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Skeletal muscle excitation-contraction coupling is independent of a conserved heptad repeat motif in the C-terminus of the DHPRbeta(1a) subunit.

ABSTRACT: In skeletal muscle excitation-contraction (EC) coupling the sarcolemmal L-type Ca(2+) channel or 1,4-dihydropyridine receptor (DHPR) transduces the membrane depolarization signal to the sarcoplasmic Ca(2+) release channel RyR1 via protein-protein interaction. While it is evident that the pore-forming and voltage-sensing DHPRalpha(1S) subunit is essential for this process, the intracellular DHPRbeta(1a) subunit was also shown to be indispensable. We previously found that the beta(1a) subunit is essential to target the DHPR into groups of four (tetrads) opposite the RyR1 homotetramers, a prerequisite for skeletal muscle EC coupling. Earlier, a unique hydrophobic heptad repeat motif (Lcdots, three dots, centeredVcdots, three dots, centeredV) in the C-terminus of beta(1a) was postulated by others to be essential for skeletal muscle EC coupling, as substitution of these residues with alanines resulted in 80% reduction of RyR1 Ca(2+) release. Therefore, we wanted to address the question if the proposed beta(1a) heptad repeat motif could be an active element of the DHPR-RyR1 signal transduction mechanism or already contributes at the ultrastructural level i.e. DHPR tetrad arrangement. Surprisingly, our experiments revealed full tetrad formation and an almost complete restoration of EC coupling in beta(1)-null zebrafish relaxed larvae and isolated myotubes upon expression of a beta(1a)-specific heptad repeat mutant (LVV to AAA) and thus contradict the earlier results.

SUBMITTER: Dayal A

PROVIDER: S-EPMC2896708 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Skeletal muscle excitation-contraction coupling is independent of a conserved heptad repeat motif in the C-terminus of the DHPRbeta(1a) subunit.

Dayal Anamika A Schredelseker Johann J Franzini-Armstrong Clara C Grabner Manfred M

Cell calcium 20100506 6

In skeletal muscle excitation-contraction (EC) coupling the sarcolemmal L-type Ca(2+) channel or 1,4-dihydropyridine receptor (DHPR) transduces the membrane depolarization signal to the sarcoplasmic Ca(2+) release channel RyR1 via protein-protein interaction. While it is evident that the pore-forming and voltage-sensing DHPRalpha(1S) subunit is essential for this process, the intracellular DHPRbeta(1a) subunit was also shown to be indispensable. We previously found that the beta(1a) subunit is e ...[more]

PMID: 20451250

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Project description:Chimeras consisting of the homologous skeletal dihydropyridine receptor (DHPR) beta1a subunit and the heterologous cardiac/brain beta2a subunit were used to determine which regions of beta1a were responsible for the skeletal-type excitation-contraction (EC) coupling phenotype. Chimeras were transiently transfected in beta1 knockout myotubes and then voltage-clamped with simultaneous measurement of confocal fluo-4 fluorescence. All chimeras expressed a similar density of DHPR charge movements, indicating that the membrane density of DHPR voltage sensors was not a confounding factor in these studies. The data indicates that a beta1a-specific domain present in the carboxyl terminus, namely the D5 region comprising the last 47 residues (beta1a 478-524), is essential for expression of skeletal-type EC coupling. Furthermore, the location of beta1aD5 immediately downstream from conserved domain D4 is also critical. In contrast, chimeras in which beta1aD5 was swapped by the D5 region of beta2a expressed Ca(2+) transients triggered by the Ca(2+) current, or none at all. A hydrophobic heptad repeat is present in domain D5 of beta1a (L478, V485, V492). To determine the role of this motif, residues in the heptad repeat were mutated to alanines. The triple mutant beta1a(L478A/V485A/V492A) recovered weak skeletal-type EC coupling (DeltaF/F(max) = 0.4 +/- 0.1 vs. 2.7 +/- 0.5 for wild-type beta1a). However, a triple mutant with alanine substitutions at positions out of phase with the heptad repeat, beta1a(S481A/L488A/S495A), was normal (DeltaF/F(max) = 2.1 +/- 0.4). In summary, the presence of the beta1a-specific D5 domain, in its correct position after conserved domain D4, is essential for skeletal-type EC coupling. Furthermore, a heptad repeat in beta1aD5 controls the EC coupling activity. The carboxyl terminal heptad repeat of beta1a might be involved in protein-protein interactions with ryanodine receptor type 1 required for DHPR to ryanodine receptor type 1 signal transmission.

Project description: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.

Project description: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.

Project description:In skeletal muscle, excitation-contraction (EC) coupling requires depolarization-induced conformational rearrangements in L-type Ca(2+) channel (Ca(V)1.1) to be communicated to the type 1 ryanodine-sensitive Ca(2+) release channel (RYR1) of the sarcoplasmic reticulum (SR) via transient protein-protein interactions. Although the molecular mechanism that underlies conformational coupling between Ca(V)1.1 and RYR1 has been investigated intensely for more than 25 years, the question of whether such signaling occurs via a direct interaction between the principal, voltage-sensing ?(1S) subunit of Ca(V)1.1 and RYR1 or through an intermediary protein persists. A substantial body of evidence supports the idea that the auxiliary ?(1a) subunit of Ca(V)1.1 is a conduit for this intermolecular communication. However, a direct role for ?(1a) has been difficult to test because ?(1a) serves two other functions that are prerequisite for conformational coupling between Ca(V)1.1 and RYR1. Specifically, ?(1a) promotes efficient membrane expression of Ca(V)1.1 and facilitates the tetradic ultrastructural arrangement of Ca(V)1.1 channels within plasma membrane-SR junctions. In this paper, we demonstrate that overexpression of the RGK protein Rem, an established ? subunit-interacting protein, in adult mouse flexor digitorum brevis fibers markedly reduces voltage-induced myoplasmic Ca(2+) transients without greatly affecting Ca(V)1.1 targeting, intramembrane gating charge movement, or releasable SR Ca(2+) store content. In contrast, a ?(1a)-binding-deficient Rem triple mutant (R200A/L227A/H229A) has little effect on myoplasmic Ca(2+) release in response to membrane depolarization. Thus, Rem effectively uncouples the voltage sensors of Ca(V)1.1 from RYR1-mediated SR Ca(2+) release via its ability to interact with ?(1a). Our findings reveal Rem-expressing adult muscle as an experimental system that may prove useful in the definition of the precise role of the ?(1a) subunit in skeletal-type EC coupling.

Project description: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.

Dataset Information

Skeletal muscle excitation-contraction coupling is independent of a conserved heptad repeat motif in the C-terminus of the DHPRbeta(1a) subunit.

Publications

Skeletal muscle excitation-contraction coupling is independent of a conserved heptad repeat motif in the C-terminus of the DHPRbeta(1a) subunit.

Similar Datasets

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