REGULATION OF THE EEL ELECTROPLAX NA CHANNEL AND PHOSPHORYLATION OF RESIDUES ON AMINO-TERMINAL AND CARBOXYL-TERMINAL DOMAINS BY CAMP-DEPENDENT PROTEIN-KINASE

Previous studies have shown that the short-motif electroplax Na channe l is phosphorylated in vitro by cyclic AMP-dependent protein kinase (P KA) at serines 6 or 7 and 17 76 and threonine 17 (Emerick & Agnew, 198 9). We here show that phosphatase treatment of solubilized, purified N a channels enhanced subsequent PKA labeling of four of five tryptic ph osphopeptides, indicating that these sites are phosphorylated in vivo. Microsequencing and analysis of PTH-amino acid products revealed endo genous labeling of serines 6, 444, 1680, and 1776. Serines 1680 and 17 76 lie in the carboxyl-terminal cytoplasmic domain, while serine 6 lie s in the amino terminus and serine 444 is in the cytoplasmic loop betw een domains I and II. Endogenous phosphorylation of serine 6 establish es experimentally that the Na channel amino terminus is cytoplasmic. I n electrophysiological experiments, brief exposure of inside-out membr ane patches excised from Sachs-organ cells to MgATP and purified PKA c atalytic subunit produced rapid, sustained reduction of Na current amp litude by approximately 80% and a hyperpolarizing shift in the conduct ance/voltage relation by 10-12 mV. The effect was absent in controls o mitting either PKA or MgATP. Serines 6 and 1776 and threonine 17 are l abeled rapidly and extensively in vitro, and only threonine 17 appears to be unphosphorylated in vivo. We suggest that phosphorylation of th e amino and carboxyl domains, perhaps especially at threonine 17, unde rlies the demonstrated downregulation of the electroplax Na channel.