Biophys J, December 2001, p. 3294-3307, Vol. 81, No. 6

A Component of Excitation-Contraction Coupling Triggered in the Absence of the T671-L690 and L720-Q765 Regions of the II-III Loop of the Dihydropyridine Receptor _1s Pore Subunit

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

We conducted a deletion analysis of two regions identified in the II-III loop of _1S, residues 671-690, which were shown to bind to ryanodine receptor type 1 (RyR1) and stimulate RyR1 channels in vitro, and residues 720-765 or the narrower 724-743 region, which confer excitation-contraction (EC) coupling function to chimeric dihydropyridine receptors (DHPRs). Deletion mutants were expressed in dysgenic _1S-null myotubes and analyzed by voltage-clamp and confocal fluo-4 fluorescence. Immunostaining of the mutant subunits using an N-terminus tag revealed abundant protein expression in all cases. Furthermore, the maximum recovered charge movement density was >80% of that recovered by full-length _1S in all cases. 671-690 had no effect on the magnitude of voltage-evoked Ca²⁺ transients or the L-type Ca²⁺ current density. In contrast, 720-765 or 724-743 abolished Ca²⁺ transients entirely, and L-type Ca²⁺ current was reduced or absent. Surprisingly, Ca²⁺ transients and Ca²⁺ currents of a moderate magnitude were recovered by the double deletion mutant 671-690/720-765. A simple explanation for this result is that 720-765 induces a conformation change that disrupts EC coupling, and this conformational change is partially reverted by 671-690. To test for Ca²⁺-entry independent EC coupling, a pore mutation (E1014K) known to entirely abolish the inward Ca²⁺ current was introduced. _1S 671-690/720-765/E1014K expressed Ca²⁺ transients with Boltzmann parameters identical to those of the Ca²⁺-conducting double deletion construct. The data strongly suggest that skeletal-type EC coupling is not uniquely controlled by _1S 720-765. Other regions of _1S or other DHPR subunits must therefore directly contribute to the activation of RyR1 during EC coupling.

Biophys J, December 2001, p. 3294-3307, Vol. 81, No. 6
© 2001 by the Biophysical Society   0006-3495/01/12/3294/14  $2.00

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