PHOSPHORYLATION AND DEPHOSPHORYLATION MODULATE A CA2-ACTIVATED K+ CHANNEL IN RAT PEPTIDERGIC NERVE-TERMINALS()

1. Ca2+-activated K+ channels regulate the excitability of many nerve terminals. A Ca2+-activated K+ channel present in the membranes of rat posterior pituitary nerve terminals runs down following the formation of excised patches. This run-down process reflects enzymatic dephosph orylation. 2. Both Mg-ATP and the protein phosphatase inhibitor okadai c acid prevented run-down of channel activity in excised patches. The okadaic acid sensitivity suggests that run-down resulted from dephosph orylation by a type 1 protein phosphatase. 3. Guanosine 5'-O-(3-thiotr iphosphate) (GTPgammaS) accelerated run-down by accelerating okadaic a cid-sensitive dephosphorylation. GTPgammaS had no effect on the activi ty of the protein kinase in these patches. These results suggest a dir ect coupling between a G-protein and a protein phosphatase. 4. After r un-down, channel activity could be restored by Mg-ATP; restoration dep ended on ATP hydrolysis, but did not require Ca2+ or a second messenge r. Restoration of channel activity by ATP was blocked by staurosporine and 1-(5-isoquinolinylsulphonyl)-3-methylpiperizine, but not by more specific inhibitors of protein kinases. 5. Restoration of channel acti vity by phosphorylation was very sensitive to membrane potential; incr easing the voltage by as little as 1.0 mV could dramatically enhance r ecovery. 6. Ca2+ and voltage acted synergistically to enhance phosphor ylation; higher [Ca2+] permitted phosphorylation at more negative pote ntials. 7. During trains of high frequency stimulation under current c lamp, action potentials were influenced by both the protein phosphatas e and protein kinase, indicating that enzymatic modulation of channel gating occurs under physiological conditions. An important implication of these results is that voltage-dependent phosphorylation could play a role in use-dependent depression of secretion from nerve terminals.