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Involvement of a Heptad Repeat in the Carboxyl Terminus of the Dihydropyridine Receptor ß1a Subunit in the Mechanism of Excitation-Contraction Coupling in Skeletal Muscle

Department of Physiology, University of Wisconsin School of Medicine, Madison, Wisconsin 53706

Correspondence: Address reprint requests to Roberto Coronado, Dept. of Physiology, University of Wisconsin, 1300 University Ave., Madison, WI 53706. Tel.: 608-263-7487; Fax: 608-265-5512; E-mail: coronado{at}physiology.wisc.edu.

Chimeras consisting of the homologous skeletal dihydropyridine receptor (DHPR) ß1a subunit and the heterologous cardiac/brain ß2a subunit were used to determine which regions of ß1a were responsible for the skeletal-type excitation-contraction (EC) coupling phenotype. Chimeras were transiently transfected in ß1 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 ß1a-specific domain present in the carboxyl terminus, namely the D5 region comprising the last 47 residues (ß1a 478–524), is essential for expression of skeletal-type EC coupling. Furthermore, the location of ß1aD5 immediately downstream from conserved domain D4 is also critical. In contrast, chimeras in which ß1aD5 was swapped by the D5 region of ß2a expressed Ca²⁺ transients triggered by the Ca²⁺ current, or none at all. A hydrophobic heptad repeat is present in domain D5 of ß1a (L478, V485, V492). To determine the role of this motif, residues in the heptad repeat were mutated to alanines. The triple mutant ß1a(L478A/V485A/V492A) recovered weak skeletal-type EC coupling (F/F_max = 0.4 ± 0.1 vs. 2.7 ± 0.5 for wild-type ß1a). However, a triple mutant with alanine substitutions at positions out of phase with the heptad repeat, ß1a(S481A/L488A/S495A), was normal (F/F_max = 2.1 ± 0.4). In summary, the presence of the ß1a-specific D5 domain, in its correct position after conserved domain D4, is essential for skeletal-type EC coupling. Furthermore, a heptad repeat in ß1aD5 controls the EC coupling activity. The carboxyl terminal heptad repeat of ß1a might be involved in protein-protein interactions with ryanodine receptor type 1 required for DHPR to ryanodine receptor type 1 signal transmission.

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