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

Abstract

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 α1S (Ca V1.1) II-III loop when alone or in combination with the rest of the cytosolic domains of α1S. Chimeras consisting of α1C (Ca V1.2) with α1S 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 ( α1S-null) myotubes for phenotypic expression of skeletal-type EC coupling. Myotubes were voltage-clamped, and Ca 2+ transients were measured by confocal line-scan imaging of fluo-4 fluorescence. In agreement with previous results, the α1C/ α1S 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 Ca 2+ transients in the absence of inward Ca 2+ current, but without changing the external solution, a mutation, E736K, was introduced into the P-loop of repeat II of α1C. The Ca 2+ transients expressed by the α1C(E736K)/ α1S II-III loop chimera were ∼70% smaller than those expressed by the Ca 2+-conducting α1C/ α1S II-III variant. The low skeletal-type EC coupling expressed by the α1C/ α1S II-III loop chimera was confirmed in the Ca 2+-conducting α1C/ α1S II-III loop variant using Cd 2+ (10 −4 M) as the Ca 2+ current blocker. In contrast to the behavior of the II-III loop chimera, Ca 2+ transients expressed by an α1C/ α1S chimera carrying all tested skeletal α1S domains (all α1S interrepeat loops, N- and C-terminus) were similar in shape and amplitude to wild-type α1S, and did not change in the presence of the E736K mutation or in the presence of 10 −4 M Cd 2+. Controls indicated that similar dihydropyridine receptor charge movements were expressed by the non-Ca 2+ permeant α1S(E1014K) variant, the α1C(E736K)/ α1S II-III loop chimera, and the α1C(E736K)/ α1S chimera carrying all tested α1S domains. The data indicate that the functional recovery produced by the α1S II-III loop is incomplete and that multiple cytosolic domains of α1S 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 α1S that influence the efficiency of EC coupling.