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 rapi dly inactivating R-type (alpha (1E)) calcium channels to slowly inactivatin g L-type (alpha (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. I n this region, alpha (1C) and alpha (1E) channels differ in seven residues, and exchanging five of those residues individually or in combination did n ot significantly affect inactivation kinetics. By contrast, replacement of residues Phe-823 or Ile-829 of alpha (1C) with the corresponding alpha (1E) : residues significantly accelerated inactivation rates and, when substitut ed concomitantly, approached the rapid inactivation kinetics of R-type chan nels. A systematic substitution of these residues with a series of other am ino acids revealed that decreasing side chain size at position 823 accelera tes 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 c hannel, they had little to no effect on the rate of recovery from inactivat ion. This suggests that the development of and recovery from inactivation a re governed by separate structural determinants. Finally, the effects of mu tations that accelerated alpha (1C) inactivation could still be antagonized following coexpression of the rat beta (2a). subunit or by domain I-II lin ker substitutions that produce ultra slow inactivation of wild type channel s, indicating that the inactivation kinetics seen with the mutants remain s ubject to regulation by the domain I-II linker. Overall, our results provid e novel insights into a complex process underlying calcium channel inactiva tion.