KATP channel inhibition by ATP requires distinct functional domains of the cytoplasmic C terminus of the pore-forming subunit

Changes in phosphorylation regulate the activity of various ClC anion transport proteins. However, the physiological context under which such regulation occurs and the signaling cascades that mediate phosphorylation are poorly understood. We have exploited the genetic model organism Caenorhabditis elegans to characterize ClC regulatory mechanisms and signaling networks. CLH-3b is a ClC anion channel that is expressed in the worm oocyte and excretory cell. Channel activation occurs in response to oocyte meiotic maturation and swelling via serine/threonine dephosphorylation mediated by the type I phosphatases GLC-7α and GLC-7β. A Ste20 kinase, germinal center kinase (GCK)-3, binds to the cytoplasmic C terminus of CLH-3b and inhibits channel activity in a phosphorylation-dependent manner. Analysis of hyperpolarization-induced activation kinetics suggests that phosphorylation may inhibit the ClC fast gating mechanism. GCK-3 is an ortholog of mammalian SPAK and OSR1, kinases that bind to, phosphorylate, and regulate the cell volume–dependent activity of mammalian cation-Cl− cotransporters. Using mass spectrometry and patch clamp electrophysiology, we demonstrate here that CLH-3b is a target of regulatory phosphorylation. Concomitant phosphorylation of S742 and S747, which are located 70 and 75 amino acids downstream from the GCK-3 binding site, are required for kinase-mediated channel inhibition. In contrast, swelling-induced channel activation occurs with dephosphorylation of S747 alone. Replacement of both S742 and S747 with glutamate gives rise to kinase- and swelling-insensitive channels that exhibit activity and biophysical properties similar to those of wild-type CLH-3b inhibited by GCK-3. Our studies provide novel insights into ClC regulation and mechanisms of cell volume signaling, and provide the foundation for studies aimed at defining how conformational changes in the cytoplasmic C terminus alter ClC gating and function in response to intracellular signaling events.

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The effect of ATP-sensitive potassium (KATP) channel inhibition on metabolic vasodilation in human skeletal muscle vasculature

Heart Lung and Circulation ◽

10.1046/j.1443-9506.2000.06814.x ◽

2000 ◽

Vol 9 (3) ◽

pp. A110

Author(s):

H.M.O. Farouque ◽

R.A.P. Skyrme-Jones ◽

M.J. Zhang ◽

R.C. O'Brien ◽

I.T. Meredith

Keyword(s):

Skeletal Muscle ◽

Katp Channel ◽

Human Skeletal Muscle ◽

Channel Inhibition ◽

Metabolic Vasodilation

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Concerted Gating Mechanism Underlying KATP Channel Inhibition by ATP

Biophysical Journal ◽

10.1016/s0006-3495(04)74269-1 ◽

2004 ◽

Vol 86 (4) ◽

pp. 2101-2112 ◽

Cited By ~ 25

Author(s):

Peter Drain ◽

Xuehui Geng ◽

Lehong Li

Keyword(s):

Katp Channel ◽

Gating Mechanism ◽

Channel Inhibition

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Selective cardiac plasma-membrane KATP channel inhibition is defibrillatory and improves survival during acute myocardial ischemia and reperfusion

European Journal of Pharmacology ◽

10.1016/j.ejphar.2007.08.016 ◽

2007 ◽

Vol 577 (1-3) ◽

pp. 115-123 ◽

Cited By ~ 33

Author(s):

Szilvia Vajda ◽

István Baczkó ◽

István Leprán

Keyword(s):

Plasma Membrane ◽

Myocardial Ischemia ◽

Katp Channel ◽

Acute Myocardial Ischemia ◽

Ischemia And Reperfusion ◽

Myocardial Ischemia And Reperfusion ◽

Channel Inhibition

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Mechanism of ATP-sensitive K Channel Inhibition by Sulfhydryl Modification

The Journal of General Physiology ◽

10.1085/jgp.112.3.325 ◽

1998 ◽

Vol 112 (3) ◽

pp. 325-332 ◽

Cited By ~ 26

Author(s):

Stefan Trapp ◽

Stephen J. Tucker ◽

Frances M. Ashcroft

Keyword(s):

Katp Channel ◽

Membrane Surface ◽

Katp Channels ◽

Cysteine Residue ◽

K Channel ◽

Atp Binding Site ◽

Channel Inhibition ◽

Cysteine Scanning ◽

Sulfhydryl Modification ◽

Cysteine Scanning Mutagenesis

ATP-sensitive potassium (KATP) channels are reversibly inhibited by intracellular ATP. Agents that interact with sulfhydryl moieties produce an irreversible inhibition of KATP channel activity when applied to the intracellular membrane surface. ATP appears to protect against this effect, suggesting that the cysteine residue with which thiol reagents interact may either lie within the ATP-binding site or be inaccessible when the channel is closed. We have examined the interaction of the membrane-impermeant thiol-reactive agent p-chloromercuriphenylsulphonate (pCMPS) with the cloned β cell KATP channel. This channel comprises the pore-forming Kir6.2 and regulatory SUR1 subunits. We show that the cysteine residue involved in channel inhibition by pCMPS resides on the Kir6.2 subunit and is located at position 42, which lies within the NH2 terminus of the protein. Although ATP protects against the effects of pCMPS, the ATP sensitivity of the KATP channel was unchanged by mutation of C42 to either valine (V) or alanine (A), suggesting that ATP does not interact directly with this residue. These results are consistent with the idea that C42 is inaccessible to the intracellular solution, and thereby protected from interaction with pCMPS when the channel is closed by ATP. We also observed that the C42A mutation does not affect the ability of SUR1 to endow Kir6.2 with diazoxide sensitivity, and reduces, but does not prevent, the effects of MgADP and tolbutamide, which are mediated via SUR1. The Kir6.2-C42A (or V) mutant channel may provide a suitable background for cysteine-scanning mutagenesis studies.

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Differential Cav2.1 and Cav2.3 channel inhibition by baclofen and α-conotoxin Vc1.1 via GABAB receptor activation

The Journal of General Physiology ◽

10.1085/jgp.201311104 ◽

2014 ◽

Vol 143 (4) ◽

pp. 465-479 ◽

Cited By ~ 33

Author(s):

Géza Berecki ◽

Jeffrey R. McArthur ◽

Hartmut Cuny ◽

Richard J. Clark ◽

David J. Adams

Keyword(s):

Gabab Receptor ◽

Point Mutations ◽

Receptor Activation ◽

Gabab Receptors ◽

Mutant Form ◽

Phosphorylation Sites ◽

Human Embryonic Kidney Cells ◽

Channel Inhibition ◽

C Terminus ◽

Voltage Dependent

Neuronal Cav2.1 (P/Q-type), Cav2.2 (N-type), and Cav2.3 (R-type) calcium channels contribute to synaptic transmission and are modulated through G protein–coupled receptor pathways. The analgesic α-conotoxin Vc1.1 acts through γ-aminobutyric acid type B (GABAB) receptors (GABABRs) to inhibit Cav2.2 channels. We investigated GABABR-mediated modulation by Vc1.1, a cyclized form of Vc1.1 (c-Vc1.1), and the GABABR agonist baclofen of human Cav2.1 or Cav2.3 channels heterologously expressed in human embryonic kidney cells. 50 µM baclofen inhibited Cav2.1 and Cav2.3 channel Ba2+ currents by ∼40%, whereas c-Vc1.1 did not affect Cav2.1 but potently inhibited Cav2.3, with a half-maximal inhibitory concentration of ∼300 pM. Depolarizing paired pulses revealed that ∼75% of the baclofen inhibition of Cav2.1 was voltage dependent and could be relieved by strong depolarization. In contrast, baclofen or Vc1.1 inhibition of Cav2.3 channels was solely mediated through voltage-independent pathways that could be disrupted by pertussis toxin, guanosine 5′-[β-thio]diphosphate trilithium salt, or the GABABR antagonist CGP55845. Overexpression of the kinase c-Src significantly increased inhibition of Cav2.3 by c-Vc1.1. Conversely, coexpression of a catalytically inactive double mutant form of c-Src or pretreatment with a phosphorylated pp60c-Src peptide abolished the effect of c-Vc1.1. Site-directed mutational analyses of Cav2.3 demonstrated that tyrosines 1761 and 1765 within exon 37 are critical for inhibition of Cav2.3 by c-Vc1.1 and are involved in baclofen inhibition of these channels. Remarkably, point mutations introducing specific c-Src phosphorylation sites into human Cav2.1 channels conferred c-Vc1.1 sensitivity. Our findings show that Vc1.1 inhibition of Cav2.3, which defines Cav2.3 channels as potential targets for analgesic α-conotoxins, is caused by specific c-Src phosphorylation sites in the C terminus.

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Fip1 Regulates the Activity of Poly(A) Polymerase through Multiple Interactions

Molecular and Cellular Biology ◽

10.1128/mcb.21.6.2026-2037.2001 ◽

2001 ◽

Vol 21 (6) ◽

pp. 2026-2037 ◽

Cited By ~ 35

Author(s):

Steffen Helmling ◽

Alexander Zhelkovsky ◽

Claire L. Moore

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Rna Binding ◽

Physiological Function ◽

Regulatory Function ◽

Functional Domains ◽

Essential Component ◽

C Terminus ◽

Mrna Precursor ◽

Multiple Interactions

ABSTRACT Fip1 is an essential component of the Saccharomyces cerevisiae polyadenylation machinery and the only protein known to interact directly with poly(A) polymerase (Pap1). Its association with Pap1 inhibits the extension of an oligo(A) primer by limiting access of the RNA substrate to the C-terminal RNA binding domain (C-RBD) of Pap1. We present here the identification of separate functional domains of Fip1. Amino acids 80 to 105 are required for binding to Pap1 and for the inhibition of Pap1 activity. This region is also essential for viability, suggesting that Fip1-mediated repression of Pap1 has a crucial physiological function. Amino acids 206 to 220 of Fip1 are needed for the interaction with the Yth1 subunit of the complex and for specific polyadenylation of the cleaved mRNA precursor. A third domain within amino acids 105 to 206 helps to limit RNA binding at the C-RBD of Pap1. Our data demonstrate that the C terminus of Fip1 is required to relieve the Fip1-mediated repression of Pap1 in specific polyadenylation. In the absence of this domain, Pap1 remains in an inhibited state. These findings show that Fip1 has a crucial regulatory function in the polyadenylation reaction by controlling the activity of poly(A) tail synthesis through multiple interactions within the polyadenylation complex.

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Sulfonylureas suppress the stimulatory action of Mg-nucleotides on Kir6.2/SUR1 but not Kir6.2/SUR2A KATP channels: A mechanistic study

The Journal of General Physiology ◽

10.1085/jgp.201411222 ◽

2014 ◽

Vol 144 (5) ◽

pp. 469-486 ◽

Cited By ~ 19

Author(s):

Peter Proks ◽

Heidi de Wet ◽

Frances M. Ashcroft

Keyword(s):

Katp Channel ◽

Neonatal Diabetes ◽

Katp Channels ◽

Nucleotide Binding ◽

Mechanistic Study ◽

Xenopus Laevis Oocytes ◽

Sulfonylurea Receptor ◽

Β Cell ◽

Stimulatory Action ◽

Channel Inhibition

Sulfonylureas, which stimulate insulin secretion from pancreatic β-cells, are widely used to treat both type 2 diabetes and neonatal diabetes. These drugs mediate their effects by binding to the sulfonylurea receptor subunit (SUR) of the ATP-sensitive K+ (KATP) channel and inducing channel closure. The mechanism of channel inhibition is unusually complex. First, sulfonylureas act as partial antagonists of channel activity, and second, their effect is modulated by MgADP. We analyzed the molecular basis of the interactions between the sulfonylurea gliclazide and Mg-nucleotides on β-cell and cardiac types of KATP channel (Kir6.2/SUR1 and Kir6.2/SUR2A, respectively) heterologously expressed in Xenopus laevis oocytes. The SUR2A-Y1206S mutation was used to confer gliclazide sensitivity on SUR2A. We found that both MgATP and MgADP increased gliclazide inhibition of Kir6.2/SUR1 channels and reduced inhibition of Kir6.2/SUR2A-Y1206S. The latter effect can be attributed to stabilization of the cardiac channel open state by Mg-nucleotides. Using a Kir6.2 mutation that renders the KATP channel insensitive to nucleotide inhibition (Kir6.2-G334D), we showed that gliclazide abolishes the stimulatory effects of MgADP and MgATP on β-cell KATP channels. Detailed analysis suggests that the drug both reduces nucleotide binding to SUR1 and impairs the efficacy with which nucleotide binding is translated into pore opening. Mutation of one (or both) of the Walker A lysines in the catalytic site of the nucleotide-binding domains of SUR1 may have a similar effect to gliclazide on MgADP binding and transduction, but it does not appear to impair MgATP binding. Our results have implications for the therapeutic use of sulfonylureas.

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