Biogenesis of the Sodium-Activated Potassium Channel Slack-B is Controlled by the Dephosphorylation of N-Terminal Serines

Abstract

Slack-B channels are expressed predominantly in brainstem regions and the olfactory bulb. Using liquid-chromatography tandem mass spectrometry, we determined that two Ser residues (S34 and S44) close to the N-terminus of Slack-B are basally phosphorylated. To test the biological role of these sites we performed site-directed mutagenesis, creating mutations that mimic either phosphorylation (S->E) or dephosphorylation (S->A) at each or both site. We found that correlated with a 20-fold increase in whole-cell currents, levels of the nonphosphorylated mutant channel are greatly increased in cRNA-injected Xenopus oocytes as compared to wild type (WT) Slack. Next, comparing the rate of protein accumulation in oocytes injected with equal amounts of WT or mutant-encoding cRNA, we confirmed that the initial rate of protein accumulation is significantly increased for the nonphosphorylated channel. Additionally, the time constant for protein accumulation was much faster for nonphosphorylated channel, becoming saturated within two days. In contrast, levels of the WT channel accumulated with linear kinetics. We observed similar changes in Slack-B channel levels in HEK293 cells transiently transfected with bicistronic vectors carrying DsRed (transfected cell indicator) and WT or mutant Slack-Bl. To rule out the possibility that changes in current result from effects of the mutations on electrophysiologic parameters, we performed both macroscopic and single channel evaluation of each of the mutants. We found no significant differences among the WT and mutant Slack-B channels. Taken together, the results indicate that dephosphorylation of these sites in nascent peptide chains is a required step for permitting the translation of Slack mRNA to go to completion during channel synthesis. Our findings also suggest that regulation of the phosphorylation state of S34 and S44 may allow neurons to alter channel abundance rapidly in response to stimulation.