THE A-KINASE ANCHORING PROTEIN IS REQUIRED FOR MEDIATING THE EFFECT OF PROTEIN-KINASE-A ON ROMK1 CHANNELS

In the present study, we have used the two-electrode voltage-clamp and patch-clamp techniques to study the effects of forskolin and cAMP on the ROMK1 channels, which are believed to be the native K+ secretory c hannels in the kidney. Addition of 1 mu M forskolin or 100 mu M 8-brom o-cAMP, within 10 min, has no significant effect on the current of ROM K1 channels expressed in Xenopus oocytes, In contrast, application of 1 mu M forskolin, within 3 min, significantly increased whole-cell Kcurrent by 35%, when ROMK1 channels were coexpressed with the A kinase anchoring protein AKAP79, which was cloned from neuronal tissue. Two lines of evidence indicate that the effect of forskolin is mediated by a cAMP-dependent pathway: (i) Addition of 100 mu M 8-bromo-cAMP mimic s the effect of forskolin and (ii) the effect of forskolin and cAMP is not additive. That AKAP is required for the effect of cAMP is further supported by experiments in which addition of ATP (100 mu M) and cAMP (100 mu M) restored the activity of run-down ROMK1 channels in inside -out patches in oocytes that coexpressed ROMK1 and AKAP79 but not in t hose that expressed ROMK1 alone. Moreover, when we used RII, the regul atory subunit of type II protein kinase A, in an overlay assay, we ide ntified a RII-binding protein in membranes obtained from the kidney co rtex but not in membranes from oocytes. This suggests that the insensi tivity of ROMK1 channels to forskolin and cAMP is due to the absence o f AKAPs. We conclude that AKAP may be a critical component that mediat es the effect of protein kinase A on the ROMK channels in the kidney.