Identification of palmitoylation sites within the L-type calcium channel beta2a subunit and effects on channel function.
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.
Chien
A J
AJ
Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois 60611, USA.
Carr
K M
KM
Shirokov
R E
RE
Rios
E
E
Hosey
M M
MM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015220:Calcium Channels / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D008969:Molecular Sequence Data; D019308:Palmitic Acid / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D015220:Calcium Channels; D011994:Recombinant Proteins; D019308:Palmitic Acid