The A kinase anchoring protein is required for mediating the effect of protein kinase A on ROMK1 channels

Abstract

In the present study, we have used the twoelectrode 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 channels in the kidney. Addition of 1 M forskolin or 100 M 8-bromocAMP, within 10 min, has no significant effect on the current of ROMK1 channels expressed in Xenopus oocytes. In contrast, application of 1 M forskolin, within 3 min, significantly increased whole-cell K ؉ current 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 M 8-bromo-cAMP mimics 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 M) and cAMP (100 M) restored the activity of run-down ROMK1 channels in inside-out patches in oocytes that coexpressed ROMK1 and AKAP79 but not in those that expressed ROMK1 alone. Moreover, when we used RII, the regulatory subunit of type II protein kinase A, in an overlay assay, we identified a RIIbinding protein in membranes obtained from the kidney cortex but not in membranes from oocytes. This suggests that the insensitivity of ROMK1 channels to forskolin and cAMP is due to the absence of AKAPs. We conclude that AKAP may be a critical component that mediates the effect of protein kinase A on the ROMK channels in the kidney.We have previously demonstrated that stimulation of protein kinase A (PKA) activates the ROMK-like K ϩ secretory channel in the thick ascending limb (TAL) and cortical collecting duct (CCD) (1). The ROMK channel is an important member of the family of inwardly rectifying K ϩ channels with two transmembrane segments (2). Several lines of evidence indicate that the ROMK channels are closely related to the renal low-conductance ATP-sensitive K ϩ channels located in the TAL and in the CCD (1). (i) In situ hybridization has shown the presence of mRNA encoding ROMK channels in the CCD and the TAL. Immunocytochemical studies have further revealed the presence of ROMK channels in the apical membrane where renal ATP-sensitive K ϩ channels have been identified (3, 4). (ii) The conductance (30-40 pS) and kinetics of ROMK channels expressed in oocytes are identical to those of the native low-conductance ATP-sensitive K ϩ channels in the CCD and the TAL (5, 6). (iii) Both the low-conductance ATP-sensitive K ϩ channel and the ROMK channel are stimulated by PKA-induced phosphorylation (7).Recently, it has been suggested that the A kinase anchoring protein (AKAP) is required for mediating PKA-induced phosphorylation of cellular proteins (8 -10). AKAPs are involved in the localization of the type II isoform of PKA in a variety of cell membranes (9). More than 70% of the type II of PKA is bound to the cell membrane (11). Several studies have recently shown that A...

Organization of kinases, phosphatases, and receptor signaling complexes

“…These effects are inhibited by an anchoring inhibitor peptide that is patterned after the conserved AKAP sequence and uncouples PKA localization. Similar experiments have implicated a role for anchored pools of PKA in the modulation of smooth muscle calcium-activated potassium channels, kidney ROMK channels, and neuronal AMPA/kainate glutamate receptors (9,10).…”

Inhibition of MAPK stimulates the Ca 2+ -dependent big-conductance K channels in cortical collecting duct

“…Because of their high open probability and channel density, SK channels (ROMK) are thought to be largely responsible for K secretion in the connecting tubule and CCD under physiological conditions. SK channels are sensitive to cytosolic changes in pH (22,23) and regulated by protein kinase A (24,25), protein kinase C (26), and protein tyrosine kinase. The latter is involved in mediating the effect of K restriction on SK channels (27).…”

Cell Surface Expression of the ROMK (Kir 1.1) Channel Is Regulated by the Aldosterone-induced Kinase, SGK-1, and Protein Kinase A

“…Like the native secretory channel (10), ROMK activity is dependent on direct PKA phosphorylation (11,12), possibly facilitated by A-kinase-associated proteins (13). In fact, activation of ROMK by PKA is thought to underlie the regulation of renal potassium transport by vasopressin (14).…”