Chris A. Ahern

Involvement of the Carboxy-Terminus Region of the Dihydropyridine Receptor β1a 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.

Comparison of Ca2+ Sparks Produced Independently by Two Ryanodine Receptor Isoforms (Type 1 or Type 3)

The molecular determinants of a Ca(2+) spark, those events that determine the sudden opening and closing of a small number of ryanodine receptor (RyR) channels limiting Ca(2+) release to a few milliseconds, are unknown. As a first step we investigated which of two RyR isoforms present in mammalian embryonic skeletal muscle, RyR type 1(RyR-1) or RyR type 3 (RyR-3) has the ability to generate Ca(2+) sparks. Their separate contributions were investigated in intercostal muscle cells of RyR-1 null and RyR-3 null mouse embryos. A comparison of Ca(2+) spark parameters of RyR-1 null versus RyR-3 null cells measured at rest with fluo-3 showed that neither the peak fluorescence intensity (DeltaF/F(o) = 1.25 +/- 0.7 vs. 1.55 +/- 0.6), spatial width at half-max intensity (FWHM = 2.7 +/- 1.2 vs. 2.6 +/- 0.6 microm), nor the duration at half-max intensity (FTHM = 45 +/- 49 vs. 43 +/- 25 ms) was significantly different. Sensitivity to caffeine (0.1 mM) was remarkably different, with sparks in RyR-1 null myotubes becoming brighter and longer in duration, whereas those in RyR-3 null cells remained unchanged. Controls performed in double RyR-1/RyR-3 null cells obtained by mice breeding showed that sparks were not observed in the absence of both isoforms in >150 cells imaged. In conclusion, 1) RyR-1 and RyR-3 appear to be the only intracellular Ca(2+) channels that participate in Ca(2+) spark activity in embryonic skeletal muscle; 2) except in their responsiveness to caffeine, both isoforms have the ability to produce Ca(2+) sparks with nearly identical properties, so it is rather unlikely that a single RyR isoform, when others are also present, would be responsible for Ca(2+) sparks; and 3) because RyR-1 null cells are excitation-contraction (EC) uncoupled and RyR-3 null cells exhibit a normal phenotype, Ca(2+) sparks result from the inherent activity of small clusters of RyRs regardless of the participation of these RyRs in EC coupling.

Truncation of the Carboxyl Terminus of the DihydropyridineReceptor β1a Subunit Promotes Ca2+ Dependent Excitation-Contraction Coupling in Skeletal Myotubes

We investigated the contribution of the carboxyl terminus region of the beta1a subunit of the skeletal dihydropyridine receptor (DHPR) to the mechanism of excitation-contraction (EC) coupling. cDNA-transfected beta1 KO myotubes were voltage clamped, and Ca(2+) transients were analyzed by confocal fluo-4 fluorescence. A chimera with an amino terminus half of beta2a and a carboxyl terminus half of beta1a (beta2a 1-287/beta1a 325-524) recapitulates skeletal-type EC coupling quantitatively and was used to generate truncated variants lacking 7 to 60 residues from the beta1a-specific carboxyl terminus (Delta7, Delta21, Delta29, Delta35, and Delta60). Ca(2+) transients recovered by the control chimera have a sigmoidal Ca(2+) fluorescence (DeltaF/F) versus voltage curve with saturation at potentials more positive than +30 mV, independent of external Ca(2+) and stimulus duration. In contrast, the amplitude of Ca(2+) transients expressed by the truncated variants varied with the duration of the pulse, and for Delta29, Delta35, and Delta60, also varied with external Ca(2+) concentration. For Delta7 and Delta21, a 50-ms depolarization produced a sigmoidal DeltaF/F versus voltage curve with a lower than control maximum fluorescence. Moreover, for Delta29, Delta35, and Delta60, a 200-ms depolarization increased the maximum fluorescence and changed the shape of the DeltaF/F versus voltage curve, from sigmoidal to bell-shaped, with a maximum at approximately +30 mV. The change in voltage dependence, together with the external Ca(2+) dependence and additional controls with ryanodine, indicated a loss of skeletal-type EC coupling and the emergence of an EC coupling component triggered by the Ca(2+) current. Analyses of d(DeltaF/F)/dt showed that the rate of cytosolic Ca(2+) increase during the Ca(2+) transient was fivefold faster for the control chimera than for the severely truncated variants (Delta29, Delta35, and Delta60) and was consistent with the kinetics of the DHPR Ca(2+) current. In summary, absence of the beta1a-specific carboxyl terminus (last 29 to 60 residues of the control chimera) results in a loss of the fast component of the Ca(2+) transient, bending of the DeltaF/F versus voltage curve, and emergence of EC coupling triggered by the Ca(2+) current. The studies underscore the essential role of the carboxyl terminus region of the DHPR beta1a subunit in fast voltage dependent EC coupling in skeletal myotubes.

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 α1s 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.

Differential Regulation of Skeletal Muscle L-Type Ca2+ Current and Excitation-Contraction Coupling by the Dihydropyridine Receptor β Subunit

The dihydropyridine receptor (DHPR) of skeletal muscle functions as a Ca2+ channel and is required for excitation-contraction (EC) coupling. Here we show that the DHPR beta subunit is involved in the regulation of these two functions. Experiments were performed in skeletal mouse myotubes selectively lacking a functional DHPR beta subunit. These beta-null cells have a low-density L-type current, a low density of charge movements, and lack EC coupling. Transfection of beta-null cells with cDNAs encoding for either the homologous beta1a subunit or the cardiac- and brain-specific beta2a subunit fully restored the L-type Ca2+ current (161 +/- 17 pS/pF and 139 +/- 9 pS/pF, respectively, in 10 mM Ca2+). We compared the Boltzmann parameters of the Ca2+ conductance restored by beta1a and beta2a, the kinetics of activation of the Ca2+ current, and the single channel parameters estimated by ensemble variance analysis and found them to be indistinguishable. In contrast, the maximum density of charge movements in cells expressing beta2a was significantly lower than in cells expressing beta1a (2.7 +/- 0.2 nC/microF and 6.7 +/- 0. 4 nC/microF, respectively). Furthermore, the amplitude of Ca2+ transient measured by confocal line-scans of fluo-3 fluorescence in voltage-clamped cells were 3- to 5-fold lower in myotubes expressing beta2a. In summary, DHPR complexes that included beta2a or beta1a restored L-type Ca2+ channels. However, a DHPR complex with beta1a was required for complete restoration of charge movements and skeletal-type EC coupling. These results suggest that the beta1a subunit participates in key regulatory events required for the EC coupling function of the DHPR.

Intramembrane charge movements and excitation– contraction coupling expressed by two-domain fragments of the Ca2+ channel

To investigate the molecular basis of the voltage sensor that triggers excitation–contraction (EC) coupling, the four-domain pore subunit of the dihydropyridine receptor (DHPR) was cut in the cytoplasmic linker between domains II and III. cDNAs for the I-II domain (α1S 1–670) and the III-IV domain (α1S 701-1873) were expressed in dysgenic α1S-null myotubes. Coexpression of the two fragments resulted in complete recovery of DHPR intramembrane charge movement and voltage-evoked Ca2+ transients. When fragments were expressed separately, EC coupling was not recovered. However, charge movement was detected in the I-II domain expressed alone. Compared with I-II and III-IV together, the charge movement in the I-II domain accounted for about half of the total charge (Qmax = 3 ± 0.23 vs. 5.4 ± 0.76 fC/pF, respectively), and the half-activation potential for charge movement was significantly more negative (V1/2 = 0.2 ± 3.5 vs. 22 ± 3.4 mV, respectively). Thus, interactions between the four internal domains of the pore subunit in the assembled DHPR profoundly affect the voltage dependence of intramembrane charge movement. We also tested a two-domain I-II construct of the neuronal α1A Ca2+ channel. The neuronal I-II domain recovered charge movements like those of the skeletal I-II domain but could not assist the skeletal III-IV domain in the recovery of EC coupling. The results demonstrate that a functional voltage sensor capable of triggering EC coupling in skeletal myotubes can be recovered by the expression of complementary fragments of the DHPR pore subunit. Furthermore, the intrinsic voltage-sensing properties of the α1A I-II domain suggest that this hemi-Ca2+ channel could be relevant to neuronal function.

Ca2+ Current and Charge Movements in Skeletal Myotubes Promoted by the β-Subunit of the Dihydropyridine Receptor in the Absence of Ryanodine Receptor Type 1

The beta-subunit of the dihydropyridine receptor (DHPR) enhances the Ca(2+) channel and voltage-sensing functions of the DHPR. In skeletal myotubes, there is additional modulation of DHPR functions imposed by the presence of ryanodine receptor type-1 (RyR1). Here, we examined the participation of the beta-subunit in the expression of L-type Ca(2+) current and charge movements in RyR1 knock-out (KO), beta1 KO, and double beta1/RyR1 KO myotubes generated by mating heterozygous beta1 KO and RyR1 KO mice. Primary myotube cultures of each genotype were transfected with various beta-isoforms and then whole-cell voltage-clamped for measurements of Ca(2+) and gating currents. Overexpression of the endogenous skeletal beta1a isoform resulted in a low-density Ca(2+) current either in RyR1 KO (36 +/- 9 pS/pF) or in beta1/RyR1 KO (34 +/- 7 pS/pF) myotubes. However, the heterologous beta2a variant with a double cysteine motif in the N-terminus (C3, C4), recovered a Ca(2+) current that was entirely wild-type in density in RyR1 KO (195 +/- 16 pS/pF) and was significantly enhanced in double beta1/RyR1 KO (115 +/- 18 pS/pF) myotubes. Other variants tested from the four beta gene families (beta1a, beta1b, beta1c, beta3, and beta4) were unable to enhance Ca(2+) current expression in RyR1 KO myotubes. In contrast, intramembrane charge movements in beta2a-expressing beta1a/RyR1 KO myotubes were significantly lower than in beta1a-expressing beta1a/RyR1 KO myotubes, and the same tendency was observed in the RyR1 KO myotube. Thus, beta2a had a preferential ability to recover Ca(2+) current, whereas beta1a had a preferential ability to rescue charge movements. Elimination of the double cysteine motif (beta2a C3,4S) eliminated the RyR1-independent Ca(2+) current expression. Furthermore, Ca(2+) current enhancement was observed with a beta2a variant lacking the double cysteine motif and fused to the surface membrane glycoprotein CD8. Thus, tethering the beta2a variant to the myotube surface activated the DHPR Ca(2+) current and bypassed the requirement for RyR1. The data suggest that the Ca(2+) current expressed by the native skeletal DHPR complex has an inherently low density due to inhibitory interactions within the DHPR and that the beta1a-subunit is critically involved in process.

Modulation of L-type Ca2+ current but not activation of Ca2+ release by the gamma1 subunit of the dihydropyridine receptor of skeletal muscle

BackgroundThe multisubunit (α1S,α2-δ, β1a and γ1) skeletal muscle dihydropyridine receptor (DHPR) transduces membrane depolarization into release of Ca2+ from the sarcoplasmic reticulum (SR) and also acts as an L-type Ca2+ channel. To more fully investigate the function of the γ1 subunit in these two processes, we produced mice lacking this subunit by gene targeting.ResultsMice lacking the DHPR γ1 subunit (γ1 null) survive to adulthood, are fertile and have no obvious gross phenotypic abnormalities. The γ1 subunit is expressed at approximately half the normal level in heterozygous mice (γ1 het). The density of the L-type Ca2+ current in γ1 null and γ1 het myotubes was higher than in controls. Inactivation of the Ca2+ current produced by a long depolarization was slower and incomplete in γ1 null and γ1 het myotubes, and was shifted to a more positive potential than in controls. However, the half-activation potential of intramembrane charge movements was not shifted, and the maximum density of the total charge was unchanged. Also, no shift was observed in the voltage-dependence of Ca2+ transients. γ1 null and γ1 het myotubes had the same peak Ca2+ amplitude vs. voltage relationship as control myotubes.ConclusionsThe L-type Ca2+ channel function, but not the SR Ca2+ release triggering function of the skeletal muscle dihydropyridine receptor, is modulated by the γ1 subunit.

Functional equivalence of dihydropyridine receptor a1S and b1a 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.

Ca2+-Dependent Excitation-Contraction Coupling Triggered by the Heterologous Cardiac/Brain DHPR β2a-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.