Multiple loops of the dihydropyridine receptor pore subunit are required for full-scale excitation-contraction coupling in skeletal muscle

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 alpha 1S (Ca(V)1.1) II-III loop when alone or in combination with the rest of the cytosolic domains of alpha 1S. Chimeras consisting of alpha 1C (Ca(V)1.2) with alpha 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 (alpha 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 alpha 1C/alpha 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 alpha 1C. The Ca2+ transients expressed by the alpha 1C(E736K)/alpha 1S II-III loop chimera were similar to 70% smaller than those expressed by the Ca2+-conducting alpha 1C/alpha 1S II-III variant. The low skeletal-type EC coupling expressed by the alpha 1C/alpha 1S II-III loop chimera was confirmed in the Ca2+-conducting alpha 1C/alpha 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 alpha 1C/alpha 1S chimera carrying all tested skeletal alpha 1S domains (all alpha 1S interrepeat loops, N- and C-terminus) were similar in shape and amplitude to wild-type alpha 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 alpha 1S(E1014K) variant, the alpha 1C(E736K)/alpha 1S II-III loop chimera, and the alpha 1C(E736K)/alpha 1S chimera carrying all tested alpha 1S domains. The data indicate that the functional recovery produced by the alpha 1S II-III loop is incomplete and that multiple cytosolic domains of alpha 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 alpha 1S that influence the efficiency of EC coupling.