title: Regulation of ROMK1 K+ channel activity involves phosphorylation processes.
abstract: An inwardly rectifying, ATP-regulated K+ channel with a distinctive molecular architecture, ROMK1, was recently cloned from rat kidney. Using patch clamp techniques, we have investigated the regulation of ROMK1 with particular emphasis on phosphorylation/dephosphorylation processes. Spontaneous channel rundown occurred after excision of membrane patches into ATP-free bath solutions in the presence of Mg2+. Channel rundown was almost completely abolished after excision of patches into either Mg(2+)-free bathing solutions or after preincubation with the broad-spectrum phosphatase inhibitor, orthovanadate, in the presence of Mg2+. MgATP preincubation also inhibited channel rundown in a dose-dependent manner. In addition, the effect of the specific phosphatase inhibitors okadaic acid (1 microM) and calyculin A (1 microM) was also investigated. The presence of either okadaic acid or calyculin A failed to inhibit channel rundown. Taken together, these data suggest that rundown of ROMK1 involves a Mg(2+)-dependent dephosphorylation process. Channel activity was also partially restored after the addition of MgATP to the bath solution. Addition of exogenous cAMP-dependent protein kinase A (PKA) catalytic subunit led to a further increase in channel open probability. Addition of Na2ATP, in the absence of Mg2+, was ineffective, suggesting that restoration of channel activity is a Mg(2+)-dependent process. Addition of the specific PKA inhibitor, PKI, to the bath solution led to a partial, reversible inhibition in channel activity. Thus, PKA-dependent phosphorylation processes are involved in the modulation of channel activity. This observation is consistent with the presence of potential PKA phosphorylation sites on ROMK1.
authors.affiliation: Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06510.
authors.forename: C M
authors.initials: CM
authors.lastname: McNicholas
authors.forename: W
authors.initials: W
authors.lastname: Wang
authors.forename: K
authors.initials: K
authors.lastname: Ho
authors.forename: S C
authors.initials: SC
authors.lastname: Hebert
authors.forename: G
authors.initials: G
authors.lastname: Giebisch
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004988:Ethers, Cyclic / Q000494:pharmacology; D005260:Female; D015640:Ion Channel Gating; D007668:Kidney / Q000502:physiology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008387:Marine Toxins; D008564:Membrane Potentials / Q000187:drug effects; D019319:Okadaic Acid; D009865:Oocytes / Q000502:physiology; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D015221:Potassium Channels / Q000096:biosynthesis / Q000187:drug effects / Q000502:physiology*; D024661:Potassium Channels, Inwardly Rectifying; D051381:Rats; D013997:Time Factors; D014638:Vanadates / Q000494:pharmacology; D014981:Xenopus
chemical_list: D004988:Ethers, Cyclic; C510692:Kcnj1 protein, rat; D008387:Marine Toxins; D010080:Oxazoles; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D019319:Okadaic Acid; D014638:Vanadates; C059041:calyculin A; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D008274:Magnesium