Identification of Inactivation Determinants in the Domain IIS6 Region of High Voltage-activated Calcium Channels*

We have recently reported that transfer of the domain IIS6 region from rapidly inactivating R-type (α_1E) calcium channels to slowly inactivating L-type (α_1C) calcium channel confers rapid inactivation (Stotz, S. C., Hamid, J., Spaetgens, R. L., Jarvis, S. E., and Zamponi, G. W. (2000) J. Biol. Chem. 275, 24575–24582). Here we have identified individual amino acid residues in the IIS6 regions that are responsible for these effects. In this region, α_1C and α_1Echannels differ in seven residues, and exchanging five of those residues individually or in combination did not significantly affect inactivation kinetics. By contrast, replacement of residues Phe-823 or Ile-829 of α_1C with the corresponding α_1E residues significantly accelerated inactivation rates and, when substituted concomitantly, approached the rapid inactivation kinetics of R-type channels. A systematic substitution of these residues with a series of other amino acids revealed that decreasing side chain size at position 823 accelerates inactivation, whereas a dependence of the inactivation kinetics on the degree of hydrophobicity could be observed at position 829. Although these point mutations facilitated rapid entry into the inactivated state of the channel, they had little to no effect on the rate of recovery from inactivation. This suggests that the development of and recovery from inactivation are governed by separate structural determinants. Finally, the effects of mutations that accelerated α_1C inactivation could still be antagonized following coexpression of the rat β_2a subunit or by domain I–II linker substitutions that produce ultra slow inactivation of wild type channels, indicating that the inactivation kinetics seen with the mutants remain subject to regulation by the domain I–II linker. Overall, our results provide novel insights into a complex process underlying calcium channel inactivation.