Abstract
The hydrophilic beta2a 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 beta2a 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 beta2a subunits that were unable to incorporate palmitic acid. Interestingly, a palmitoylation-deficient beta2a mutant still localized to membrane particulate fractions and was still able to target functional channel complexes to the plasma membrane similar to wild-type beta2a. However, channels formed with a palmitoylation-deficient beta2a subunit exhibited a dramatic decrease in ionic current per channel, indicating that although mutations eliminating palmitoylation did not affect channel targeting by the beta2a subunit, they were important determinants of channel modulation by the beta2a 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.
Highlights
The L-type calcium channel 2a subunit is a highly hydrophilic protein with no predicted membrane-spanning regions [1]
We demonstrate that one modification of the 2a subunit involves palmitoylation, a post-translational modification that has been shown to facilitate the membrane localization of other hydrophilic proteins
Palmitoylation involves the addition of palmitic acid to cysteine residues through a thioester linkage [3, 4]
Summary
The L-type calcium channel 2a subunit is a highly hydrophilic protein with no predicted membrane-spanning regions [1]. The residues involved in palmitoylation of the 2a subunit were identified, and the functional roles of these amino acids were investigated in biochemical and electrophysiological studies using mutant proteins. In order to investigate the possibility that  subunits may be palmitoylated, transiently transfected tsA201 cells expressing different  isoforms were metabolically labeled with [3H]palmitic acid, immunoprecipitated with the GEN antiserum, and subsequently analyzed for acylation of  proteins.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
