Fast inactivation of voltage-dependent calcium channels - A hinged-lid mechanism?

We recently described domains II and III as important determinants of fast, voltage-dependent inactivation of R-type calcium channels (Spaetgens, R, L ., and Zamponi, G. W. (1999) J. Biol. Chem. 274, 22428-22438). Here we exam ine in greater detail the structural determinants of inactivation using a s eries of chimeras comprising various regions of wild type alpha(1C) and alp ha(1E) calcium channels. Substitution of the II S6 and/or III S6 segments o f alpha(1E) into the alpha(1C) backbone resulted in rapid inactivation rate s that closely approximated those of wild type alpha(1E) channels. However, neither individual or combined substitution of the II S6 and III S6 segmen ts could account for the 60 mV more negative half-inactivation potential se en with wild type alpha(1E) channels, indicating that the S6 regions contri bute only partially to the voltage dependence of inactivation. Interestingl y, the converse replacement of alpha(1E) S6 segments of domains II, III, or II + III with those of cu,, was insufficient to significantly slow inactiv ation rates. Only when the I-II linker region and the domain II and III S6 regions of alpha(1E) were concomitantly replaced with alpha(1C) sequence co uld inactivation be abolished. Conversely, introduction of the cu,, domain I-II linker sequence into alpha(1C) conferred alpha(1E)-like inactivation r ates, indicating that the domain I-II linker is a key contributor to calciu m channel inactivation. Overall, our data are consistent with a mechanism i n which inactivation of voltage-dependent calcium channels may occur via do cking of the I-II linker region to a site comprising, at least in part, the domain II and III S6 segments.