Key words dihydropyridine receptor, L-type calcium channel, voltage sensor, subunits, patch clamp, site-directed mutagenesis
Springer Online Journal Archives 1860-2000
Abstract A histidine substitution for the outermost arginine in II/S4 of the α1 subunit of the human skeletal muscle dihydropyridine (DHP) receptor has been reported to cause hypokalaemic periodic paralysis (HypoPP). This mutation shifts the voltage dependence of L-type Ca curent inactivation in myotubes from HypoPP patients by −40 mV without affecting activation. Based on the strong homology of II/S4 in cardiac and skeletal muscle α1, we introduced the corresponding mutation into the rabbit cardiac α1 subunit (R650H). Wild type (WT) and mutant constructs were transiently transfected in HEK cells together with β and α2δ subunits and Ca and Ba currents were studied using the whole-cell patch-clamp technique. In contrast to the results obtained from human myotubes, R650H produced a small (−5 mV) but significant shift of both the steady-state activation and inactivation curves. When external pH was increased from 7.4 to 8.4 in order to favour deprotonization of H650, the only difference between WT and mutant channels was a slightly reduced steepness of the inactivation curve. Additional cotransfection of the γ subunit which is only found in skeletal but not in heart muscle, shifted the inactivation curves of both WT and R650H by −20 mV. We conclude that R650 plays a different role in voltage-dependent gating of the cardiac L-type Ca channel than the corresponding residue in the human skeletal muscle L-type channel, since a distinct and selective effect on the midpoint voltage of steady-state inactivation could not be found for R650H.
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