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Results for “Potassium Channels, Inwardly Rectifying”

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At least 19 recordsLinked to original sources

PubMed2001

Multiple PIP2 binding sites in Kir2.1 inwardly rectifying potassium channels.

Inwardly rectifying potassium channels require binding of phosphatidylinositol-4,5-bisphosphate (PIP2) for channel activity. Three independent sites (aa 175-206, aa 207-246, aa 324-365) were located in the C-terminal domain of Kir2.1 channels by assaying the binding of overlapping fragments to PIP2 containing liposomes. Mutations in the first site, which abolished channel activity, reduced PIP2 binding of this fragment but not of the complete C-terminus. Point mutations in the third site also reduced both, channel activity and PIP2 binding of this segment. The relevance of the third PIP2 binding site provides a basis for the understanding of constitutively active Kir2 channels.

Animals↗

PubMed1996

Regions responsible for the assembly of inwardly rectifying potassium channels.

Inwardly rectifying potassium channels have an important role in determining the resting potential of the cell. They are tetrameric proteins with two transmembrane segments (M1 and M2), a pore-forming loop (H5), a cytoplasmic N-terminal, and longer C-terminal domain. We have used biochemical and electrophysiological methods to identify regions required for homotypic interactions and those responsible for the incompatibility between IRK1 and two other members of the same subfamily (IRK2 and IRK3) and two members from other subfamilies (ROMK1 and 6.1 uK(ATP)). The data indicate that, in contrast to the voltage-gated class of potassium channel, the proximal C-terminus and the transmembrane segment M2 determine homo-and heteromultimerization and that heteromultimerization between members of the same or different subfamilies is case specific.

Animals↗

PubMed1999

Transmembrane structure of an inwardly rectifying potassium channel.

Inwardly rectifying potassium channels (K(ir)), comprising four subunits each with two transmembrane domains, M1 and M2, regulate many important physiological processes. We employed a yeast genetic screen to identify functional channels from libraries of K(ir) 2.1 containing mutagenized M1 or M2 domains. Patterns in the allowed sequences indicate that M1 and M2 are helices. Protein-lipid and protein-water interaction surfaces identified by the patterns were verified by sequence minimization experiments. Second-site suppressor analyses of helix packing indicate that the M2 pore-lining inner helices are surrounded by the M1 lipid-facing outer helices, arranged such that the M1 helices participate in subunit-subunit interactions. This arrangement is distinctly different from the structure of a bacterial potassium channel with the same topology and identifies helix-packing residues as hallmark sequences common to all K(ir) superfamily members.

Amino Acid Sequence↗

PubMed2000

The role of members of the pertussis toxin-sensitive family of G proteins in coupling receptors to the activation of the G protein-gated inwardly rectifying potassium channel.

Inwardly rectifying potassium (K(+)) channels gated by G proteins (Kir3.x family) are widely distributed in neuronal, atrial, and endocrine tissues and play key roles in generating late inhibitory postsynaptic potentials, slowing the heart rate and modulating hormone release. They are directly activated by G(betagamma) subunits released from G protein heterotrimers of the G(i/o) family upon appropriate receptor stimulation. Here we examine the role of isoforms of pertussis toxin (PTx)-sensitive G protein alpha subunits (G(ialpha1-3) and G(oalphaA)) in mediating coupling between various receptor systems (A(1), alpha(2A), D(2S), M(4), GABA(B)1a+2, and GABA(B)1b+2) and the cloned counterpart of the neuronal channel (Kir3.1+3.2A). The expression of mutant PTx-resistant G(i/oalpha) subunits in PTx-treated HEK293 cells stably expressing Kir3.1+3.2A allows us to selectively investigate that coupling. We find that, for those receptors (A(1), alpha(2A)) known to interact with all isoforms, G(ialpha1-3) and G(oalphaA) can all support a significant degree of coupling to Kir3.1+3.2A. The M(4) receptor appears to preferentially couple to G(ialpha2) while another group of receptors (D(2S), GABA(B)1a+2, GABA(B)1b+2) activates the channel predominantly through G(betagamma) liberated from G(oA) heterotrimers. Interestingly, we have also found a distinct difference in G protein coupling between the two splice variants of GABA(B)1. Our data reveal selective pathways of receptor activation through different G(i/oalpha) isoforms for stimulation of the G protein-gated inwardly rectifying K(+) channel.

Cell Line↗

PubMed1999

Inwardly rectifying potassium channels.

Inwardly rectifying potassium (Kir) channels regulate the resting membrane potential of the cell and thereby modulate the electrical activity of cardiac and neuronal cells, insulin secretion and epithelial K(+) transport. Considerable progress in understanding the molecular structure of Kir channels and the way in which they are regulated by extracellular and intracellular modulators has been made during the past year.

Animals↗

PubMed1995

Determination of the subunit stoichiometry of an inwardly rectifying potassium channel.

Inwardly rectifying K+ channels are distantly related to their voltage-gated counterparts and possess a structural motif of only two putative transmembrane segments in each subunit. They are formed by the assembly of an unknown number of subunits. We have examined the subunit stoichiometry of a strongly rectifying K+ channel, IRK1, by linking together the coding sequence of three or four subunits and distinguishing channels with different numbers of subunits carrying a double mutation that alters inward rectification and single-channel properties. We find that IRK1 channels, like voltage-gated K+ channels, are tetrameric channels. Interestingly, the high sensitivity to Mg2+ and polyamines, cations that produce inward rectification by blocking the channel pore from the cytoplasmic side is largely retained in a channel containing only one wild-type subunit and three subunits bearing mutations that abolish high affinity Mg2+ and polyamine block.

Animals↗

PubMed1995

Contributions of the C-terminal domain to gating properties of inward rectifier potassium channels.

Two inward rectifier potassium channels, the G protein-dependent GIRK1 and the G protein-independent BIR10, display large differences in rectification and macroscopic kinetics. A chimeric channel was constructed in which the putative intracellular carboxy-terminal domain of the G protein-dependent channel replaced the corresponding domain of the G protein-independent channel. The chimeric channel conducted potassium ions without the requirement of activated G proteins, yet displayed activation and deactivation kinetics and rectification properties similar to those of the G protein-dependent channel. The results demonstrate that structural elements in the C-terminus can independently control gating but not G protein signal transduction. The voltage dependence, time course, and kinetics of gating suggest a mechanism in which the pore may be occluded by reversible interactions with charged residues in the C-terminus.

Amino Acid Sequence↗

PubMed1995

Identification and molecular localization of a pH-sensing domain for the inward rectifier potassium channel HIR.

Inward rectifier potassium channels are found in the heart and CNS, where they are critical for the modulation and maintenance of cellular excitability. We present evidence that the inward rectifier potassium channel HIR is modulated by extracellular pH in the physiological range. We show that proton-induced changes in HIR single-channel conductance underlie the HIR pH sensitivity seen on the macroscopic level. We used chimeric and mutant channels to localize the molecular determinant of HIR pH sensitivity to a single residue, H117, in the M1-to-H5 linker region. This residue provides a molecular context that allows a titratable group to influence pore properties. We present evidence that this titratable group is one of two cysteines located in the M1-to-H5 and H5-to-M2 linkers.

Amino Acid Sequence↗

PubMed2004

An alternatively spliced isoform of PSD-93/chapsyn 110 binds to the inwardly rectifying potassium channel, Kir2.1.

Inwardly rectifying potassium (Kir) channels are prime determinants of resting membrane potential in neurons. Their subcellular distribution and surface density thus help shape neuronal excitability, yet mechanisms governing the membrane targeting and localization of Kir channels are poorly understood. Here we report a direct interaction between the strong inward rectifier, Kir2.1, and a recently identified splice variant of postsynaptic density-93 (PSD-93), a protein involved the subcellular targeting of ion channels and glutamate receptors at excitatory synapses. Yeast two-hybrid screening of a human brain cDNA library using the carboxyl terminus of Kir2.1 as bait yielded cDNA encoding the first two PDZ domains of PSD-93, but with an extended N-terminal region that diverged from other PSD-93 isoforms. This clone represented the human homologue of the mouse PSD-93 splice variant, PSD-93delta. Reverse transcription-polymerase chain reaction analysis showed diffuse low level PSD-93delta expression throughout the brain, with significantly higher levels in spinal cord. In vitro binding studies revealed that a type I PDZ recognition motif at the extreme C terminus of the Kir2.1 mediates interaction with all three PDZ domains of PSD-93delta, and association between Kir2 channels and PSD-93delta was confirmed further by the ability of anti-Kir2.1 antibodies to coimmunoprecipitate PSD-93delta from rat spinal cord lysates. Functionally, coexpression of Kir2.1 and PSD-93delta had no discernible effect upon channel kinetics but resulted in cell surface Kir2.1 clustering and suppression of channel internalization. We conclude that PSD-93delta is potentially an important regulator of the spatial and temporal distribution of Kir2 channels within neuronal membranes of the central nervous system.

Alternative Splicing↗

PubMed2000

Effects of inhibitors of small- and intermediate-conductance calcium-activated potassium channels, inwardly-rectifying potassium channels and Na(+)/K(+) ATPase on EDHF relaxations in the rat hepatic artery.

1. In the rat hepatic artery, the SK(Ca) inhibitors UCL 1684 (300 nM) completely blocked, and scyllatoxin (1 microM) and d-tubocurarine (100 microM) partially inhibited EDHF relaxations when each of them was combined with charybdotoxin (300 nM). 2. The IK(Ca) inhibitors clotrimazole (3 microM) and 2-chlorophenyl-bisphenyl-methanol (3 microM) strongly depressed EDHF relaxations when each of them was combined with apamin (300 nM). The cytochrome P450 mono-oxygenase inhibitor ketoconazole (10 microM) had no effect in the presence of apamin. 3. Ciclazindol (10 microM), which abolishes EDHF relaxations in the presence of apamin, almost completely prevented the calcium ionophore (A23187) stimulated (86)Rb(+) influx via the Gardos channel (IK(Ca)) in human erythrocytes. 4. The Na(+)/K(+) ATPase inhibitor ouabain (500 microM) and the K(IR) blocker Ba(2+) (30 microM) neither alone nor in combination inhibited EDHF relaxations. Ba(2+) was also without effect in the presence of either apamin or charybdotoxin. 5. In contrast to EDHF, an increase in extracellular [K(+)] from 4.6 mM to 9.6, 14.6 and 19.6 mM inconsistently relaxed arteries. In K(+)-free physiological salt solution, re-admission of K(+) always caused complete and sustained relaxations which were abolished by ouabain but unaffected by Ba(2+). 6. The present study provides pharmacological evidence for the involvement of SK(Ca) and IK(Ca) in the action of EDHF in the rat hepatic artery. Our results are not consistent with the idea that EDHF is K(+) activating Na(+)/K(+) ATPase and K(IR) in this blood vessel.

Acetylcholine↗

PubMed2003

Role of the small GTPase Rho in modulation of the inwardly rectifying potassium channel Kir2.1.

The inwardly rectifying potassium channel Kir2.1 is inhibited by a variety of G-protein-coupled receptors (GPCRs). However, the mechanisms underlying the inhibition have not been fully elucidated. In this study the role of the small GTPase, Rho, in mediating this inhibition was determined. Stimulation of the m1 muscarinic receptor inhibited Kir2.1, when both receptor and channel were coexpressed in tsA201 cells. The inhibition of Kir2.1 by carbachol was reversible and atropine-sensitive. Cotransfection with a dominant-negative mutant of the small GTPase Rho abolished the inhibition of Kir2.1 with current amplitudes remaining at control levels in the presence of carbachol. Conversely, cotransfection with the constitutively activated mutant of Rho resulted in a reduction in basal Kir2.1 current amplitudes, suggesting that Rho inhibits Kir2.1. To further confirm the involvement of Rho in the signal transduction pathway, cotransfection with C3 transferase (EFC3), a selective inhibitor of Rho, abolished the reduction in Kir2.1 currents noted upon application of carbachol under control conditions. Preincubation with the phosphatidylinositol 3-kinase inhibitor wortmannin or the Rho kinase inhibitor (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, 2 HCl (Y-27632) had no effect on agonist-induced inhibition of Kir2.1, precluding these kinases as downstream effectors of Rho in mediation of the signal. In addition, 2'-amino-3'-methoxyflavone (PD98059), an inhibitor of mitogen-activated protein (MAP) kinase kinase (MEK), had no effect on the m1 receptor-induced inhibition of Kir2.1, suggesting that MAP kinases are not involved in the signaling pathway. In conclusion, these data indicate that the small GTPase, Rho, transduces the m1 muscarinic receptor-induced inhibition of Kir2.1 via an unidentified mechanism.

Cells, Cultured↗

PubMed1996

[K+] dependence of open-channel conductance in cloned inward rectifier potassium channels (IRK1, Kir2.1).

Potassium conduction through unblocked inwardly rectifying (IRK1, Kir2.1) potassium channels was measured in inside-out-patches from Xenopus oocytes, after removal of polyamine-induced strong inward rectification. Unblocked IRK1 channel current-voltage (I-V) relations show very mild inward rectification in symmetrical solutions, are linearized in nonsymmetrical solutions that bring the K+ reversal potential to extreme negative values, and follow Goldman-Hodgkin-Katz constant field equation at extreme positive E alpha. When intracellular K+ concentration (KIN) was varied, at constant extracellular K+ concentration (KOUT) the conductance at the reversal potential (GREV) followed closely the predictions of the Goldman-Hodgkin-Katz constant field equation at low concentrations and saturated sharply at concentrations of > 150 mM. Similarly, when KOUT was varied, at constant KIN, GREV saturated at concentrations of > 150 mM. A square-root dependence of conductance on KOUT is a well-known property of inward rectifier potassium channels and is a property of the open channel. A nonsymmetrical two-site three-barrier model can qualitatively explain both the I-V relations and the [K+] dependence of conductance of open IRK1 (Kir2.1) channels.

Animals↗

PubMed1998

Tetrameric subunit structure of the native brain inwardly rectifying potassium channel Kir 2.2.

Strongly inwardly rectifying potassium channels of the Kir 2 subfamily (IRK1, IRK2, and IRK3) are involved in maintenance and modulation of cell excitability in brain and heart. Electrophysiological studies of channels expressed in heterologous systems have suggested that the pore-conducting pathway contains four subunits. However, inferences from electrophysiological studies have not been tested on native channels and do not address the possibility of nonconducting auxiliary subunits. Here, we investigate the subunit stoichiometry of endogenous inwardly rectifying potassium channel Kir 2.2 (IRK2) from rat brain. Using chemical cross-linking, immunoprecipitiation, and velocity sedimentation, we report physical evidence demonstrating the tetrameric organization of the native channel. Kir 2.2 was sequentially cross-linked to produce bands on SDS-polyacrylamide gel electrophoresis corresponding in size to monomer, dimer, trimer, and three forms of tetramer. Fully cross-linked channel was present as a single band of tetrameric size. Immunoprecipitation of biotinylated membranes revealed a single band corresponding to Kir 2.2, suggesting that the channel is composed of a single type of subunit. Hydrodynamic properties of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonic acid-solubilized channel were used to calculate the molecular mass of the channel. Velocity sedimentation in H2O or D2O gave a sharp peak with a sedimentation coefficient of 17.3 S. Gel filtration yielded a Stokes radius of 5.92 nm. These data indicate a multisubunit protein with a molecular mass of 193 kDa, calculated to contain 3.98 subunits. Together, these results demonstrate that Kir 2.2 channels are formed by the homotetrameric association of Kir 2.2 subunits and do not contain tightly associated auxiliary subunits. These studies suggest that Kir 2.2 channels differ in structure from related heterooctomeric ATP-sensitive K channels and heterotetrameric G-protein-regulated inward rectifier K channels.

Amino Acid Sequence↗

PubMed2001

Coupling Gbetagamma-dependent activation to channel opening via pore elements in inwardly rectifying potassium channels.

G protein-coupled inwardly rectifying potassium channels, GIRK/Kir3.x, are gated by the Gbetagamma subunits of the G protein. The molecular mechanism of gating was investigated by employing a novel yeast-based random mutagenesis approach that selected for channel mutants that are active in the absence of Gbetagamma. Mutations in TM2 were found that mimicked the Gbetagamma-activated state. The activity of these channel mutants was independent of receptor stimulation and of the availability of heterologously expressed Gbetagamma subunits but depended on PtdIns(4,5)P(2). The results suggest that the TM2 region plays a key role in channel gating following Gbetagamma binding in a phospholipid-dependent manner. This mechanism of gating in inwardly rectifying K+ channels may be similar to the involvement of the homologous region in prokaryotic KcsA potassium channel and, thus, suggests evolutionary conservation of the gating structure.

Amino Acid Sequence↗

PubMed2000

Single channel studies of inward rectifier potassium channel regulation by muscarinic acetylcholine receptors.

Negative regulation of the heartbeat rate involves the activation of an inwardly rectifying potassium current (I(KACh)) by G protein-coupled receptors such as the m2 muscarinic acetylcholine receptor. Recent studies have shown that this process involves the direct binding of G(betagamma) subunits to the NH(2)- and COOH-terminal cytoplasmic domains of the proteins termed GIRK1 and GIRK4 (Kir3.1 and Kir3.4/CIR), which mediate I(KACh). Because of the very low basal activity of native I(KACh), it has been difficult to determine the single channel effect of G(betagamma) subunit binding on I(KACh) activity. Through analysis of a novel G protein-activated chimeric inward rectifier channel that displays increased basal activity relative to I(KACh), we find that single channel activation can be explained by a G protein-dependent shift in the equilibrium of open channel transitions in favor of a bursting state of channel activity over a long-lived closed state.

Animals↗

PubMed2004

Endocannabinoids modulate N-type calcium channels and G-protein-coupled inwardly rectifying potassium channels via CB1 cannabinoid receptors heterologously expressed in mammalian neurons.

Endocannabinoids may serve as retrograde messengers to inhibit neurotransmitter release during depolarization-induced suppression of inhibition (DSI) or excitation (DSE). We therefore tested whether endocannabinoids inhibit N-type voltage-dependent Ca2+ channels by activating G(i/o)-protein-coupled CB1 cannabinoid receptors (CB1R)--a possible mechanism underlying DSI/DSE. Three putative endocannabinoids [2-arachidonylglycerol (2-AG), 2-arachidonyl glycerol ether (2-AGE), and anandamide (AEA)] and the cannabimimetic aminoalkylindole WIN 55,212-2 (WIN) inhibited whole-cell Ca2+ currents in rat sympathetic neurons previously injected with cDNA encoding a human CB1R. Agonist-mediated Ca2+ current inhibition was blocked by a selective CB1R antagonist [SR141716A, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride] and pertussis toxin (PTX) pretreatment. The rank order of potency was WIN (IC50=2 nM)>2-AGE (350 nM) approximately 2-AG (480 nM)>AEA (approximately 3 microM), with each agonist displaying similar efficacy (approximately 50% maximal inhibition). Increasing CB1R expression level significantly enhanced AEA potency. AEA (10 microM) also inhibited Ca2+ channels in a voltage-independent, CB1R-independent, and PTX-insensitive manner, whereas 2-AG and 2-AGE were devoid of this activity. All three endocannabinoids activated G-protein-coupled inwardly rectifying potassium (GIRK) channels, GIRK1/4, heterologously expressed in sympathetic neurons. These results suggest a mechanism by which endocannibinoids might influence presynaptic function.

Animals↗

PubMed2002

Characterization of Dir: a putative potassium inward rectifying channel in Drosophila.

Potassium channels vary in their function and regulation, yet they maintain a number of important features - they are involved in the control of potassium flow, cell volume, cell membrane resting potential, cell excitability and hormone release. The potassium (K(+)) inward rectifier (Kir) superfamily of channels are potassium selective channels, that are sensitive to the concentration of K(+) ions. They are termed inward rectifiers since they allow a much greater K(+) influx than efflux. There are at least seven subfamilies of Kir channels, grouped according to sequence and functional similarities (Curr. Opin. Neurobiol. 5 (1995) 268; Annu. Rev. Physiol. 59 (1997) 171). While numerous Kir channels have been discovered in a variety of organisms, Drosophila inward rectifier (Dir) is the first putative inward rectifier to be studied in Drosophila. In fact, there are only three genes (including Dir) encoding putative inward rectifiers in the Drosophila genome. Though there are other known potassium channels in Drosophila such as ether-a-go-go and shaker, most are voltage-gated channels. As an important first step in characterizing Kir channels in Drosophila, we initiated studies on Dir.

Amino Acid Sequence↗

PubMed2000

Functional evidence for inward-rectifier potassium channels in rat cremaster muscle arterioles.

Moderate increases in extracellular K(+) produce vasodilation in fourth order cremasteric arterioles in the anesthetized rat. We studied the contribution of different subtypes of K(+) channels to this response. Cremaster muscle arteriolar diameters were observed during superfusion with buffer containing 5-30 mM K(+) in the absence (control) and presence of barium (Ba(2+), 50 microM), glibenclamide (GLIB, 1 microM), or iberiotoxin (IBTX, 100 nM) to block inward-rectifier, ATP-sensitive, or Ca(2+)-activated K(+) channels, respectively. Under control conditions, vessels dilated in response to 10-25 mM K(+) and constricted at higher concentrations. At 5 mM K(+), vessel diameters were significantly decreased by GLIB and Ba(2+), but not IBTX, suggesting that basal diameter was regulated by inward-rectifier and ATP-sensitive K(+) channels. In contrast, Ba(2+), but not GLIB or IBTX, prevented K(+)-induced dilation. The data indicate that the inward-rectifier K(+) channel (blocked by low concentrations of Ba(2+), but not GLIB or IBTX) was most likely responsible for the K(+)-induced arteriolar dilation.

Adenosine↗