Functional and structural characterization of PKA-mediated pHi gating of ROMK1 channels.
Hyperprostaglandin E syndrome/antenatal Bartter syndrome (HPS/aBS) is a severe salt-losing renal tubular disorder and results from the mutation of renal outer medullary K(+) (ROMK1) channels. The aberrant ROMK1 function induces alterations in intracellular pH (pH(i)) gating under physiological conditions. We investigate the role of protein kinase A (PKA) in the pH(i) gating of ROMK1 channels. Using giant patch clamp with Xenopus oocytes expressing wild-type and mutant ROMK1 channels, PKA-mediated phosphorylation decreased the sensitivity of ROMK1 channels to pH(i). A homology model of ROMK1 reveals that a PKA phosphorylation site (S219) is spatially juxtaposed to the phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding residues (R188, R217, and K218). Molecular dynamics simulations suggest a stable transition state, in which the shortening of distance between S219 and R217 and the movement of K218 towards the membrane after the PKA-phosphorylation can be observed. Such conformational change may bring the PIP(2) binding residues (K218) more accessible to the membrane-bound PIP(2). In addition, PIP(2) dose-dependently reactivates the acidification-induced rundown channels only when ROMK1 channels have been phosphorylated by PKA. This implies a sequence regulatory episode reflecting the role of PIP(2) in the pH(i) gating of ROMK1 channels by PKA-mediated phosphorylation. Our results provide new insights into the molecular mechanisms underlying the ROMK1 channel regulation associated with HPS/aBS.
Lee
Chien-Hsing
CH
Department of Pharmacology, College of Medicine, National Taiwan University, Taiwan.
Huang
Po-Tsang
PT
Lou
Kuo-Long
KL
Liou
Horng-Huei
HH
D000595:Amino Acid Sequence; D000818:Animals; D003198:Computer Simulation; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D005260:Female; D006863:Hydrogen-Ion Concentration; D042541:Intracellular Space / Q000378:metabolism*; D015640:Ion Channel Gating; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010766:Phosphorylation; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry* / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D014982:Xenopus laevis
D050505:Mutant Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D024661:Potassium Channels, Inwardly Rectifying; D017868:Cyclic AMP-Dependent Protein Kinases