Lu M , Hebert SC , Giebisch G .

R01 DK054998 NIDDK NIH HHS , R01 DK054999 NIDDK NIH HHS

	Figure 1. . Small-conductance K+ channel run-down in a representative excised patch. In the cell-attached patch, inward K+ current was recorded showing five active SK channels. Since the pipette solution contained 140 mM KCl, the channel shows inward current displaying typical SK channel kinetics with high open probability. When the patch was excised into a 5 mM K+ bath solution, the current amplitude immediately decreased due to loss of the membrane potential. Then pipette holding potential (−Vpipette) was elevated from 0 to −40 mV, current amplitude was increased. SK channels completely closed in 1.7 min in the Ringer's solution after excision. The downward deflection of the traces shows inward current and the line “C” indicates the zero current level (channel completely closed state).
	Figure 2. . Depletion of intracellular ATP concentration decreased SK channel activity. In a representative cell-attached patch, the recording showed seven open channels before addition of 20 μM antimycin A and 20 mM 2-deoxy-D-glucose to the bath solution. In 7.4 min after application of the metabolic inhibitors, the SK channel activity was reversibly inhibited. NPo decreased from 6.4 ± 0.6 to 1.9 ± 0.3. Pipette holding potential (−Vpipette) = 0 mV. C = channel-closed state.
	Figure 3. . Hydrolyzable ATP prevented SK channel run-down in inside-out patches. (A) In the absence of Mg-ATP, channel activity rapidly decreased in the Ca2+/Mg2+-free Ringer's solution. 0.5 mM Mg-ATP reactivated the run-down channel. (B) SK channel activity recorded from an inside-out patch shows run-down in the presence of ATPγS (0.5 mM)-containing, Ca2+-free bath solution. The SK channel activity could be reactivated by 0.5 mM Mg-ATP in a Ca2+-free Ringer solution. The time to run-down was similar to those shown in Figs. 1 and 3 A. C = channel-closed state. −Vpipette = −40 mV.
	Figure 4. . A representative inside-out patch recording showing that alkaline phosphatase (cAP, 20 U/ml) reversibly inhibited SK channel activity in the presence of 0.5 mM Mg-ATP. After removal of cAP, SK channel activity was rapidly restored. C = channel-closed state. −Vpipette = −40 mV.
	Figure 5. . PIP2 reactivated run-down SK channels in a representative inside-out patch. (A) After run-down in the Ca2+-free, ATP-free bath solution, SK channel activity was restored by application of 50 μM PIP2. (B) PIP2 antibodies (anti-PIP2, 20 μM) caused SK channel run-down in the presence of Mg-ATP. (C) After removal of PIP2 antibodies, SK channel activity was not restored by Mg-ATP. However, SK channel activity was observed following addition of reducing agent, 100 μM DL-dithiothreitol (DTT). C = channel-closed state. −Vpipette = −40 mV.
	Figure 6. . (A) The PI kinase inhibitor, LY294002 (20 μM), reversibly reduced SK channel activity in the presence of 0.5 mM Mg-ATP/Ca2+-free bath solution in an inside-out patch. (B) PIP2 (50 μM) reactivated SK channel activity inhibited by the PI kinase inhibitor, LY294002 (20 μM). Channel current was recorded in an inside-out patch bathed in 0.5 mM Mg-ATP/Ca2+-free solution. C = channel-closed state. −Vpipette = −40 mV.
	Figure 7. . Summary of SK channel activity inhibited by the PI kinase inhibitors, LY294002 (20 μM), quercetin (60 μM), and wortmannin (20 μM). Channel activity (NPo) was normalized to the control value. Ca2+-free bath solution contained 0.5 mM Mg-ATP. PIP2 concentrations was 50 μM. ** indicates P < 0.01 versus control.
	Figure 8. . (A) PKI reduced SK channel activity in the presence of 0.5 mM Mg-ATP by 36% in 10 min. Data were from inside-out patch configuration. * indicates P < 0.05 compared with control (without PKI). (B) A representative trace showing that alkaline phosphatase (cAP) + PKI reduced SK channel activity in the presence of 0.5 mM Mg-ATP. After cAP wash-out, ∼65% of channel activity was restored in the presence of PKI + 0.5 mM Mg-ATP. C = channel-closed state. −Vpipette = −40 mV.
	Figure 9. . PIP2 and PKA are required for restoration of SK channel activity after purinergic receptors mediated inhibition by extracellular Mg-ATP. Cell-attached apical membrane patches were obtained, extracellular Mg-ATP (0.2 mM) was added to the bath, and then patches were excised in the inside-out configuration. Representative SK channel traces are shown demonstrating inhibition of SK activity by extracellular Mg-ATP in the cell-attached configuration. In the inside-out configuration, no channel activity was observed in the absence of Mg-ATP or PIP2 (A). However, with addition of 0.5 mM Mg-ATP followed by 50 U of the catalytic subunit of PKA (B) SK channels were nearly completely reactivated. C = channel-closed state. −Vpipette = pipette-holding potential.
	Figure 10. . PIP2 reduces Mg-ATP sensitivity of SK channels. (A) Representative SK channel activity recording of the concentration dependence of Mg-ATP inhibition in the presence of 50 μM PIP2 in the bath solution. 1 mM Mg-ATP had little effect on channel activity in the presence of PIP2 (B, open circle), whereas this Mg-ATP concentration abolished channel activity in the absence of PIP2 (B, solid circle). C = channel-closed state. −Vpipette = −40 mV. (B) Mg-ATP concentration-dependence of SK channel activity in the presence of 50 μM PIP2 (open circles). EC50 = 1.8 mM; Curve was fitted to the equation: Y = 1/{1 + 10^[(logEC50 − X)*Hill]}. Hill = 1.2 ± 0.1.

Anderson, Phosphatidylinositol phosphate kinases, a multifaceted family of signaling enzymes. 1999, Pubmed

Anderson, Phosphatidylinositol phosphate kinases, a multifaceted family of signaling enzymes. 1999, Pubmed
Ashcroft, Properties and functions of ATP-sensitive K-channels. 1990, Pubmed
Ashcroft, The beta-cell K(ATP) channel. 2000, Pubmed
Baukrowitz, PIP2 and PIP as determinants for ATP inhibition of KATP channels. 1998, Pubmed , Xenbase
Boim, ROMK inwardly rectifying ATP-sensitive K+ channel. II. Cloning and distribution of alternative forms. 1995, Pubmed , Xenbase
Corvera, Phosphoinositides in membrane traffic. 1999, Pubmed
Eckstein, Nucleoside phosphorothioates. 1985, Pubmed
Enkvetchakul, The kinetic and physical basis of K(ATP) channel gating: toward a unified molecular understanding. 2000, Pubmed
Fan, Anionic phospholipids activate ATP-sensitive potassium channels. 1997, Pubmed
Frindt, Low-conductance K channels in apical membrane of rat cortical collecting tubule. 1989, Pubmed
Giebisch, Physiological roles of renal potassium channels. 1999, Pubmed
Hilgemann, Cytoplasmic ATP-dependent regulation of ion transporters and channels: mechanisms and messengers. 1997, Pubmed
Hilgemann, The complex and intriguing lives of PIP2 with ion channels and transporters. 2001, Pubmed
Ho, Cloning and expression of an inwardly rectifying ATP-regulated potassium channel. 1993, Pubmed , Xenbase
Huang, Direct activation of inward rectifier potassium channels by PIP2 and its stabilization by Gbetagamma. 1998, Pubmed , Xenbase
Jeong, Kinase assay based on thiophosphorylation and biotinylation. 1999, Pubmed
Kobrinsky, Receptor-mediated hydrolysis of plasma membrane messenger PIP2 leads to K+-current desensitization. 2000, Pubmed , Xenbase
Kohda, Localization of the ROMK potassium channel to the apical membrane of distal nephron in rat kidney. 1998, Pubmed
Kubokawa, Role of Ca2+/CaMK II in Ca(2+)-induced K+ channel inhibition in rat CCD principal cell. 1995, Pubmed
Kubokawa, Regulation of ATP-sensitive K+ channel by membrane-bound protein phosphatases in rat principal tubule cell. 1995, Pubmed
Lee, ROMK inwardly rectifying ATP-sensitive K+ channel. I. Expression in rat distal nephron segments. 1995, Pubmed
Leevers, Signalling through phosphoinositide 3-kinases: the lipids take centre stage. 1999, Pubmed
Leung, Phosphatidylinositol 4,5-bisphosphate and intracellular pH regulate the ROMK1 potassium channel via separate but interrelated mechanisms. 2000, Pubmed , Xenbase
Liou, Regulation of ROMK1 channel by protein kinase A via a phosphatidylinositol 4,5-bisphosphate-dependent mechanism. 1999, Pubmed , Xenbase
Loussouarn, Dynamic sensitivity of ATP-sensitive K(+) channels to ATP. 2001, Pubmed
Lu, Extracellular ATP inhibits the small-conductance K channel on the apical membrane of the cortical collecting duct from mouse kidney. 2000, Pubmed
MacGregor, Partially active channels produced by PKA site mutation of the cloned renal K+ channel, ROMK2 (kir1.2). 1998, Pubmed , Xenbase
MacGregor, Nucleotides and phospholipids compete for binding to the C terminus of KATP channels. 2002, Pubmed
Mandel, ATP depletion: a novel method to study junctional properties in epithelial tissues. II. Internalization of Na+,K(+)-ATPase and E-cadherin. 1994, Pubmed
Martin, Phosphoinositide lipids as signaling molecules: common themes for signal transduction, cytoskeletal regulation, and membrane trafficking. 1998, Pubmed
Marx, Requirement of a macromolecular signaling complex for beta adrenergic receptor modulation of the KCNQ1-KCNE1 potassium channel. 2002, Pubmed
McNicholas, Molecular site for nucleotide binding on an ATP-sensitive renal K+ channel (ROMK2). 1996, Pubmed , Xenbase
Palmer, Is the secretory K channel in the rat CCT ROMK? 1997, Pubmed , Xenbase
Palmer, Potassium secretion and the regulation of distal nephron K channels. 1999, Pubmed
Ribalet, Regulation of cloned ATP-sensitive K channels by phosphorylation, MgADP, and phosphatidylinositol bisphosphate (PIP(2)): a study of channel rundown and reactivation. 2000, Pubmed
Ruknudin, Novel subunit composition of a renal epithelial KATP channel. 1998, Pubmed , Xenbase
Schulte, Gating of inward-rectifier K+ channels by intracellular pH. 2000, Pubmed , Xenbase
Schulte, pH-dependent gating of ROMK (Kir1.1) channels involves conformational changes in both N and C termini. 1998, Pubmed , Xenbase
Shyng, Membrane phospholipid control of nucleotide sensitivity of KATP channels. 1998, Pubmed
Shyng, Structural determinants of PIP(2) regulation of inward rectifier K(ATP) channels. 2000, Pubmed
Shyng, Modulation of nucleotide sensitivity of ATP-sensitive potassium channels by phosphatidylinositol-4-phosphate 5-kinase. 2000, Pubmed
Tanabe, Direct photoaffinity labeling of the Kir6.2 subunit of the ATP-sensitive K+ channel by 8-azido-ATP. 1999, Pubmed , Xenbase
Tanabe, Direct photoaffinity labeling of Kir6.2 by [gamma-(32)P]ATP-[gamma]4-azidoanilide. 2000, Pubmed , Xenbase
Wang, Regulation of small-conductance K+ channel in apical membrane of rat cortical collecting tubule. 1990, Pubmed
Wang, Mechanism of apical K+ channel modulation in principal renal tubule cells. Effect of inhibition of basolateral Na(+)-K(+)-ATPase. 1993, Pubmed
Wang, Renal potassium channels and their regulation. 1992, Pubmed
Wang, Renal K+ channels: structure and function. 1997, Pubmed
Wang, Dual modulation of renal ATP-sensitive K+ channel by protein kinases A and C. 1991, Pubmed
Wang, Dual effect of adenosine triphosphate on the apical small conductance K+ channel of the rat cortical collecting duct. 1991, Pubmed
Xie, Wortmannin, an inhibitor of phosphatidylinositol kinases, blocks the MgATP-dependent recovery of Kir6.2/SUR2A channels. 1999, Pubmed
Xie, Phospholipase C-linked receptors regulate the ATP-sensitive potassium channel by means of phosphatidylinositol 4,5-bisphosphate metabolism. 1999, Pubmed
Yokoshiki, ATP-sensitive K+ channels in pancreatic, cardiac, and vascular smooth muscle cells. 1998, Pubmed
Zhang, Activation of inwardly rectifying K+ channels by distinct PtdIns(4,5)P2 interactions. 1999, Pubmed , Xenbase
Zhou, Primary structure and functional properties of an epithelial K channel. 1994, Pubmed , Xenbase