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

The hydrophilic beta(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 beta(2a) subunit was palmitoylated through a hydroxylamine sensitive thioester linkage, Palmitoylation required a pair of cysteines in the N terminus, Cys(3) and Cys(4); mutation of these residues to serines resulted in mutant beta(2a) subunits that were unable to incorporate palmitic acid, Interestingly, a palmitoylation-deficient beta(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 beta(2a). However, channels formed with a palmitoylation-deficient beta(2a) subunit exhibited a dramatic decrease in ionic current per channel, indicating that although mutations eliminating palmitoylation did not affect channel targeting by the beta(2a) subunit, they were important determinants of channel modulation by the beta(2a) subunit, Three other known beta 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 beta isoform.