Phosphorylation Regulates Gating and Channel-Protein Interaction in the Slack Kna Channel

Abstract

Na+-activated potassium (KNa) channels encoded by the Slack and Slick genes contribute to neuronal adaptation during sustained stimulation and regulate the accuracy of timing of action potentials. Activation of protein kinase C (PKC) increases the amplitude of Slack-B currents and slows their rate of activation. We have now identified a specific residue that is required for modulation by PKC and have found that phosphorylation alters the interaction of Slack channels with other components at the plasma membrane. Using Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS) on immunopurified Slack-B protein, in combination with site-directed mutagenesis, we have found that at least three specific residues in the Slack protein are phosphorylated in the basal state, and that only one site (S407) is absolutely required for PKC activation to alter the gating of Slack-B channels. The Slack protein is known to interact with a variety of cytoplasmic signaling molecules, including FMRP, the Fragile-X Mental Retardation Protein. We have now also found that phosphorylation may modify interactions of Slack with these cytoplasmic components, Using resonance wavelength grating optical biosensors (the Corning Epic system and the SRU Biosciences BIND system), we have determined that the pharmacological activation of Slack channels by bithionol produces a sustained decrease in mass distribution close to the plasma membrane. Changes in mass distribution are specific to Slack channel because activation of the very closely related Slick channel with bithionol does not alter mass distribution. Moreover, when Slack channels are treated with bithionol after pretreatment with an activator of PKC, the change in mass distribution is very significantly reduced. Our findings suggest that, in addition to regulating channel gating, phosphorylation of Slack modulates its protein-protein interactions with cytoplasmic components.