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Multiple Loops of the Dihydropyridine Receptor Pore Subunit Are Required for Full-Scale Excitation-Contraction Coupling in Skeletal Muscle

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

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.

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 1S (Ca_V1.1) II-III loop when alone or in combination with the rest of the cytosolic domains of 1S. Chimeras consisting of 1C (Ca_V1.2) with 1S 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 (1S-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 1C/1S 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 1C. The Ca²⁺ transients expressed by the 1C(E736K)/1S II-III loop chimera were 70% smaller than those expressed by the Ca²⁺-conducting 1C/1S II-III variant. The low skeletal-type EC coupling expressed by the 1C/1S II-III loop chimera was confirmed in the Ca²⁺-conducting 1C/1S 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 1C/1S chimera carrying all tested skeletal 1S domains (all 1S interrepeat loops, N- and C-terminus) were similar in shape and amplitude to wild-type 1S, 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 1S(E1014K) variant, the 1C(E736K)/1S II-III loop chimera, and the 1C(E736K)/1S chimera carrying all tested 1S domains. The data indicate that the functional recovery produced by the 1S II-III loop is incomplete and that multiple cytosolic domains of 1S 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 1S that influence the efficiency of EC coupling.

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