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.     

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.     

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.     

Ryanodine receptor type 1 (RyR1) mutations C4958S and C4961S reveal excitation-coupled calcium entry (ECCE) is independent of sarcoplasmic reticulum store depletion

Bi-directional signaling between ryanodine receptor type 1 (RyR1) and dihydropyridine receptor (DHPR) in skeletal muscle serves as a prominent example of conformational coupling. Evidence for a physiological mechanism that upon depolarization of myotubes tightly couples three calcium channels, DHPR, RyR1, and a Ca(2+) entry channel with SOCC-like properties, has recently been presented. This form of conformational coupling, termed excitation-coupled calcium entry (ECCE) is triggered by the alpha(1s)-DHPR voltage sensor and is highly dependent on RyR1 conformation. In this report, we substitute RyR1 cysteines 4958 or 4961 within the TXCFICG motif, common to all ER/SR Ca(2+) channels, with serine. When expressed in skeletal myotubes, C4958S- and C4961S-RyR1 properly target and restore L-type current via the DHPR. However, these mutants do not respond to RyR activators and do not support skeletal type EC coupling. Nonetheless, depolarization of cells expressing C4958S- or C4961S-RyR1 triggers calcium entry via ECCE that resembles that for wild-type RyR1, except for substantially slowed inactivation and deactivation kinetics. ECCE in these cells is completely independent of store depletion, displays a cation selectivity of Ca(2+)>Sr(2+) approximately Ba(2+), and is fully inhibited by SKF-96365 or 2-APB. Mutation of other non-CXXC motif cysteines within the RyR1 transmembrane assembly (C3635S, C4876S, and C4882S) did not replicate the phenotype observed with C4958S- and C4961S-RyR1. This study demonstrates the essential role of Cys(4958) and Cys(4961) within an invariant CXXC motif for stabilizing conformations of RyR1 that influence both its function as a release channel and its interaction with ECCE channels.     

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.     

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.     

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.     

Structural requirements of the dihydropyridine receptor alpha1S II-III loop for skeletal-type excitation-contraction coupling

Residues Leu720-Leu764 within the II-III loop of the skeletal muscle dihydropyridine receptor (DHPR) alpha1S subunit represent a critical domain for the orthograde excitation-contraction coupling as well as for retrograde DHPR L-current-enhancing coupling with the ryanodine receptor (RyR1). To better understand the molecular mechanism underlying this bidirectional DHPR-RyR1 signaling interaction, we analyzed the critical domain to the single amino acid level. To this end, constructs based on the highly dissimilar housefly DHPR II-III loop in an otherwise skeletal DHPR as an interaction-inert sequence background were expressed in dysgenic (alpha1S-null) myotubes for simultaneous recordings of depolarization-induced intracellular Ca2+ transients (orthograde coupling) and whole-cell Ca2+ currents (retrograde coupling). In the minimal skeletal II-III loop sequence (Asp734-Asp748 required for full bidirectional coupling, eight amino acids heterologous between skeletal and cardiac DHPR were exchanged for the corresponding cardiac residues. Four of these skeletal-specific residues (Ala739, Phe741, Pro742, and Asp744) turned out to be essential for orthograde and two of them (Ala739 and Phe741) for retrograde coupling, indicating that orthograde coupling does not necessarily correlate with retrograde signaling. Secondary structure predictions of the critical domain show that an alpha-helical (cardiac sequence-type) conformation of a cluster of negatively charged residues (Asp744-Glu751 of alpha1S) corresponds with significantly reduced Ca2+ transients. Conversely, a predicted random coil structure (skeletal sequence-type) seems to be prerequisite for the restoration of skeletal-type excitation-contraction coupling. Thus, not only the primary but also the secondary structure of the critical domain is an essential determinant of the tissue-specific mode of EC coupling.     

Molecular origin of the L-type Ca2+ current of skeletal muscle myotubes selectively deficient in dihydropyridine receptor beta1a subunit.

The origin of Ibetanull, the Ca2+ current of myotubes from mice lacking the skeletal dihydropyridine receptor (DHPR) beta1a subunit, was investigated. The density of Ibetanull was similar to that of Idys, the Ca2+ current of myotubes from dysgenic mice lacking the skeletal DHPR alpha1S subunit (-0.6 +/- 0.1 and -0.7 +/- 0.1 pA/pF, respectively). However, Ibetanull activated at significantly more positive potentials. The midpoints of the GCa-V curves were 16.3 +/- 1.1 mV and 11.7 +/- 1.0 mV for Ibetanull and Idys, respectively. Ibetanull activated significantly more slowly than Idys. At +30 mV, the activation time constant for Ibetanull was 26 +/- 3 ms, and that for Idys was 7 +/- 1 ms. The unitary current of normal L-type and beta1-null Ca2+ channels estimated from the mean variance relationship at +20 mV in 10 mM external Ca2+ was 22 +/- 4 fA and 43 +/- 7 fA, respectively. Both values were significantly smaller than the single-channel current estimated for dysgenic Ca2+ channels, which was 84 +/- 9 fA under the same conditions. Ibetanull and Idys have different gating and permeation characteristics, suggesting that the bulk of the DHPR alpha1 subunits underlying these currents are different. Ibetanull is suggested to originate primarily from Ca2+ channels with a DHPR alpha1S subunit. Dysgenic Ca2+ channels may be a minor component of this current. The expression of DHPR alpha1S in beta1-null myotubes and its absence in dysgenic myotubes was confirmed by immunofluorescence labeling of cells.     

Numerical analysis of ryanodine receptor activation by L-type channel activity in the cardiac muscle diad.

Computer simulations were used to examine the response of ryanodine receptors (RyRs) to the sarcolemmal calcium influx via L-type calcium channels (DHPRs). The effects of ryanodine receptor organization, diad geometry, DHPR single-channel current, and DHPR gating were examined. In agreement with experimental findings, the simulations showed that RyRs can respond rapidly (approximately 0.4 ms) to calcium influx via DHPRs. The responsiveness of the RyR depends on the geometrical arrangement between the RyRs and the DHPR in the diad, with wider diads being generally less responsive. When the DHPR single-channel current is small (approximately 25 fA), the organization of RyRs into small clusters results in an improved responsiveness. With experimentally observed DHPR mean open and closed times (0.17 ms and 4 ms, respectively) it is the first opening of the DHPR that is most likely to activate the RyR. A measure of the efficiency (Q) by which DHPR gating evokes sarcoplasmic reticulum release is defined. Q is at maximum for tau approximately 0.3 ms, and we interpret this finding in terms of the "tuning" of DHPR gating to RyR response. If certain cardiac myopathies are associated with a mismatch in the "tuning," then modification of DHPR gating with drugs to "retune" calcium-induced calcium release should be possible.     

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.     

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.     

RyR1/RyR3 chimeras reveal that multiple domains of RyR1 are involved in skeletal-type E-C coupling

Skeletal-type E-C coupling is thought to require a direct interaction between RyR1 and the alpha(1S)-DHPR. Most available evidence suggests that the cytoplasmic II-III loop of the dihydropyridine receptor (DHPR) is the primary source of the orthograde signal. However, identification of the region(s) of RyR1 involved in bidirectional signaling with the alpha(1S)-DHPR remains elusive. To identify these regions we have designed a series of chimeric RyR cDNAs in which different segments of RyR1 were inserted into the corresponding region of RyR3 and expressed in dyspedic 1B5 myotubes. RyR3 provides a preferable background than RyR2 for defining domains essential for E-C coupling because it possesses less sequence homology to RyR1 than the RyR2 backbone used in previous studies. Our data show that two regions of RyR1 (chimera Ch-10 aa 1681-2641 and Ch-9 aa 2642-3770), were independently able to restore skeletal-type E-C coupling to RyR3. These two regions were further mapped and the critical RyR1 residues were 1924-2446 (Ch-21) and 2644-3223 (Ch-19). These results both support and refine the previous hypothesis that multiple domains of RyR1 combine to functionally interact with the DHPR during E-C coupling.     

Multiple regions of RyR1 mediate functional and structural interactions with alpha(1S)-dihydropyridine receptors in skeletal muscle

Excitation-contraction (e-c) coupling in muscle relies on the interaction between dihydropyridine receptors (DHPRs) and RyRs within Ca(2+) release units (CRUs). In skeletal muscle this interaction is bidirectional: alpha(1S)DHPRs trigger RyR1 (the skeletal form of the ryanodine receptor) to release Ca(2+) in the absence of Ca(2+) permeation through the DHPR, and RyR1s, in turn, affect the open probability of alpha(1S)DHPRs. alpha(1S)DHPR and RyR1 are linked to each other, organizing alpha(1S)-DHPRs into groups of four, or tetrads. In cardiac muscle, however, alpha(1C)DHPR Ca(2+) current is important for activation of RyR2 (the cardiac isoform of the ryanodine receptor) and alpha(1C)-DHPRs are not organized into tetrads. We expressed RyR1, RyR2, and four different RyR1/RyR2 chimeras (R4: Sk1635-3720, R9: Sk2659-3720, R10: Sk1635-2559, R16: Sk1837-2154) in 1B5 dyspedic myotubes to test their ability to restore skeletal-type e-c coupling and DHPR tetrads. The rank-order for restoring skeletal e-c coupling, indicated by Ca(2+) transients in the absence of extracellular Ca(2+), is RyR1 > R4 > R10 > R16 > R9 > RyR2. The rank-order for restoration of DHPR tetrads is RyR1 > R4 = R9 > R10 = R16 > RyR2. Because the skeletal segment in R9 does not overlap with that in either R10 or R16, our results indicate that multiple regions of RyR1 may interact with alpha(1S)DHPRs and that the regions responsible for tetrad formation do not correspond exactly to the ones required for functional coupling.     

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.     

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.     

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 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.     

Metabolic biotinylation as a probe of supramolecular structure of the triad junction in skeletal muscle

Excitation-contraction coupling in skeletal muscle involves conformational coupling between dihydropyridine receptors (DHPRs) in the plasma membrane and ryanodine receptors (RyRs) in the sarcoplasmic reticulum. However, it remains uncertain what regions, if any, of the two proteins interact with one another. Toward this end, it would be valuable to know the spatial interrelationships of DHPRs and RyRs within plasma membrane/sarcoplasmic reticulum junctions. Here we describe a new approach based on metabolic incorporation of biotin into targeted sites of the DHPR. To accomplish this, cDNAs were constructed with a biotin acceptor domain (BAD) fused to selected sites of the DHPR, with fluorescent protein (XFP) attached at a second site. All of the BAD-tagged constructs properly targeted to junctions (as indicted by small puncta of XFP) and were functional for excitation-contraction coupling. To determine whether the introduced BAD was biotinylated and accessible to avidin (approximately 60 kDa), myotubes were fixed, permeablized, and exposed to fluorescently labeled avidin. Upon expression in beta1-null or dysgenic (alpha1S-null) myotubes, punctate avidin fluorescence co-localized with the XFP puncta for BAD attached to the beta1a N- or C-terminals, or the alpha1S N-terminal or II-III loop. However, BAD fused to the alpha1S C-terminal was inaccessible to avidin in dysgenic myotubes (containing RyR1). In contrast, this site was accessible to avidin when the identical construct was expressed in dyspedic myotubes lacking RyR1. These results indicate that avidin has access to a number of sites of the DHPR within fully assembled (RyR1-containing) junctions, but not to the alpha1S C-terminal, which appears to be occluded by the presence of RyR1.