| 1994 |
ROMK1 channel activity undergoes rundown via a Mg2+-dependent dephosphorylation process; PKA catalytic subunit restores and increases channel open probability, demonstrating that PKA-dependent phosphorylation modulates ROMK1 activity. The effect requires MgATP (not Na2ATP alone), and PKA inhibitor PKI partially reverses channel activity. |
Patch clamp (excised inside-out patches), pharmacological inhibitors (orthovanadate, okadaic acid, calyculin A, PKI), exogenous PKA application |
Proceedings of the National Academy of Sciences of the United States of America |
High |
8058760
|
| 1999 |
PKA activates ROMK1 not by direct channel activation but by enhancing the channel's interaction with membrane PIP2: PKA phosphorylation lowers the PIP2 concentration required for channel activation, and mutation of PKA phosphorylation sites on ROMK1 reduces PIP2-channel interaction. |
Inside-out patch clamp, ATP[γS] as non-hydrolyzable PKA substrate, solution-binding assay with anti-PIP2 antibodies, site-directed mutagenesis of PKA phosphorylation sites |
Proceedings of the National Academy of Sciences of the United States of America |
High |
10318968
|
| 2003 |
PKC inhibits ROMK1 channels by reducing membrane PIP2 content, thereby reducing channel open probability. ROMK1 mutants with reduced PIP2 affinity are hypersensitive to PKC activation; exogenous PIP2 restores channel activity after PKC inhibition. |
Cell-attached and inside-out patch clamp, PKC activator (PMA), muscarinic receptor activation in oocytes, PIP2 measurement, site-directed mutagenesis |
The Journal of biological chemistry |
High |
12615924
|
| 2002 |
PKC phosphorylates ROMK1 at serine residues 4 and 201 (identified by in vitro phosphorylation of His-tagged protein and synthetic peptides); double mutation S4A/S201A nearly abolishes surface expression and K+ current, while phosphomimetic S4D/S201D restores both, demonstrating that PKC phosphorylation at these sites is essential for plasma membrane expression. |
In vitro phosphorylation assay with [32P]ATP, two-electrode voltage clamp in oocytes, confocal microscopy, biotin labeling of surface protein, site-directed mutagenesis |
The Journal of biological chemistry |
High |
12221079
|
| 2007 |
WNK1 and WNK4 stimulate clathrin-dependent endocytosis of ROMK1 by interacting with the scaffold protein intersectin (ITSN); this interaction requires specific proline-rich motifs of WNKs but not WNK kinase activity. Disease-causing WNK4 mutations enhance interactions with ITSN and ROMK1, increasing ROMK1 endocytosis. |
Co-immunoprecipitation, endocytosis assays, domain mutagenesis, kinase-dead constructs, interaction studies with proline-rich motif mutants |
The Journal of clinical investigation |
High |
17380208
|
| 2002 |
SGK1 and NHERF2 synergize to increase ROMK1 activity by enhancing the abundance of channel protein in the plasma membrane; neither SGK1 nor NHERF2 alone affects ROMK1, but co-expression of both markedly increases K+ current and reduces decay of channel activity after inhibition of vesicle insertion. |
Two-electrode voltage clamp in Xenopus oocytes, co-expression experiments, brefeldin A treatment, NHERF2/SGK1 co-expression |
Journal of the American Society of Nephrology : JASN |
Medium |
12444200
|
| 2003 |
SGK1 phosphorylates ROMK1 at serine 44; the PDZ-binding motif of ROMK1 and the second PDZ domain of NHERF2 are required for SGK1/NHERF2-mediated stimulation of ROMK1 plasma membrane abundance and K+ current. Phosphomimetic S44D shifts channel pH sensitivity to more acidic values. |
Pull-down assays (NHERF2-SGK1 interaction via PDZ domain), deletion mutagenesis of PDZ domains, two-electrode voltage clamp in oocytes, chemiluminescence surface expression assay |
Biochemical and biophysical research communications |
Medium |
14623317
|
| 2000 |
Protein tyrosine kinase (c-Src) and tyrosine phosphatase (PTP-1D) regulate ROMK1 plasma membrane abundance: inhibiting PTP increases tyrosine phosphorylation of ROMK1 and stimulates dynamin-dependent endocytosis, while inhibiting PTK increases channel surface expression via enhanced exocytosis. The key residue is tyrosine 337; mutation Y337A abolishes both effects. |
Two-electrode voltage clamp, patch clamp, Western blot, co-expression of c-Src in oocytes/HEK293 cells, pharmacological inhibitors (PAO, herbimycin A), mutagenesis (Y337A) |
The Journal of biological chemistry |
High |
11114300
|
| 2001 |
Inhibition of PTP by phenylarsine oxide (PAO) increases ROMK1 tyrosine phosphorylation, reduces surface ROMK1 by 65% (biotin labeling), and induces dynamin-dependent endocytosis; dominant negative dynamin (K44A) completely blocks PAO-induced internalization, establishing that tyrosine phosphorylation drives dynamin-dependent endocytosis of ROMK1. |
Confocal microscopy, biotin surface labeling, patch clamp, dominant negative dynamin co-expression, phosphorylation assay |
The Journal of biological chemistry |
High |
11719519
|
| 1998 |
AKAP79 (A kinase anchoring protein) is required for PKA-dependent regulation of ROMK1; co-expression of ROMK1 with AKAP79 renders channels responsive to forskolin/cAMP, an effect absent with ROMK1 alone. Endogenous RII-binding protein is present in kidney cortex membranes but not oocyte membranes, explaining the native requirement for AKAPs. |
Two-electrode voltage clamp, patch clamp, overlay assay with RII regulatory subunit, co-expression in Xenopus oocytes, pharmacological validation with H89 (PKA inhibitor) |
Proceedings of the National Academy of Sciences of the United States of America |
High |
9707637
|
| 1998 |
pH-dependent gating of Kir1.1 (ROMK1) involves conformational changes in both N terminus (Cys49) and C terminus (Cys308): both cysteines are accessible to water-soluble thiol reagents only in the closed (acidified) state, not in the open state, indicating movement of these protein domains during gating. |
Site-directed mutagenesis of all intracellular cysteines, state-dependent modification with water-soluble oxidants and sulfhydryl reagents, patch clamp |
The Journal of biological chemistry |
High |
9852128
|
| 1996 |
Kir1.1 channels are directly regulated by extracellular K+ concentration, and this regulation is coupled to intracellular pH. The core region (M1, M2, P) determines K+ regulation, while the N terminus determines pH coupling; swapping the N terminus with the pH-insensitive IRK1 decouples pH from K+ regulation. |
Chimeric channel construction, site-directed mutagenesis, two-electrode voltage clamp in Xenopus oocytes |
The Journal of biological chemistry |
High |
8663367
|
| 2005 |
Leucine 160 (Kir1.1b) at the cytoplasmic apex of the inner transmembrane helices constitutes the structural pH gate: replacing L160 with small (glycine) or polar (serine, threonine) residues abolishes pH gating, while alanine (intermediate size) retains gating. Homology modeling based on KirBac1.1 supports steric occlusion of the permeation path by four leucines as the closure mechanism. |
Site-directed mutagenesis, patch clamp electrophysiology, homology modeling based on KirBac1.1 crystal structure |
Biophysical journal |
High |
15653740
|
| 2006 |
Cytoplasmic acidification induces a conformational change that closes the Kir1.1 pH gate at the helix bundle crossing (near position 175 in TM2): introduction of cysteine at 175-Kir1.1a allows MTS reagent blockade in the open state but low pH protects from modification, while the cytoplasmic pore residue G223C is accessible in both open and closed states. |
Cysteine-scanning mutagenesis, MTS reagent modification in inside-out macropatches, Xenopus oocyte expression system |
Biophysical journal |
High |
16891366
|
| 2000 |
C-terminal histidine residues (His-225, His-274, His-342, His-354) in ROMK1 are critical for CO2 and pH sensing: mutation of any one reduces CO2 and pH sensitivity by 20–50%, and simultaneous mutation of all four eliminates CO2 sensitivity and markedly reduces pH sensitivity. |
Systematic site-directed mutagenesis of all histidine residues, two-electrode voltage clamp and patch clamp in Xenopus oocytes |
The Journal of biological chemistry |
High |
10713095
|
| 2005 |
ROMK1 is monoubiquitinated at Lys-22; monoubiquitination regulates channel activity by reducing surface plasma membrane expression. Mutation K22R abolishes ubiquitination, increases surface expression, and increases K+ current without altering biophysical properties. |
Immunoprecipitation from renal cortex tissue, mutagenesis of all intracellular lysines, two-electrode voltage clamp, confocal microscopy, biotin surface labeling, Western blot for ubiquitin |
Proceedings of the National Academy of Sciences of the United States of America |
High |
15767585
|
| 2009 |
POSH (a scaffold E3 ubiquitin ligase) binds ROMK1 at its N terminus (shown by GST pulldown and co-IP), stimulates ROMK1 ubiquitination (including in vitro ubiquitination assay), and enhances dynamin-dependent, clathrin-independent endocytosis of the channel, reducing its surface expression and K+ current. |
Immunoprecipitation, GST pulldown, electrophysiology (two-electrode voltage clamp, patch clamp), biotin surface labeling, in vitro ubiquitination assay, dominant negative dynamin, deletion of POSH RING domain |
The Journal of biological chemistry |
High |
19710010
|
| 2004 |
c-Src (Src family PTK) is co-expressed with ROMK in the thick ascending limb, cortical collecting duct (CCD), and outer medullary collecting duct; stimulation of PTK by PAO increases ROMK localization in intracellular compartments and decreases apical/subapical membrane staining in CCD (but not TAL), demonstrating segment-specific PTK regulation of ROMK trafficking. |
Immunofluorescence staining, confocal microscopy, biotinylation surface assay, PAO and herbimycin A treatment of isolated tubules |
American journal of physiology. Renal physiology |
Medium |
15075184
|
| 2002 |
Tetanus toxin abolishes the herbimycin A-induced increase in surface ROMK1, demonstrating that SNARE proteins are required for the PTK-inhibition-induced exocytosis of ROMK1. Tyrosine residue 337 is essential for the PTK-regulated trafficking, and dephosphorylation at Y337 is required for enhanced exocytosis. |
Confocal microscopy, biotin surface labeling, patch clamp in cell-attached configuration, tetanus toxin (SNARE inhibitor) treatment, mutagenesis (Y337A) |
American journal of physiology. Renal physiology |
Medium |
12556363
|
| 2005 |
WNK3 inhibits ROMK1 channel activity by reducing its plasmalemmal surface expression (without affecting conductance or open probability); this inhibition is independent of WNK3 catalytic activity and is mediated by WNK3's carboxyl terminus. A kinase-inactivating mutation or a disease-homologous missense mutation enhances inhibition >2.5-fold. |
Two-electrode voltage clamp in Xenopus oocytes, co-expression studies, kinase-dead and disease-mutant constructs, domain deletion analysis |
The Journal of physiology |
Medium |
16357011
|
| 2010 |
Angiotensin II inhibits ROMK1 channels via two mechanisms: (1) PKC-dependent stimulation of c-Src tyrosine kinase (phosphorylation of Tyr416 in c-Src), increasing tyrosine phosphorylation of ROMK1 (requiring Y337), and (2) synergizing WNK4-mediated inhibition. SGK1 reversal of WNK4 inhibition is attenuated by angiotensin II. |
Perforated whole-cell patch clamp, Western blot (tyrosine phosphorylation), pharmacological inhibitors (losartan, PKC inhibitor, PTK inhibitor), mutagenesis (R1Y337A), co-transfection studies in HEK293 cells |
Kidney international |
Medium |
20927043
|
| 2015 |
c-Src phosphorylates WNK4 at Tyr1092, Tyr1094, and Tyr1143 (identified by Western blot and mass spectrometry); c-Src and PTP-1D co-immunoprecipitate with WNK4 at these sites. WNK4 Y1092/1094F double mutant eliminates ROMK1 inhibition by WNK4, and c-Src prevents SGK1-induced phosphorylation of WNK4 at Ser1196, thus modulating the WNK4–SGK1–ROMK1 regulatory axis. |
Western blot, mass spectrometry for phosphorylation sites, co-immunoprecipitation, site-directed mutagenesis, electrophysiology |
Proceedings of the National Academy of Sciences of the United States of America |
High |
25805816
|
| 2006 |
CFTR is required for PKA-regulated ATP sensitivity of Kir1.1 (ROMK) channels in the TAL: ATP and glibenclamide sensitivity of the 30 pS K channel are absent in CFTR knockout and ΔF508 mice, and increasing PKA activity abrogates CFTR's effect. CFTR provides a PKA-regulated functional switch determining the distribution of open and ATP-inhibited K channels. |
Patch clamp electrophysiology in native TAL cells from CFTR knockout and ΔF508 mice, curcumin treatment, PKA manipulation |
The Journal of clinical investigation |
High |
16470247
|
| 2002 |
ENaC up-regulates Kir1.1 (ROMK1) surface expression and currents in a CFTR-dependent manner; this effect is lost without CFTR, is reduced by Liddle syndrome ENaC mutant, and does not occur with the closely related Kir4.1 channel, indicating a specific regulatory linkage between ENaC, CFTR, and ROMK1 surface expression. |
Two-electrode voltage clamp in Xenopus oocytes, co-expression of ENaC, CFTR, and ROMK1, surface expression assay |
The Journal of biological chemistry |
Medium |
11994290
|
| 2001 |
An amino acid triplet 'IRA' in the NH2 terminus of ROMK1 (and ROMK3) blocks SUR2B interaction, preventing glibenclamide sensitivity. In vitro co-translation and immunoprecipitation confirmed direct ROMK1–SUR2B protein interaction is required for glibenclamide sensitivity. ROMK2 (lacking this NH2 extension) forms glibenclamide-sensitive channels with SUR2B. |
In vitro co-translation and immunoprecipitation, NH2-terminal deletion and substitution mutagenesis, two-electrode voltage clamp in oocytes |
The Journal of biological chemistry |
High |
11567030
|
| 2002 |
SUR2B does not traffic to the plasma membrane when co-expressed with Kir1.1b (ROMK2), and is not required to confer glibenclamide sensitivity to Kir1.1b, which has intrinsic glibenclamide sensitivity. This contradicts earlier models requiring SUR2B for the renal ATP-regulated K channel. |
Co-expression in Xenopus oocytes, surface expression assay, electrophysiological glibenclamide sensitivity measurements |
The Journal of biological chemistry |
Medium |
11927600
|
| 2002 |
The ATP- and PIP2-binding site of Kir1.1 is localized to the first 39 amino acid residues of the COOH terminus (distal to TM2); three conserved arginines (R188, R203, R217) are required for TNP-ATP binding, and PIP2 competes with ATP for this site, providing a mechanism for PIP2 antagonism of ATP inhibition. |
MBP fusion protein binding assay with fluorescent TNP-ATP, truncation and point mutagenesis of COOH terminus, PIP2 competition assay |
The Journal of biological chemistry |
High |
12381730
|
| 1999 |
A Bartter's syndrome-causing C-terminal truncation mutation (331X) locks Kir1.1a channels in a closed state despite normal plasma membrane expression; the extreme COOH terminus (amino acids 332–351) is required for channel opening and subunit oligomerization efficiency. A single truncated subunit in a tetrameric concatemer confers a complete dominant negative effect. |
Co-expression of wild-type and truncated channels, GFP-fusion localization by confocal microscopy, incremental COOH-terminus reconstruction, tetrameric concatemer experiments, patch clamp |
The Journal of general physiology |
High |
10532965
|
| 1995 |
The second transmembrane segment (M2) of ROMK1 is a straight alpha-helix with three distinct structural environments: lipid-facing (very tolerant of substitution), protein-interior-facing (tolerant), and pore-facing (intolerant) positions, identified by systematic Trp/Ala substitution mutagenesis of 18 consecutive residues. |
Systematic site-directed mutagenesis (Trp and Ala substitutions at each of 18 positions in M2), functional expression and electrophysiology in Xenopus oocytes |
Proceedings of the National Academy of Sciences of the United States of America |
High |
8618841
|
| 1998 |
The NH2 terminus of ROMK1 (specifically serine-4 phosphorylation) is essential for arachidonic acid (AA)-mediated inhibition; ROMK2 and ROMK3 (lacking this NH2 region) are insensitive to AA, and the S4A mutant also loses AA sensitivity. The effect of AA does not involve membrane-bound PKC. |
Patch clamp in Xenopus oocytes, splice variant comparison (ROMK1/2/3), NH2 terminal deletion and point mutagenesis (R1ND37, R1S4A), exogenous PKC application, PKC inhibitors |
The American journal of physiology |
Medium |
9458837
|
| 2009 |
The immunoglobulin-like domain (IgLD) in the cytoplasmic region of Kir1.1 is essential for thermodynamic stability, trafficking, and gating: two Bartter syndrome mutations (A198T, Y314C) in the IgLD core impair biosynthesis and trafficking to the cell surface in mammalian cells; a fraction reaching the surface is electrically silent and fails to rectify. |
Homology modeling and thermodynamic calculation, mammalian cell expression, biochemical trafficking assay, patch clamp electrophysiology, compensatory mutagenesis |
Channels (Austin, Tex.) |
Medium |
19221509
|
| 2014 |
SPAK and OSR1 (kinases regulated by WNK kinases) down-regulate ROMK1 activity and membrane protein abundance in a kinase-activity-dependent manner; constitutively active forms down-regulate ROMK1, while catalytically inactive forms do not. |
Two-electrode voltage clamp and chemiluminescence of HA-tagged ROMK1 in Xenopus oocytes, constitutively active and kinase-dead constructs |
Kidney & blood pressure research |
Medium |
25322850
|
| 2008 |
WNK1 inhibits ROMK1 via its NH2-terminal proline-rich domain (N-PRD, aa 1–119), which is necessary and sufficient; the N-linker (aa 120–220) antagonizes N-PRD activity; the kinase domain reverses this antagonism through charge-charge interactions at conserved catalytic residues (K233, D368) independent of kinase activity; the autoinhibitory domain and first coiled-coil domain further modulate the effect. |
Domain deletion and mutagenesis constructs, two-electrode voltage clamp in Xenopus oocytes, kinase-dead and charge-reversal mutants |
American journal of physiology. Renal physiology |
Medium |
18550644
|
| 2000 |
Valine 66 (equivalent position to Lys-53 in Kir4.1) is a molecular determinant of ROMK1's high pH sensitivity; V66K mutation decreases pH sensitivity, while K53V in Kir4.1 markedly increases it toward Kir1.1 levels. C-terminal histidine residues (His-342, His-354 in Kir1.1) also contribute to the difference in pH sensitivity between Kir1.1 and Kir4.1. |
Site-directed mutagenesis, two-electrode voltage clamp in Xenopus oocytes, pH sensitivity measurements (pKa) |
American journal of physiology. Cell physiology |
Medium |
11029294
|
| 2006 |
Conserved glycines G148 and G157 in the inner transmembrane helix (TM2) of Kir1.1 facilitate pH gating by reducing the energetic barrier to channel closure: Ala substitutions shift pKa toward alkaline values (additive effect in double mutant), and G148P shifts pKa acidic; these glycines are not absolutely required for gating. The putative pH sensor K61 shifts pKa without abolishing gating, indicating it is not the exclusive sensor. |
Site-directed mutagenesis, two-electrode voltage clamp and inside-out patch clamp in Xenopus oocytes, pKa measurements |
Biophysical journal |
Medium |
16533837
|
| 2009 |
An intersubunit salt bridge (R128-E132) in the P-loop near the selectivity filter stabilizes the active state of Kir1.1; mutation of either residue causes inactivation that is not reversed by alkalization unless high external K is present. External tertiapin-Q (TPNQ) binding to the outer mouth also protects against inactivation, suggesting conformational changes near the selectivity filter underlie Kir1.1 inactivation. |
Site-directed mutagenesis, inside-out and cell-attached patch clamp in oocytes, external K manipulation, TPNQ application, Ba pretreatment |
Biophysical journal |
Medium |
19686653
|
| 2017 |
VU590 inhibits Kir1.1 by binding within the pore (voltage- and K+-dependent block); asparagine 171 (N171) is the only pore-lining residue required for high-affinity block, and substitution with negatively charged residues dramatically weakens block. This defines the pore polarity determinant of Kir1.1 inhibitor pharmacology. |
Site-directed mutagenesis, patch clamp electrophysiology, molecular modeling |
Molecular pharmacology |
Medium |
28619748
|
| 2011 |
High-K diet causes a large increase in apical ROMK surface expression and mature glycosylation in DCT2, CNT, and CD (but not DCT1), demonstrating that dietary K controls ROMK plasma membrane density by differential, segment-specific trafficking regulation. |
Immunofluorescence with new verified ROMK antibody, ROMK knockout mice as control, segmental markers, glycosylation state analysis |
American journal of physiology. Renal physiology |
Medium |
21454252
|
| 2016 |
ROMK1-specific knockout mice do not develop the Bartter phenotype (no reduction in NKCC2 activity, no altered NCC), indicating ROMK1 is not functionally linked to NKCC2 in the TAL. However, ROMK1 is required for high-K intake-stimulated K+ secretion in the collecting tubule: high-K diet increased apical SK channel number and ROMK intensity only in wild-type, not ROMK1-knockout mice (which developed hyperkalemia). |
Cre-LoxP conditional knockout, patch clamp (SK channel activity), immunofluorescence, Western blot for NKCC2 and NCC |
The Journal of biological chemistry |
High |
26728465
|
| 2017 |
NHERF1 knockdown in M-1 cortical collecting duct cells reduces ROMK1 surface expression (biotin labeling) and Ba2+-sensitive K+ current, demonstrating that NHERF1 is required for efficient plasma membrane targeting of ROMK1 in native collecting duct cells. |
siRNA knockdown of NHERF1, cell biotinylation assay, patch clamp electrophysiology in M-1 cells |
Biochemical and biophysical research communications |
Medium |
28533091
|
| 2010 |
Hypertension-resistance polymorphisms R193P, H251Y, and T313FS prevent ROMK channel surface expression (retained in ER), while P166S and R169H have normal surface expression but reduced PIP2 binding affinity, making channels susceptible to G protein-coupled receptor-stimulated PIP2 hydrolysis-induced inhibition. |
Two-microelectrode voltage clamp in Xenopus oocytes, surface antibody binding to external epitope-tagged channels, glycosylation state analysis, giant excised patch clamp PIP2 affinity measurements |
American journal of physiology. Renal physiology |
Medium |
20926634
|
| 2009 |
During pH-gating of Kir1.1, the conformation of the cytoplasmic pore changes: FRET efficiency between N-terminal ECFP and C-terminal EYFP increases (from 25% to 40%) when pH is decreased from 10 to 7.4 (channel closure), indicating that the N and C termini move apart from each other in the open state at physiological pH. |
FRET measurements using ECFP-Kir1.1-EYFP fusion proteins in Xenopus oocytes, pH manipulation |
Journal of biomedical science |
Low |
19272129
|
| 2014 |
Kir1.1 (ROMK) channels are required for gastric acid secretion: Kir1.1-deficient mice have absent secretagogue-stimulated gastric acid secretion despite normal parietal cell morphology and number; luminal K+ restores acid secretion. Kir1.1 co-localizes with H+/K+-ATPase β-subunit in gastric parietal cells, and TPNQ (Kir1.1 inhibitor) reduces proton secretion in wild-type mice. |
Kir1.1 knockout mice, acid secretion assays (whole stomach and perfused gastric glands), immunolocalization (co-localization with H+/K+-ATPase), pharmacological inhibition with TPNQ and XE991 |
Pflugers Archiv : European journal of physiology |
Medium |
25127675
|