Identification of Palmitoylation Sites within the L-type Calcium Channel β2a Subunit and Effects on Channel Function

The hydrophilic β2a 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., Puri, 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 β2a 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 β2a subunits that were unable to incorporate palmitic acid. Interestingly, a palmitoylation-deficient β2a mutant still localized to membrane particulate fractions and was still able to target functional channel complexes to the plasma membrane similar to wild-type β2a. However, channels formed with a palmitoylation-deficient β2a subunit exhibited a dramatic decrease in ionic current per channel, indicating that although mutations eliminating palmitoylation did not affect channel targeting by the β2a subunit, they were important determinants of channel modulation by the β2a 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. The hydrophilic β2a 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., Puri, 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 β2a 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 β2a subunits that were unable to incorporate palmitic acid. Interestingly, a palmitoylation-deficient β2a mutant still localized to membrane particulate fractions and was still able to target functional channel complexes to the plasma membrane similar to wild-type β2a. However, channels formed with a palmitoylation-deficient β2a subunit exhibited a dramatic decrease in ionic current per channel, indicating that although mutations eliminating palmitoylation did not affect channel targeting by the β2a subunit, they were important determinants of channel modulation by the β2a 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.