Functional and structural characterization of PKA-mediated pHi gating of ROMK1 channels

Lee, Chien-Hsing; Huang, Po-Tsang; Lou, Kuo-Long; Liou, Horng-Huei

doi:10.1016/j.jmgm.2008.06.001

Cited by 5 publications

(17 citation statements)

References 48 publications

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“…Homology modeling of ROMK1 Homology modeling was performed following previously described procedures [9]. Briefly, residues of the ROMK1 channel chosen according to the results of GCG paired sequence alignment were superimposed onto the structure coordinates of the Cα atoms of the corresponding structural conserved regions (SCRs) from the template channel structure (PDB ID: 3JYC).…”

Section: Methodsmentioning

confidence: 99%

“…MD simulations were performed using InsightII 2005 and the Chemistry at Harvard Molecular Mechanics (CHARMM) special forcefield c27b4 as previously described [9]. The system is in an appropriate environment with the TIP3P water model.…”

Section: Molecular Dynamics Simulationsmentioning

confidence: 99%

“…During this equilibration process, the water molecules and ions were free to move. All simulations were performed following previously described procedures [9]. The MD simulations were performed only in implicit membranes not real biomembranes.…”

Section: Molecular Dynamics Simulationsmentioning

confidence: 99%

“…ROMK1 channel activity is not only regulated by PKCmediated phosphorylation, but is also highly sensitive to changes in cytosolic pH (pH i ) [8][9][10][11][12]. Intracellular acidification reversibly reduces the open probability (P o ) of native K + channels in CCDs as well as that of ROMK1 channels expressed in oocytes, with an effective pK a value of ∼6.8 [8][9][10][11][12]. This pH i sensitivity provides an important mechanism for linking renal K + excretion with acid-base balance [13].…”

Section: Introductionmentioning

confidence: 99%

“…This pH i sensitivity provides an important mechanism for linking renal K + excretion with acid-base balance [13]. We have previously shown that the molecular mechanisms underlying PKA-mediated phosphorylation in regulating ROMK1 channels and pH i gating defects are associated with hyperprostaglandin E syndrome/antenatal Bartter syndrome [9]. PKC has been reported to antagonize PKA activation of ROMK1 channels in CCDs [2,14], but the molecular mechanisms for the regulation of pH i in ROMK1 mediated by PKC remain poorly understood.…”

Section: Introductionmentioning

confidence: 99%

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Protein kinase C mediated pH i -regulation of ROMK1 channels via a phosphatidylinositol-4,5-bisphosphate-dependent mechanism

et al. 2011

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The protein kinase C (PKC) pathway is important for the regulation of K(+) transport. The renal outer medullar K(+) (ROMK1) channels show an exquisite sensitivity to intracellular protons (pH(i)) (effective pK(a) approximately 6.8) and play a key role in K(+) homeostasis during metabolic acidosis. Our molecular dynamic simulation results suggest that PKC-mediated phosphorylation on Thr-193 may disrupt the PIP(2)-channel interaction via a charge-charge interaction between Thr-193 and Arg-188. Therefore, we investigated the role of PKC and pH(i) in regulation of ROMK1 channel activity using a giant patch clamp with Xenopus oocytes expressing wild-type and mutant ROMK1 channels. ROMK1 channels pre-incubated with the PKC activator phorbol-12-myristate-13-acetate exhibited increased sensitivity to pH(i) (effective pK(a) shifted to pH approximately 7.0). In the presence of GF109203X--a PKC selective inhibitor--the effective pK(a) for inhibition of ROMK1 channels by pH(i) decreased (effective pK(a) shifted to pH approximately 6.5). The pH(i) sensitivity of ROMK1 channels mediated by PKC appeared to be dependent of PIP(2) depletion. The giant patch clamp together with site direct mutagenesis revealed that Thr-193 is the phosphorylation site on PKC that regulates the pH(i) sensitivity of ROMK1 channels. Mutation of PKC-induced phosphorylation sites (T193A) decreases the pH(i) sensitivity and increases the interaction of channel-PIP(2). Taken together, these results provide new insights into the molecular mechanisms underlying the pH(i) gating of ROMK1 channel regulation by PKC.

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Section: Methodsmentioning

confidence: 99%

Section: Molecular Dynamics Simulationsmentioning

confidence: 99%

Section: Molecular Dynamics Simulationsmentioning

confidence: 99%