Functional and structural characterization of PKA-mediated pH i gating of ROMK1 channels

Abstract

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.