ROLE OF THE NH2 TERMINUS OF THE CLONED RENAL K-MEDIATED INHIBITION( CHANNEL, ROMK1, IN ARACHIDONIC ACID)

We have previously demonstrated that the ROMK channel maintains the pr operty of arachidonic acid (AA) sensitivity observed originally in the native ATP-sensitive K+ channel of the rat cortical collecting duct ( 16). We used the patch-clamp technique to extend these studies to othe r NH2-terminal splice variants of the ROMK channel family, ROMK2 and R OMK3, expressed in Xenopus oocytes to determine the mechanism by which AA inhibits channel activity. Although the conductance, channel open probability, and open/closed times of the three homologs were determin ed to be similar, addition of 5-10 mu M AA caused only a moderate inhi bition of ROMK2 (15 +/- 8%) and ROMK3 (13 +/- 9%) activity, indicating that differences in the NH2 termini of ROMK channels strongly influen ce the AA action. We consequently examined the effect of AA on a ROMK1 variant, R1ND37, in which the NH2 terminal amino acids 2-37 were dele ted, and on a mutant ROMK1, R1S4A, in which the serine-4 residue was m utated to alanine. Like ROMK2 and ROMK3, AA had a diminished effect on these variants. Addition of 1 nM exogenous protein kinase C (PKC) inh ibited ROMK1 but not the mutant, R1S4A. However, the effect of AA is n ot a result of stimulation of a membrane bound PKC, since PKC inhibito rs, calphostin C and chelerythrine, failed to abolish the AA-induced i nhibition. In contrast, application of 5 mu M staurosporine, a nonspec ific protein kinase inhibitor at high concentration, abolished the eff ect of AA. We conclude that phosphorylation of serine-4 residue in the NH2 terminus plays a key role in determination of AA effect on ROMK c hannels.