Identification of palmitoylation sites within the L-type calcium channel β(2a) subunit and effects on channel function

The hydrophilic β(2a) subunit of the L-type calcium channel was recently shown to be a membrane-localized, post-translationally modified protein (Chien, A. J., Zhao, X. L., Shirokov, R. E., Purl, T. S., Chang, C. F., Sun, D. D., Rios, E., and Hosey, M. M. (1995) J. Biol. Chem. 270, 30036- 30044). In this study, we demonstrate that the rat β(2a) subunit was palmitoylated through a hydroxylamine-sensitive thioester linkage. Palmitoylation required a pair of cysteines in the N terminus, Cys³ and Cys⁴; mutation of these residues to serines resulted in mutant β(2a) subunits that were unable to incorporate palmitic acid. Interestingly, a palmitoylation-deficient β(2a) mutant still localized to membrane particulate fractions and was still able to target functional channel complexes to the plasma membrane similar to wild-type β(2a). However, channels formed with a palmitoylation-deficient β(2a) subunit exhibited a dramatic decrease in ionic current per channel, indicating that although mutations eliminating palmitoylation did not affect channel targeting by the β(2a) subunit, they were important determinants of channel modulation by the β(2a) subunit. Three other known β subunits that were analyzed were not palmitoylated, suggesting that palmitoylation could provide a basis for the regulation of L-type channels through modification of a specific β isoform.