PKA-induced stimulation of ROMK1 channel activity is governed by both tethering and non-tethering domains of an A Kinase Anchor Protein

We have used the patch-clamp technique to explore the role of A Kinase Anch or Proteins (AKAP) in mediating the effect of cAMP on ROMK1 channels expres sed in the Xenopus oocytes. Addition of membrane permeant cAMP analogs incr eased channel activity only in oocytes injected with ROMK1 and AKAP79 cRNA but had no effect on channel activity in oocytes injected with ROMK1 alone, Using the two-electrode voltage clamp technique, we determined that applic ation of H89, a potent inhibitor of protein kinase A (PKA), abolished the s timulatory effect of cAMP/forskolin. To investigate the role of AKAP specif icity in conferring cAMP responses to ROMK1 channels, we examined channel a ctivity in oocytes expressing ROMK1 and either AKAP18, AKAP-KL or AKAP75. A ddition of cAMP failed to increase channel current in oocytes expressing RO MK1 and either AKAP18 or AKAP-KL. In contrast, cAMP increased ROMK1 channel activity by 33%, in oocytes coexpressing AKAP75, the bovine homologue of A KAP79. The effect of cAMP on ROMK1 in oocytes coexpressing AKAP75 is inhibi ted by H89. Since all three AKAPs bind PKAII, the results suggest that a un ique structural domain in AKAP75/79 collaborates with the PKAII binding sit e and enables a productive association of Pt(A with ROMK1 channels. Deletio n of either the membrane targeting region of AKAP75 (AKAP45) or PKAII bindi ng domain of AKAP75 (AKAP75 DeltaC) abolished the effects of forskolin on R OMK1 channels. This suggests that the membrane targeting and the PKA bindin g domains of AKAP75 are essential for the effect of cAMP However, the natur e of the AKAP, that interacts with ROMK1 in the native tissue, remains to b e determined because AKAP75/79 are not expressed in the kidney. We conclude that the regulation of ROMK1 channels by PKA requires the involvement of t he cell membrane-directed AKAPs that are able to specifically link PKA to t he target channel protein. Copyright (C) 2001 S. Karger AG, Basel.