient bidirectional coupling as determined by the measurement of L-type current and myoplasmic Ca²⁺ transients. In contrast, insertion of a CFP–YFP tandem within the N-terminal portion of the critical domain (between residues 726 and 727) abolished bidirectional signaling. Bidirectional coupling was partially preserved when only a single YFP was inserted between residues 726 and 727. However, insertion of YFP near the C-terminal boundary of the critical domain (between residues 760 and 761) or in the conserved C-terminal portion of the α_1S II–III loop (between residues 785 and 786) eliminated bidirectional coupling. None of the fluorescent protein insertions, even those that interfered with signaling, significantly altered membrane expression or targeting. Thus, bidirectional signaling is ablated by insertions at two different sites in the C-terminal portion of the α_1S II–III loop. Significantly, our results indicate that the conserved portion of the α_1S II–III loop C terminal to the critical domain plays an important role in bidirectional coupling either by conveying conformational changes to the critical domain from other regions of the DHPR or by serving as a site of interaction with other junctional proteins such as RYR1.     

Identification of a region of RyR1 that participates in allosteric coupling with the alpha(1S) (Ca(V)1.1) II-III loop.

In skeletal muscle, excitation-contraction (EC) coupling and retrograde signaling are thought to result from direct interactions between the ryanodine receptor (RyR1) and the alpha(1) subunit of the dihydropyridine receptor (alpha(1S)). Previous work has shown that the s53 region of alpha(1S) (residues 720-765 in the II-III loop) and regions R10 (1635-2636) and R9 (2659-3720) of RyR1 are involved in this signaling. Using the yeast two-hybrid system, we here report an interaction between s53 and the sR16 region of RyR1 (1837-2168, within R10), whereas no interaction was seen using upstream residues of the alpha(1S) II-III loop (s31, 666-709). The specificity of the s53-sR16 interaction was tested by using fragments of the cardiac RyR (RyR2) and DHPR (alpha(1C)) that correspond to sR16 and s53, respectively. No interaction was observed for sR16 x c53 (alpha(1C) 850-897), but weak interaction was occasionally observed for s53 x cR16 (RyR2 1817-2142). To test the functional significance of the s53 x sR16 interaction, we expressed in dyspedic myotubes a chimeric RyR (chimeraR16) in which sR16 was substituted for the corresponding region of RyR2. ChimeraR16 was found to mediate weak skeletal-type EC coupling. To test the necessity of sR16 sequence for coupling, we used "chimeraR16-rev," in which sR16 and a small upstream region of RyR1 were replaced by RyR2 sequence. ChimeraR16-rev did not differ from RyR1 in its ability to mediate EC coupling. Thus, interaction between residues 720-765 of alpha(1S) and residues 1837-2168 of RyR1 appears to contribute to but is not essential for EC coupling in skeletal muscle.     

Functional equivalence of dihydropyridine receptor alpha1S and beta1a subunits in triggering excitation-contraction coupling in skeletal muscle

Molecular understanding of the mechanism of excitation-contraction (EC) coupling in skeletal muscle has been made possible by cultured myotube models lacking specific dihydropyridine receptor (DHPR) subunits and ryanodine receptor type 1 (RyR1) isoforms. Transient expression of missing cDNAs in mutant myotubes leads to a rapid recovery, within days, of various Ca2+ current and EC coupling phenotypes. These myotube models have thus permitted structure-function analysis of EC coupling domains present in the DHPR controlling the opening of RyR1. The purpose of this brief review is to highlight advances made by this laboratory towards understanding the contribution of domains present in alpha1S and beta1a subunits of the skeletal DHPR to EC coupling signaling. Our main contention is that domains of the alpha1S II-III loop are necessary but not sufficient to recapitulate skeletal-type EC coupling. Rather, the structural unit that controls the EC coupling signal appears to be the alpha1S/beta1a pair.     

Conversion of an inactive cardiac dihydropyridine receptor II-III loop segment into forms that activate skeletal ryanodine receptors

A 25 amino acid segment (Glu666-Pro691) of the II-III loop of the alpha1 subunit of the skeletal dihydropyridine receptor, but not the corresponding cardiac segment (Asp788-Pro814), activates skeletal ryanodine receptors. To identify the structural domains responsible for activation of skeletal ryanodine receptors, we systematically replaced amino acids of the cardiac II-III loop with their skeletal counterparts. A cluster of five basic residues of the skeletal II-III loop (681RKRRK685) was indispensable for activation of skeletal ryanodine receptors. In the cardiac segment, a negatively charged residue (Glu804) appears to diminish the electrostatic potential created by this basic cluster. In addition, Glu800 in the group of negatively charged residues 798EEEEE802 of the cardiac II-III loop may serve to prevent the binding of the activation domain.     

A component of excitation-contraction coupling triggered in the absence of the T671-L690 and L720-Q765 regions of the II-III loop of the dihydropyridine receptor alpha(1s) pore subunit.

We conducted a deletion analysis of two regions identified in the II-III loop of alpha(1S), residues 671-690, which were shown to bind to ryanodine receptor type 1 (RyR1) and stimulate RyR1 channels in vitro, and residues 720-765 or the narrower 724-743 region, which confer excitation-contraction (EC) coupling function to chimeric dihydropyridine receptors (DHPRs). Deletion mutants were expressed in dysgenic alpha(1S)-null myotubes and analyzed by voltage-clamp and confocal fluo-4 fluorescence. Immunostaining of the mutant subunits using an N-terminus tag revealed abundant protein expression in all cases. Furthermore, the maximum recovered charge movement density was >80% of that recovered by full-length alpha(1S) in all cases. Delta671-690 had no effect on the magnitude of voltage-evoked Ca(2+) transients or the L-type Ca(2+) current density. In contrast, Delta720-765 or Delta724-743 abolished Ca(2+) transients entirely, and L-type Ca(2+) current was reduced or absent. Surprisingly, Ca(2+) transients and Ca(2+) currents of a moderate magnitude were recovered by the double deletion mutant Delta671-690/Delta720-765. A simple explanation for this result is that Delta720-765 induces a conformation change that disrupts EC coupling, and this conformational change is partially reverted by Delta671-690. To test for Ca(2+)-entry independent EC coupling, a pore mutation (E1014K) known to entirely abolish the inward Ca(2+) current was introduced. alpha(1S) Delta671-690/Delta720-765/E1014K expressed Ca(2+) transients with Boltzmann parameters identical to those of the Ca(2+)-conducting double deletion construct. The data strongly suggest that skeletal-type EC coupling is not uniquely controlled by alpha(1S) 720-765. Other regions of alpha(1S) or other DHPR subunits must therefore directly contribute to the activation of RyR1 during EC coupling.     

Cyclization of the intrinsically disordered α1S dihydropyridine receptor II-III loop enhances secondary structure and in vitro function

A key component of excitation contraction (EC) coupling in skeletal muscle is the cytoplasmic linker (II-III loop) between the second and third transmembrane repeats of the α(1S) subunit of the dihydropyridine receptor (DHPR). The II-III loop has been previously examined in vitro using a linear II-III loop with unrestrained N- and C-terminal ends. To better reproduce the loop structure in its native environment (tethered to the DHPR transmembrane domains), we have joined the N and C termini using intein-mediated technology. Circular dichroism and NMR spectroscopy revealed a structural shift in the cyclized loop toward a protein with increased α-helical and β-strand structure in a region of the loop implicated in its in vitro function and also in a critical region for EC coupling. The affinity of binding of the II-III loop binding to the SPRY2 domain of the skeletal ryanodine receptor (RyR1) increased 4-fold, and its ability to activate RyR1 channels in lipid bilayers was enhanced 3-fold by cyclization. These functional changes were predicted consequences of the structural enhancement. We suggest that tethering the N and C termini stabilized secondary structural elements in the DHPR II-III loop and may reflect structural and dynamic characteristics of the loop that are inherent in EC coupling.     

The alpha(1S) 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

In skeletal muscle, coupling between the 1,4-dihydropyridine receptor (DHPR) and the type 1 ryanodine receptor (RyR1) underlies excitation-contraction (EC) coupling. The III-IV loop of the DHPR alpha(1S) subunit binds to a segment of RyR1 in vitro, and mutations in the III-IV loop alter the voltage dependence of EC coupling, raising the possibility that this loop is directly involved in signal transmission from the DHPR to RyR1. To clarify the role of the alpha(1S) III-IV loop in EC coupling, we examined the functional properties of a chimera (GFP-alpha(1S)[III-IVa]) in which the III-IV loop of the divergent alpha(1A) isoform replaced that of alpha(1S). Dysgenic myotubes expressing GFP-alpha(1S)[III-IVa] yielded myoplasmic Ca(2+) transients that activated at approximately 10 mV more hyperpolarized potentials and that were approximately 65% smaller than those of GFP-alpha(1S). A similar reduction was observed in voltage-dependent charge movements for GFP-alpha(1S)[III-IVa], indicating that the chimeric channels trafficked less well to the membrane but that those that were in the membrane functioned as efficiently in EC coupling as GFP-alpha(1S). Relative to GFP-alpha(1S), L-type currents mediated by GFP-alpha(1S)[III-IVa] were approximately 40% smaller and activated at approximately 5 mV more hyperpolarized potentials. The altered gating of GFP-alpha(1S)[III-IVa] was accentuated by exposure to +/-Bay K 8644, which caused a much larger hyperpolarizing shift in activation compared with its effect on GFP-alpha(1S). Taken together, our observations indicate that the alpha(1S) III-IV loop is not directly involved in EC coupling but does influence DHPR gating transitions important both for EC coupling and activation of L-type conductance.     

Involvement of the carboxy-terminus region of the dihydropyridine receptor beta1a subunit in excitation-contraction coupling of skeletal muscle

Skeletal muscle knockout cells lacking the beta subunit of the dihydropyridine receptor (DHPR) are devoid of slow L-type Ca(2+) current, charge movements, and excitation-contraction coupling, despite having a normal Ca(2+) storage capacity and Ca(2+) spark activity. In this study we identified a specific region of the missing beta1a subunit critical for the recovery of excitation-contraction. Experiments were performed in beta1-null myotubes expressing deletion mutants of the skeletal muscle-specific beta1a, the cardiac/brain-specific beta2a, or beta2a/beta1a chimeras. Immunostaining was used to determine that all beta constructs were expressed in these cells. We examined the Ca(2+) conductance, charge movements, and Ca(2+) transients measured by confocal fluo-3 fluorescence of transfected myotubes under whole-cell voltage-clamp. All constructs recovered an L-type Ca(2+) current with a density, voltage-dependence, and kinetics of activation similar to that recovered by full-length beta1a. In addition, all constructs except beta2a mutants recovered charge movements with a density similar to full-length beta1a. Thus, all beta constructs became integrated into a skeletal-type DHPR and, except for beta2a mutants, all restored functional DHPRs to the cell surface at a high density. The maximum amplitude of the Ca(2+) transient was not affected by separate deletions of the N-terminus of beta1a or the central linker region of beta1a connecting two highly conserved domains. Also, replacement of the N-terminus half of beta1a with that of beta2a had no effect. However, deletion of 35 residues of beta1a at the C-terminus produced a fivefold reduction in the maximum amplitude of the Ca(2+) transients. A similar observation was made by deletion of the C-terminus of a chimera in which the C-terminus half was from beta1a. The identified domain at the C-terminus of beta1a may be responsible for colocalization of DHPRs and ryanodine receptors (RyRs), or may be required for the signal that opens the RyRs during excitation-contraction coupling. This new role of DHPR beta in excitation-contraction coupling represents a cell-specific function that could not be predicted on the basis of functional expression studies in heterologous cells.     

Ca2+-dependent excitation-contraction coupling triggered by the heterologous cardiac/brain DHPR beta2a-subunit in skeletal myotubes

Molecular determinants essential for skeletal-type excitation-contraction (EC) coupling have been described in the cytosolic loops of the dihydropyridine receptor (DHPR) alpha1S pore subunit and in the carboxyl terminus of the skeletal-specific DHPR beta1a-subunit. It is unknown whether EC coupling domains present in the beta-subunit influence those present in the pore subunit or if they act independent of each other. To address this question, we investigated the EC coupling signal that is generated when the endogenous DHPR pore subunit alpha1S is paired with the heterologous heart/brain DHPR beta2a-subunit. Studies were conducted in primary cultured myotubes from beta1 knockout (KO), ryanodine receptor type 1 (RyR1) KO, ryanodine receptor type 3 (RyR3) KO, and double RyR1/RyR3 KO mice under voltage clamp with simultaneous monitoring of confocal fluo-4 fluorescence. The beta2a-mediated Ca2+ current recovered in beta1 KO myotubes lacking the endogenous DHPR beta1a-subunit verified formation of the alpha1S/beta1a pair. In myotube genotypes which express no or low-density L-type Ca2+ currents, namely beta1 KO and RyR1 KO, beta2a overexpression recovered a wild-type density of nifedipine-sensitive Ca2+ currents with a slow activation kinetics typical of skeletal myotubes. Concurrent with Ca2+ current recovery, there was a drastic reduction of voltage-dependent, skeletal-type EC coupling and emergence of Ca2+ transients triggered by the Ca2+ current. A comparison of beta2a overexpression in RyR3 KO, RyR1 KO, and double RyR1/RyR3 KO myotubes concluded that both RyR1 and RyR3 isoforms participated in Ca2+-dependent Ca2+ release triggered by the beta2a-subunit. In beta1 KO and RyR1 KO myotubes, the Ca2+-dependent EC coupling promoted by beta2a overexpression had the following characteristics: 1), L-type Ca2+ currents had a wild-type density; 2), Ca2+ transients activated much slower than controls overexpressing beta1a, and the rate of fluorescence increase was consistent with the activation kinetics of the Ca2+ current; 3), the voltage dependence of the Ca2+ transient was bell-shaped and the maximum was centered at approximately +30 mV, consistent with the voltage dependence of the Ca2+ current; and 4), Ca2+ currents and Ca2+ transients were fully blocked by nifedipine. The loss in voltage-dependent EC coupling promoted by beta2a was inferred by the drastic reduction in maximal Ca2+ fluorescence at large positive potentials (DeltaF/Fmax) in double dysgenic/beta1 KO myotubes overexpressing the pore mutant alpha1S (E1014K) and beta2a. The data indicate that beta2a, upon interaction with the skeletal pore subunit alpha1S, overrides critical EC coupling determinants present in alpha1S. We propose that the alpha1S/beta pair, and not the alpha1S-subunit alone, controls the EC coupling signal in skeletal muscle.     

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

Excitation-contraction coupling in skeletal muscle involves conformational coupling between the dihydropyridine receptor (DHPR) and the type 1 ryanodine receptor (RyR1) at junctions between the plasma membrane and sarcoplasmic reticulum. In an attempt to find which regions of these proteins are in close proximity to one another, we have constructed a tandem of cyan and yellow fluorescent proteins (CFP and YFP, respectively) linked by a 23-residue spacer, and measured the fluorescence resonance energy transfer (FRET) of the tandem either in free solution or after attachment to sites of the alpha1S and beta1a subunits of the DHPR. For all of the sites examined, attachment of the CFP-YFP tandem did not impair function of the DHPR as a Ca2+ channel or voltage sensor for excitation-contraction coupling. The free tandem displayed a 27.5% FRET efficiency, which decreased significantly after attachment to the DHPR subunits. At several sites examined for both alpha1S (N-terminal, proximal II-III loop of a two fragment construct) and beta1a (C-terminal), the FRET efficiency was similar after expression in either dysgenic (alpha1S-null) or dyspedic (RyR1-null) myotubes. However, compared with dysgenic myotubes, the FRET efficiency in dyspedic myotubes increased from 9.9 to 16.7% for CFP-YFP attached to the N-terminal of beta1a, and from 9.5 to 16.8% for CFP-YFP at the C-terminal of alpha1S. Thus, the tandem reporter suggests that the C terminus of alpha1S and the N terminus of beta1a may be in close proximity to the ryanodine receptor.     

Skeletal and cardiac ryanodine receptors bind to the Ca(2+)-sensor region of dihydropyridine receptor alpha(1C) subunit

In striated muscles, excitation-contraction coupling is mediated by the functional interplay between dihydropyridine receptor L-type calcium channels (DHPR) and ryanodine receptor calcium-release channel (RyR). Although significantly different molecular mechanisms are involved in skeletal and cardiac muscles, bidirectional cross-talk between the two channels has been described in both tissues. In the present study using surface plasmon resonance spectroscopy, we demonstrate that both RyR1 and RyR2 can bind to structural elements of the C-terminal cytoplasmic domain of alpha(1C). The interaction is restricted to the CB and IQ motifs involved in the calmodulin-mediated Ca(2+)-dependent inactivation of the DHPR, suggesting functional interactions between the two channels.