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Article: Contribution of Kir3.1, Kir3.2A and Kir3.2C subunits to native G protein-gated inwardly rectifying potassium currents in cultured hippocampal neurons.

Leaney, Joanne L

2003 Volume 18, Issue 8, Page(s) 2110–2118

Abstract: G protein-gated inwardly rectifying potassium (GIRK) channels are found in neurons, atrial myocytes and neuroendocrine cells. A characteristic feature is their activation by stimulation of Gi/o-coupled receptors. In central neurons, for example, they are ...

Abstract	G protein-gated inwardly rectifying potassium (GIRK) channels are found in neurons, atrial myocytes and neuroendocrine cells. A characteristic feature is their activation by stimulation of Gi/o-coupled receptors. In central neurons, for example, they are activated by adenosine and GABA and, as such, they play an important role in neurotransmitter-mediated regulation of membrane excitability. The channels are tetrameric assemblies of Kir3.x subunits (Kir3.1-3.4 plus splice variants). In this study I have attempted to identify the channel subunits which contribute to the native GIRK current recorded from primary cultured rat hippocampal pyramidal neurons. Reverse transcriptase-polymerase chain reaction revealed the expression of mRNA for Kir3.1, 3.2A, 3.2C and 3.3 subunits and confocal immunofluorescence microscopy was used to investigate their expression patterns. Diffuse staining was observed on both cell somata and dendrites for Kir3.1 and Kir3.2A yet that for Kir3.2C was weaker and punctate. Whole-cell patch clamp recordings were used to record GIRK currents from hippocampal pyramidal neurons which were identified on the basis of inward rectification, dependence of reversal potential on external potassium concentration and sensitivity to tertiapin. The GIRK currents were enhanced by the stimulation of a number of Gi/o-coupled receptors and were inhibited by pertussis toxin. In order to ascertain which Kir3.x subunits were responsible for the native GIRK current I compared the properties with those of the cloned Kir3.1 + 3.2A and Kir3.1 + 3.2C channels heterologously expressed in HEK293 cells.
MeSH term(s)	Adenosine/pharmacology ; Analgesics/pharmacology ; Animals ; Animals, Newborn ; Baclofen/pharmacology ; Benzoxazines ; Blotting, Northern ; Calcium Channel Blockers/pharmacology ; Carbachol/pharmacology ; Cell Cycle Proteins/metabolism ; Cells, Cultured ; Cholinergic Agonists/pharmacology ; Dose-Response Relationship, Drug ; Drug Interactions ; Embryo, Mammalian ; Free Radical Scavengers/pharmacology ; G Protein-Coupled Inwardly-Rectifying Potassium Channels ; GABA Agonists/pharmacology ; GTP-Binding Proteins/physiology ; Hippocampus/cytology ; Hormones/pharmacology ; Humans ; Kidney ; Membrane Potentials/drug effects ; Microscopy, Confocal/instrumentation ; Microscopy, Confocal/methods ; Morpholines/pharmacology ; Naphthalenes/pharmacology ; Neurons/drug effects ; Neurons/physiology ; Patch-Clamp Techniques/methods ; Pertussis Toxin/pharmacology ; Potassium Channels/metabolism ; Potassium Channels/physiology ; Potassium Channels, Inwardly Rectifying/classification ; Potassium Channels, Inwardly Rectifying/physiology ; Protein Subunits/physiology ; RNA, Messenger/biosynthesis ; Rats ; Rats, Sprague-Dawley ; Receptors, G-Protein-Coupled/metabolism ; Reverse Transcriptase Polymerase Chain Reaction/methods ; Serotonin/pharmacology ; Somatostatin/pharmacology ; Time Factors
Chemical Substances	Analgesics ; Benzoxazines ; Calcium Channel Blockers ; Cell Cycle Proteins ; Cholinergic Agonists ; Free Radical Scavengers ; G Protein-Coupled Inwardly-Rectifying Potassium Channels ; G2A receptor ; GABA Agonists ; Hormones ; Morpholines ; Naphthalenes ; Potassium Channels ; Potassium Channels, Inwardly Rectifying ; Protein Subunits ; RNA, Messenger ; Receptors, G-Protein-Coupled ; Serotonin (333DO1RDJY) ; Somatostatin (51110-01-1) ; (3R)-((2,3-dihydro-5-methyl-3-((4-morpholinyl)methyl)pyrrolo-(1,2,3-de)-1,4-benzoxazin-6-yl)(1-naphthalenyl))methanone (5H31GI9502) ; Carbachol (8Y164V895Y) ; Pertussis Toxin (EC 2.4.2.31) ; GTP-Binding Proteins (EC 3.6.1.-) ; Baclofen (H789N3FKE8) ; Adenosine (K72T3FS567)
Language	English
Publishing date	2003-10-18
Publishing country	France
Document type	Comparative Study ; Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	645180-9
ISSN	1460-9568 ; 0953-816X
ISSN (online)	1460-9568
ISSN	0953-816X
DOI	10.1046/j.1460-9568.2003.02933.x
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Zs.A 2548: Show issues

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Article ; Online: Investigating dynamic protocol-dependence of hERG potassium channel inhibition at 37 degrees C: Cisapride versus dofetilide.

Milnes, James T / Witchel, Harry J / Leaney, Joanne L / Leishman, Derek J / Hancox, Jules C

Journal of pharmacological and toxicological methods

2010 Volume 61, Issue 2, Page(s) 178–191

Abstract: Introduction: Pharmacological inhibition of cardiac potassium channels encoded by hERG (human ether-à-go-go-related gene) is associated with QT interval prolongation and torsades de pointes arrhythmia. Electrophysiological assays of hERG channel ... ...

Abstract	Introduction: Pharmacological inhibition of cardiac potassium channels encoded by hERG (human ether-à-go-go-related gene) is associated with QT interval prolongation and torsades de pointes arrhythmia. Electrophysiological assays of hERG channel inhibition are integral to the safety testing of novel drug candidates. This study was conducted to compare, for the high affinity hERG inhibitors dofetilide and cisapride, hERG blockade between action potential (AP) and conventional (step and step-ramp) screening waveforms. Furthermore, it evaluated dynamic (pulse-by-pulse) protocol-dependence of hERG channel inhibition by these drugs. Methods: Whole-cell patch-clamp recordings were made at 37 degrees C from hERG-expressing HEK 293 cells. Half-maximal inhibitory concentrations (IC(50) values) for I(hERG) blockade were obtained using conventional voltage clamp and action potential clamp, using previously digitised ventricular and Purkinje fibre (PF) AP waveforms. Results: A more marked variation in IC(50) values with different command waveforms was observed for cisapride (ranging from 7 to 72 nM) than for dofetilide (ranging from 4 to 15 nM), with higher IC(50)s obtained with AP than step or step-ramp commands. The two drugs differed little from one another in effects on voltage-dependent activation; however, I(hERG) blockade by each drug was initially voltage-dependent, but at steady-state was only voltage-dependent for cisapride. There was comparatively little difference between the two drugs in effects on I(hERG) availability or time constants of development of inactivation. Features of time-dependence of blockade and the use of protocols employing varying rest periods in drug or commands of alternating duration highlighted a pronounced ability of cisapride, but not dofetilide, to dissociate and reassociate from hERG on a pulse-by-pulse basis. Discussion: Protocols described here that demonstrated dynamic variation (drug dissociation/reassociation) in hERG channel current blockade at 37 degrees C for cisapride may have future value for investigating drug interactions with the hERG channel. Downloadable digitised ventricular and PF AP waveforms that can be used in AP clamp experiments also accompany this article.
MeSH term(s)	Action Potentials/drug effects ; Axons/drug effects ; Cell Line ; Cisapride/metabolism ; Cisapride/pharmacology ; Data Interpretation, Statistical ; Electrophysiology ; Ether-A-Go-Go Potassium Channels/antagonists & inhibitors ; Ether-A-Go-Go Potassium Channels/metabolism ; Humans ; Patch-Clamp Techniques ; Phenethylamines/metabolism ; Phenethylamines/pharmacology ; Potassium Channel Blockers/metabolism ; Potassium Channel Blockers/pharmacology ; Sulfonamides/metabolism ; Sulfonamides/pharmacology ; Temperature
Chemical Substances	Ether-A-Go-Go Potassium Channels ; Phenethylamines ; Potassium Channel Blockers ; Sulfonamides ; dofetilide (R4Z9X1N2ND) ; Cisapride (UVL329170W)
Language	English
Publishing date	2010-03
Publishing country	United States
Document type	Comparative Study ; Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1105919-9
ISSN	1873-488X ; 1056-8719
ISSN (online)	1873-488X
ISSN	1056-8719
DOI	10.1016/j.vascn.2010.02.007
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Zs.A 1440: Show issues

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Article: Agonist unbinding from receptor dictates the nature of deactivation kinetics of G protein-gated K+ channels.

Benians, Amy / Leaney, Joanne L / Tinker, Andrew

Proceedings of the National Academy of Sciences of the United States of America

2003 Volume 100, Issue 10, Page(s) 6239–6244

Abstract: G protein-gated inwardly rectifying K(+) (Kir) channels are found in neurones, atrial myocytes, and endocrine cells and are involved in generating late inhibitory postsynaptic potentials, slowing the heart rate and inhibiting hormone release. They are ... ...

Abstract	G protein-gated inwardly rectifying K(+) (Kir) channels are found in neurones, atrial myocytes, and endocrine cells and are involved in generating late inhibitory postsynaptic potentials, slowing the heart rate and inhibiting hormone release. They are activated by G protein-coupled receptors (GPCRs) via the inhibitory family of G protein, G(i/o), in a membrane-delimited fashion by the direct binding of Gbetagamma dimers to the channel complex. In this study we are concerned with the kinetics of deactivation of the cloned neuronal G protein-gated K(+) channel, Kir3.1 + 3.2A, after stimulation of a number of GPCRs. Termination of the channel activity on agonist removal is thought to solely depend on the intrinsic hydrolysis rate of the G protein alpha subunit. In this study we present data that illustrate a more complex behavior. We hypothesize that there are two processes that account for channel deactivation: agonist unbinding from the GPCR and GTP hydrolysis by the G protein alpha subunit. With some combinations of agonist/GPCR, the rate of agonist unbinding is slow and rate-limiting, and deactivation kinetics are not modulated by regulators of G protein-signaling proteins. In another group, channel deactivation is generally faster and limited by the hydrolysis rate of the G protein alpha subunit. G protein isoform and interaction with G protein-signaling proteins play a significant role with this group of GPCRs.
MeSH term(s)	Adenosine-5'-(N-ethylcarboxamide)/pharmacology ; Animals ; Cell Line ; G Protein-Coupled Inwardly-Rectifying Potassium Channels ; GTP-Binding Proteins/metabolism ; Guanosine Triphosphate/metabolism ; Humans ; Ion Channel Gating/physiology ; Kinetics ; Membrane Potentials/physiology ; Neurons/physiology ; Patch-Clamp Techniques ; Potassium Channels/agonists ; Potassium Channels/drug effects ; Potassium Channels/physiology ; Potassium Channels, Inwardly Rectifying/agonists ; Potassium Channels, Inwardly Rectifying/drug effects ; Potassium Channels, Inwardly Rectifying/physiology ; Recombinant Fusion Proteins/agonists ; Recombinant Fusion Proteins/metabolism ; Transfection ; gamma-Aminobutyric Acid/pharmacology
Chemical Substances	G Protein-Coupled Inwardly-Rectifying Potassium Channels ; Potassium Channels ; Potassium Channels, Inwardly Rectifying ; Recombinant Fusion Proteins ; Adenosine-5'-(N-ethylcarboxamide) (35920-39-9) ; gamma-Aminobutyric Acid (56-12-2) ; Guanosine Triphosphate (86-01-1) ; GTP-Binding Proteins (EC 3.6.1.-)
Language	English
Publishing date	2003-05-13
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	209104-5
ISSN	1091-6490 ; 0027-8424
ISSN (online)	1091-6490
ISSN	0027-8424
DOI	10.1073/pnas.1037595100
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Zs.A 1148: Show issues

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Article: Differential phosphoinositide binding to components of the G protein-gated K+ channel.

Thomas, Alison M / Brown, Sean G / Leaney, Joanne L / Tinker, Andrew

The Journal of membrane biology

2006 Volume 211, Issue 1, Page(s) 43–53

Abstract: The regulation of ion channels and transporters by anionic phospholipids is currently very topical. G protein-gated K(+) channels from the Kir3.0 family are involved in slowing the heart rate, generating late inhibitory postsynaptic potentials and ... ...

Abstract	The regulation of ion channels and transporters by anionic phospholipids is currently very topical. G protein-gated K(+) channels from the Kir3.0 family are involved in slowing the heart rate, generating late inhibitory postsynaptic potentials and controlling hormone release from neuroendocrine cells. There is considerable functional precedent for the control of these channels by phosphatidylinositol 4,5-bisphosphate. In this study, we used a biochemical assay to investigate the lipid binding properties of Kir3.0 channel domains. We reveal a differential binding affinity to a range of phosphoinositides between the C termini of the Kir3.0 isoforms. Furthermore, the N terminus in addition to the C terminus of Kir3.4 is necessary to observe binding and is decreased by the mutations R72A, K195A and R196A but not K194A. Protein kinase C phosphorylation of the Kir3.1 C-terminal fusion protein decreases anionic phospholipid binding. The differential binding affinity has functional consequences as the inhibition of homomeric Kir3.1, occurring after M3 receptor activation, recovers over minutes while homomeric Kir3.2 does not.
MeSH term(s)	Amino Acid Sequence ; Carrier Proteins/genetics ; Carrier Proteins/metabolism ; G Protein-Coupled Inwardly-Rectifying Potassium Channels/genetics ; G Protein-Coupled Inwardly-Rectifying Potassium Channels/metabolism ; Humans ; Maltose-Binding Proteins ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Phosphatidylinositols/metabolism ; Protein Binding/physiology ; Protein Kinase C/physiology ; Recombinant Fusion Proteins/genetics ; Recombinant Fusion Proteins/metabolism
Chemical Substances	Carrier Proteins ; G Protein-Coupled Inwardly-Rectifying Potassium Channels ; Maltose-Binding Proteins ; Phosphatidylinositols ; Recombinant Fusion Proteins ; Protein Kinase C (EC 2.7.11.13)
Language	English
Publishing date	2006-05
Publishing country	United States
Document type	Comparative Study ; Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	3082-x
ISSN	1432-1424 ; 0022-2631
ISSN (online)	1432-1424
ISSN	0022-2631
DOI	10.1007/s00232-006-0014-5
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Zs.A 613: Show issues

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Article: Differential Phosphoinositide Binding to Components of the G Protein-Gated K⁺ Channel

Thomas, Alison M / Brown, Sean G / Leaney, Joanne L / Tinker, Andrew

Journal of membrane biology. 2006 May, v. 211, no. 1

2006

Abstract: The regulation of ion channels and transporters by anionic phospholipids is currently very topical. G protein-gated K⁺ channels from the Kir3.0 family are involved in slowing the heart rate, generating late inhibitory postsynaptic potentials and ... ...

Abstract	The regulation of ion channels and transporters by anionic phospholipids is currently very topical. G protein-gated K⁺ channels from the Kir3.0 family are involved in slowing the heart rate, generating late inhibitory postsynaptic potentials and controlling hormone release from neuroendocrine cells. There is considerable functional precedent for the control of these channels by phosphatidylinositol 4,5-bisphosphate. In this study, we used a biochemical assay to investigate the lipid binding properties of Kir3.0 channel domains. We reveal a differential binding affinity to a range of phosphoinositides between the C termini of the Kir3.0 isoforms. Furthermore, the N terminus in addition to the C terminus of Kir3.4 is necessary to observe binding and is decreased by the mutations R72A, K195A and R196A but not K194A. Protein kinase C phosphorylation of the Kir3.1 C-terminal fusion protein decreases anionic phospholipid binding. The differential binding affinity has functional consequences as the inhibition of homomeric Kir3.1, occurring after M3 receptor activation, recovers over minutes while homomeric Kir3.2 does not.
Language	English
Dates of publication	2006-05
Size	p. 43-53.
Publisher	Springer-Verlag
Publishing place	New York
Document type	Article
ZDB-ID	3082-x
ISSN	1432-1424 ; 0022-2631
ISSN (online)	1432-1424
ISSN	0022-2631
DOI	10.1007/s00232-006-0014-5
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Zs.A 613: Show issues

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Article: The dynamics of formation and action of the ternary complex revealed in living cells using a G-protein-gated K+ channel as a biosensor.

Benians, Amy / Leaney, Joanne L / Milligan, Graeme / Tinker, Andrew

The Journal of biological chemistry

2003 Volume 278, Issue 12, Page(s) 10851–10858

Abstract: Traditionally the consequences of activation of G-protein-coupled receptors (GPCRs) by an agonist are studied using biochemical assays. In this study we use live cells and take advantage of a G-protein-gated inwardly rectifying potassium channel (Kir3.1+ ... ...

Abstract	Traditionally the consequences of activation of G-protein-coupled receptors (GPCRs) by an agonist are studied using biochemical assays. In this study we use live cells and take advantage of a G-protein-gated inwardly rectifying potassium channel (Kir3.1+3.2A) that is activated by the direct binding of Gbetagamma subunit to the channel complex to report, in real-time, using the patch clamp technique the activity of the "ternary complex" of agonist/receptor/G-protein. This analysis is further facilitated by the use of pertussis toxin-resistant fluorescent and non-fluorescent Galpha(i/o) subunits and a series of HEK293 cell lines stably expressing both channel and receptors (including the adenosine A(1) receptor, the adrenergic alpha(2A) receptor, the dopamine D(2S) receptor, the M4 muscarinic receptor, and the dimeric GABA-B(1b/2) receptor). We systematically analyzed the contribution of the various inputs to the observed kinetic response of channel activation. Our studies indicate that the combination of agonist, GPCR, and G-protein isoform uniquely specify the behavior of these channels and thus support the importance of the whole ternary complex at a kinetic level.
MeSH term(s)	Biosensing Techniques ; Cell Line ; G Protein-Coupled Inwardly-Rectifying Potassium Channels ; Heterotrimeric GTP-Binding Proteins/physiology ; Humans ; Ion Channel Gating ; Kinetics ; Pertussis Toxin/pharmacology ; Potassium Channels/chemistry ; Potassium Channels/metabolism ; Potassium Channels, Inwardly Rectifying ; Receptors, Cell Surface/metabolism
Chemical Substances	G Protein-Coupled Inwardly-Rectifying Potassium Channels ; Potassium Channels ; Potassium Channels, Inwardly Rectifying ; Receptors, Cell Surface ; Pertussis Toxin (EC 2.4.2.31) ; Heterotrimeric GTP-Binding Proteins (EC 3.6.5.1)
Language	English
Publishing date	2003-01-14
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1074/jbc.M212299200
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Article: hERG K+ channel blockade by the antipsychotic drug thioridazine: An obligatory role for the S6 helix residue F656.

Milnes, James T / Witchel, Harry J / Leaney, Joanne L / Leishman, Derek J / Hancox, Jules C

Biochemical and biophysical research communications

2006 Volume 351, Issue 1, Page(s) 273–280

Abstract: The phenothiazine antipsychotic agent thioridazine has been linked with prolongation of the QT interval on the electrocardiogram, ventricular arrhythmias, and sudden death. Although thioridazine is known to inhibit cardiac hERG K(+) channels there is ... ...

Abstract	The phenothiazine antipsychotic agent thioridazine has been linked with prolongation of the QT interval on the electrocardiogram, ventricular arrhythmias, and sudden death. Although thioridazine is known to inhibit cardiac hERG K(+) channels there is little mechanistic information on this action. We have investigated in detail hERG K(+) channel current (I(hERG)) blockade by thioridazine and identified a key molecular determinant of blockade. Whole-cell I(hERG) measurements were made at 37 degrees C from human embryonic kidney (HEK-293) cells expressing wild-type and mutant hERG channels. Thioridazine inhibited I(hERG) tails at -40mV following a 2s depolarization to +20mV with an IC(50) value of 80nM. Comparable levels of I(hERG) inhibition were seen with physiological command waveforms (ventricular and Purkinje fibre action potentials). Thioridazine block of I(hERG) was only weakly voltage-dependent, though the time dependence of I(hERG) inhibition indicated contingency of blockade upon channel gating. The S6 helix point mutation F656A almost completely abolished, and the Y652A mutation partially attenuated, I(hERG) inhibition by thioridazine. In summary, thioridazine is one of the most potent hERG K(+) channel blockers amongst antipsychotics, exhibiting characteristics of a preferential open/activated channel blocker and binding at a high affinity site in the hERG channel pore.
MeSH term(s)	Antipsychotic Agents/administration & dosage ; Cell Line ; Chlorpromazine/administration & dosage ; Dose-Response Relationship, Drug ; ERG1 Potassium Channel ; Ether-A-Go-Go Potassium Channels/chemistry ; Ether-A-Go-Go Potassium Channels/physiology ; Humans ; Ion Channel Gating/physiology ; Kidney/drug effects ; Kidney/physiology ; Membrane Potentials/drug effects ; Membrane Potentials/physiology ; Protein Structure, Tertiary ; Structure-Activity Relationship
Chemical Substances	Antipsychotic Agents ; ERG1 Potassium Channel ; Ether-A-Go-Go Potassium Channels ; KCNH2 protein, human ; Chlorpromazine (U42B7VYA4P)
Language	English
Publishing date	2006-12-08
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	205723-2
ISSN	0006-291X ; 0006-291X
ISSN (online)	0006-291X
ISSN	0006-291X
DOI	10.1016/j.bbrc.2006.10.039
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Zs.A 116: Show issues

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Article: A novel strategy to engineer functional fluorescent inhibitory G-protein alpha subunits.

Leaney, Joanne L / Benians, Amy / Graves, Fiona M / Tinker, Andrew

The Journal of biological chemistry

2002 Volume 277, Issue 32, Page(s) 28803–28809

Abstract: Signaling studies in living cells would be greatly facilitated by the development of functional fluorescently tagged G-protein alpha subunits. We have designed G(i/o)alpha subunits fused to the cyan fluorescent protein and assayed their function by ... ...

Abstract	Signaling studies in living cells would be greatly facilitated by the development of functional fluorescently tagged G-protein alpha subunits. We have designed G(i/o)alpha subunits fused to the cyan fluorescent protein and assayed their function by studying the following two signal transduction pathways: the regulation of G-protein-gated inwardly rectifying K(+) channels (Kir3.0 family) and adenylate cyclase. Palmitoylation and myristoylation consensus sites were removed from G(i/o) alpha subunits (G(i1)alpha, G(i2)alpha, G(i3)alpha, and G(oA)alpha) and a mutation introduced at Cys(-4) rendering the subunit resistant to pertussis toxin. This construct was fused in-frame with cyan fluorescent protein containing a short peptide motif from GAP43 that directs palmitoylation and thus membrane targeting. Western blotting confirmed G(i/o)alpha protein expression. Confocal microscopy and biochemical fractionation studies revealed membrane localization. Each mutant G(i/o) alpha subunit significantly reduced basal current density when transiently expressed in a stable cell line expressing Kir3.1 and Kir3.2A, consistent with the sequestration of the Gbetagamma dimer by the mutant Galpha subunit. Moreover, each subunit was able to support A1-mediated and D2S-mediated channel activation when transiently expressed in pertussis toxin-treated cells. Overexpression of tagged G(i3)alpha and G(oA)alpha alpha subunits reduced receptor-mediated and forskolin-induced cAMP mobilization.
MeSH term(s)	Adenylate Cyclase Toxin ; Adenylyl Cyclases/metabolism ; Binding Sites ; Blotting, Western ; Cell Line ; Colforsin/pharmacology ; Cyclic AMP/metabolism ; DNA, Complementary/metabolism ; Dimerization ; G Protein-Coupled Inwardly-Rectifying Potassium Channels ; GTP-Binding Proteins/metabolism ; Humans ; Microscopy, Confocal ; Microscopy, Fluorescence/methods ; Mutation ; Myristic Acid/metabolism ; Open Reading Frames ; Palmitic Acid/metabolism ; Pertussis Toxin ; Potassium/metabolism ; Potassium Channels/metabolism ; Potassium Channels, Inwardly Rectifying/metabolism ; Protein Binding ; Transfection ; Virulence Factors, Bordetella/pharmacology
Chemical Substances	Adenylate Cyclase Toxin ; DNA, Complementary ; G Protein-Coupled Inwardly-Rectifying Potassium Channels ; Potassium Channels ; Potassium Channels, Inwardly Rectifying ; Virulence Factors, Bordetella ; Myristic Acid (0I3V7S25AW) ; Colforsin (1F7A44V6OU) ; Palmitic Acid (2V16EO95H1) ; Cyclic AMP (E0399OZS9N) ; Pertussis Toxin (EC 2.4.2.31) ; GTP-Binding Proteins (EC 3.6.1.-) ; Adenylyl Cyclases (EC 4.6.1.1) ; Potassium (RWP5GA015D)
Language	English
Publishing date	2002-06-04
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1074/jbc.M204683200
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Article: PKC-delta sensitizes Kir3.1/3.2 channels to changes in membrane phospholipid levels after M3 receptor activation in HEK-293 cells.

Brown, Sean G / Thomas, Alison / Dekker, Lodewijk V / Tinker, Andrew / Leaney, Joanne L

American journal of physiology. Cell physiology

2005 Volume 289, Issue 3, Page(s) C543–56

Abstract: G protein-gated inward rectifier (Kir3) channels are inhibited by activation of G(q/11)-coupled receptors and this has been postulated to involve the signaling molecules protein kinase C (PKC) and/or phosphatidylinositol 4,5-bisphosphate (PIP(2)). Their ... ...

Abstract	G protein-gated inward rectifier (Kir3) channels are inhibited by activation of G(q/11)-coupled receptors and this has been postulated to involve the signaling molecules protein kinase C (PKC) and/or phosphatidylinositol 4,5-bisphosphate (PIP(2)). Their precise roles in mediating the inhibition of this family of channels remain controversial. We examine here their relative roles in causing inhibition of Kir3.1/3.2 channels stably expressed in human embryonic kidney (HEK)-293 cells after muscarinic M(3) receptor activation. In perforated patch mode, staurosporine prevented the G(q/11)-mediated, M(3) receptor, inhibition of channel activity. Recovery from M(3)-mediated inhibition was wortmannin sensitive. Whole cell currents, where the patch pipette was supplemented with PIP(2), were still irreversibly inhibited by M(3) receptor stimulation. When adenosine A(1) receptors were co-expressed, inclusion of PIP(2) rescued the A(1)-mediated response. Recordings from inside-out patches showed that catalytically active PKC applied directly to the intracellular membrane face inhibited the channels: a reversible effect modulated by okadaic acid. Generation of mutant heteromeric channel Kir3.1S185A/Kir3.2C-S178A, still left the channel susceptible to receptor, pharmacological, and direct kinase-mediated inhibition. Biochemically, labeled phosphate is incorporated into the channel. We suggest that PKC-delta mediates channel inhibition because recombinant PKC-delta inhibited channel activity, M(3)-mediated inhibition of the channel, was counteracted by overexpression of two types of dominant negative PKC-delta constructs, and, by using confocal microscopy, we have demonstrated translocation of green fluorescent protein-tagged PKC-delta to the plasma membrane on M(3) receptor stimulation. Thus Kir3.1/3.2 channels are sensitive to changes in membrane phospholipid levels but this is contingent on the activity of PKC-delta after M(3) receptor activation in HEK-293 cells.
MeSH term(s)	Cell Line ; Cell Membrane ; G Protein-Coupled Inwardly-Rectifying Potassium Channels ; GTP-Binding Protein alpha Subunits, Gq-G11/metabolism ; Humans ; Kidney/cytology ; Membrane Potentials/physiology ; Patch-Clamp Techniques ; Phosphatidylinositol 4,5-Diphosphate/metabolism ; Phosphorylation ; Potassium Channels, Inwardly Rectifying/physiology ; Protein Kinase C/metabolism ; Protein Kinase C-delta ; Protein Kinase C-epsilon ; Receptor Cross-Talk/physiology ; Receptor, Muscarinic M3/physiology
Chemical Substances	G Protein-Coupled Inwardly-Rectifying Potassium Channels ; KCNJ3 protein, human ; Phosphatidylinositol 4,5-Diphosphate ; Potassium Channels, Inwardly Rectifying ; Receptor, Muscarinic M3 ; PRKCD protein, human (EC 2.7.11.13) ; PRKCE protein, human (EC 2.7.11.13) ; Protein Kinase C (EC 2.7.11.13) ; Protein Kinase C-delta (EC 2.7.11.13) ; Protein Kinase C-epsilon (EC 2.7.11.13) ; GTP-Binding Protein alpha Subunits, Gq-G11 (EC 3.6.5.1)
Language	English
Publishing date	2005-04-27
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	392098-7
ISSN	1522-1563 ; 0363-6143
ISSN (online)	1522-1563
ISSN	0363-6143
DOI	10.1152/ajpcell.00025.2005
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Article: Rapid desensitization of G protein-gated inwardly rectifying K(+) currents is determined by G protein cycle.

Leaney, Joanne L / Benians, Amy / Brown, Sean / Nobles, Muriel / Kelly, David / Tinker, Andrew

American journal of physiology. Cell physiology

2004 Volume 287, Issue 1, Page(s) C182–91

Abstract: Activation of G protein-gated inwardly rectifying K(+) (GIRK) channels, found in the brain, heart, and endocrine tissue, leads to membrane hyperpolarization that generates neuronal inhibitory postsynaptic potentials, slows the heart rate, and inhibits ... ...

Abstract	Activation of G protein-gated inwardly rectifying K(+) (GIRK) channels, found in the brain, heart, and endocrine tissue, leads to membrane hyperpolarization that generates neuronal inhibitory postsynaptic potentials, slows the heart rate, and inhibits hormone release. During stimulation of G(i/o)-coupled receptors and subsequent channel activation, it has been observed that the current desensitizes. In this study we examined mechanisms underlying fast desensitization of cloned heteromeric neuronal Kir3.1+3.2A and atrial Kir3.1+3.4 channels and also homomeric Kir3.0 currents in response to stimulation of several G(i/o) G protein-coupled receptors (GPCRs) expressed in HEK-293 cells (adenosine A(1), adrenergic alpha(2A), dopamine D(2S), M(4) muscarinic, and GABA(B1b/2) receptors). We found that all agonist-induced currents displayed a similar degree of desensitization except the adenosine A(1) receptor, which exhibits an additional desensitizing component. Using the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), we found that this is due to a receptor-dependent, G protein-independent process. Using Ca(2+) imaging we showed that desensitization is unlikely to be accounted for solely by phospholipase C activation and phosphatidylinositol 4,5-bisphosphate (PIP(2)) hydrolysis. We examined the contribution of the G protein cycle and found the following. First, agonist concentration is strongly correlated with degree of desensitization. Second, competitive inhibition of GDP/GTP exchange by using nonhydrolyzable guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS) has two effects, a slowing of channel activation and an attenuation of the fast desensitization phenomenon. Finally, using specific Galpha subunits we showed that ternary complexes with fast activation rates display more prominent desensitization than those with slower activation kinetics. Together our data suggest that fast desensitization of GIRK currents is accounted for by the fundamental properties of the G protein cycle.
MeSH term(s)	Cell Line ; Electric Conductivity ; G Protein-Coupled Inwardly-Rectifying Potassium Channels ; GTP-Binding Protein alpha Subunits, Gq-G11/metabolism ; GTP-Binding Proteins/metabolism ; Humans ; Ion Channel Gating ; Potassium Channels/metabolism ; Potassium Channels, Inwardly Rectifying/metabolism ; Potassium Channels, Inwardly Rectifying/physiology ; Protein Isoforms/metabolism ; Receptors, Cell Surface/metabolism
Chemical Substances	G Protein-Coupled Inwardly-Rectifying Potassium Channels ; Potassium Channels ; Potassium Channels, Inwardly Rectifying ; Protein Isoforms ; Receptors, Cell Surface ; GTP-Binding Proteins (EC 3.6.1.-) ; GTP-Binding Protein alpha Subunits, Gq-G11 (EC 3.6.5.1)
Language	English
Publishing date	2004-03-10
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	392098-7
ISSN	1522-1563 ; 0363-6143
ISSN (online)	1522-1563
ISSN	0363-6143
DOI	10.1152/ajpcell.00540.2003
Database	MEDical Literature Analysis and Retrieval System OnLINE

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