Gq/11 and PLC-beta 1 mediate the substance P-induced inhibition of an inward rectifier K+ channel in brain neurons

1996 ◽  
Vol 76 (3) ◽  
pp. 2131-2136 ◽  
Author(s):  
K. Takano ◽  
J. Yasufuku-Takano ◽  
T. Kozasa ◽  
W. D. Singer ◽  
S. Nakajima ◽  
...  
Keyword(s):  
Substance P ◽  
Single Channel ◽  
Cholinergic Neurons ◽  
Nucleus Basalis ◽  
Alpha Subunit ◽  
K Channel ◽  
Whole Cell Patch Clamp ◽  
K Current ◽  
Beta 2 ◽  
G Protein Alpha Subunit

1. Substance P (SP) induces a slow neuronal excitation in cholinergic neurons from the nucleus basalis by suppressing an inwardly rectifying K+ current (Kir). We have determined which G protein alpha-subunit mediates this SP effect. 2. After intracellularly injecting antibody against each alpha-subunit of G proteins (Gq alpha/11 alpha, G12 alpha, and G13 alpha) with an Eppendorf microinjector, we examined, by using the whole cell patch-clamp and the ON-cell mode of single-channel recording, the effect of SP on Kir in cultured neurons of the nucleus basalis. The effect of SP on Kir was substantially reduced in neurons injected with antibodies to Gq alpha/11 alpha but not with antibodies to G12 alpha or G13 alpha. 3. The effects of antibodies against three isozymes of phospholipase C (PLC-beta 1, PLC-beta 2, and PLC-beta 3) were tested. The SP-induced suppression of Kir was reduced by antibody against PLC-beta 1 but not by antibodies against PLC-beta 2 or PLC-beta 3. 4. We conclude that the SP-induced inhibition of Kir in nucleus basalis neurons is mediated by Gq/11 and PLC-beta 1.

scholarly journals Potassium channel types in arterial chemoreceptor cells and their selective modulation by oxygen.

1992 ◽  
Vol 100 (3) ◽  
pp. 401-426 ◽  
Author(s):  
M D Ganfornina ◽  
J López-Barneo
Keyword(s):  
Time Course ◽  
Single Channel ◽  
K Channel ◽  
Open Probability ◽  
K Channels ◽  
Glomus Cells ◽  
Control Value ◽  
Voltage Dependent ◽  
K Current ◽  
Chemoreceptor Cells

Single K+ channel currents were recorded in excised membrane patches from dispersed chemoreceptor cells of the rabbit carotid body under conditions that abolish current flow through Na+ and Ca2+ channels. We have found three classes of voltage-gated K+ channels that differ in their single-channel conductance (gamma), dependence on internal Ca2+ (Ca2+i), and sensitivity to changes in O2 tension (PO2). Ca(2+)-activated K+ channels (KCa channels) with gamma approximately 210 pS in symmetrical K+ solutions were observed when [Ca2+]i was greater than 0.1 microM. Small conductance channels with gamma = 16 pS were not affected by [Ca2+]i and they exhibited slow activation and inactivation time courses. In these two channel types open probability (P(open)) was unaffected when exposed to normoxic (PO2 = 140 mmHg) or hypoxic (PO2 approximately 5-10 mmHg) external solutions. A third channel type (referred to as KO2 channel), having an intermediate gamma(approximately 40 pS), was the most frequently recorded. KO2 channels are steeply voltage dependent and not affected by [Ca2+]i, they inactivate almost completely in less than 500 ms, and their P(open) reversibly decreases upon exposure to low PO2. The effect of low PO2 is voltage dependent, being more pronounced at moderately depolarized voltages. At 0 mV, for example, P(open) diminishes to approximately 40% of the control value. The time course of ensemble current averages of KO2 channels is remarkably similar to that of the O2-sensitive K+ current. In addition, ensemble average and macroscopic K+ currents are affected similarly by low PO2. These observations strongly suggest that KO2 channels are the main contributors to the macroscopic K+ current of glomus cells. The reversible inhibition of KO2 channel activity by low PO2 does not desensitize and is not related to the presence of F-, ATP, and GTP-gamma-S at the internal face of the membrane. These results indicate that KO2 channels confer upon glomus cells their unique chemoreceptor properties and that the O2-K+ channel interaction occurs either directly or through an O2 sensor intrinsic to the plasma membrane closely associated with the channel molecule.

Alpha-subunit of Gk activates atrial K+ channels of chick, rat, and guinea pig

1988 ◽  
Vol 254 (6) ◽  
pp. H1200-H1205 ◽  
Author(s):  
G. E. Kirsch ◽  
A. Yatani ◽  
J. Codina ◽  
L. Birnbaumer ◽  
A. M. Brown
Keyword(s):  
Guinea Pig ◽  
Single Channel ◽  
Neonatal Rat ◽  
Alpha Subunit ◽  
K Channel ◽  
Muscarinic Agonist ◽  
Guanine Nucleotide Binding Protein ◽  
K Channels ◽  
Open Time ◽  
Guanine Nucleotide Binding

A specific guanine nucleotide-binding protein, Gk, is the link by which muscarinic receptors activate atrial potassium channels (Science Wash. DC 235: 207-211, 1987). In adult guinea pigs, the alpha-subunit at picomolar concentrations mediates the holo-G protein effect (Science Wash. DC 236: 442-445, 1987), but in chick embryo it has been reported that the beta gamma-dimer at nanomolar concentrations rather than the alpha-subunit is the effective mediator (Nature Lond. 325: 321-326, 1987). This difference might have a phylogenetic or ontogenetic basis, and the present experiments tested these possibilities. Preactivated alpha k derived from human red blood cell Gk, when applied to the intracellular surface of inside-out membrane patches from the atria of embryonic chick, neonatal rat, and adult guinea pig activated single K+ channel currents. In each case, the alpha k-activated channels had the same single-channel conductance and mean open time as the muscarinic agonist-activated channels. Half-maximal activation was achieved at alpha k-concentrations of 2.4-13.8 pM. Hence, alpha k-activation of these K+ channels is independent of differences in age or species. The detergent 3-[3-cholamidopropyl)-dimethyammoniol]-1-propanesulfonate (CHAPS), which was used by Logothetis et al. (Nature Lond. 325: 321-326, 1987) at 184 microM to suspend the hydrophobic beta gamma-dimers, activated the same currents. We conclude that the effects of the beta gamma-dimer on these K+ channels is unknown and that as we had proposed earlier (Science Wash. DC 236: 442-445, 1987) it is the alpha-subunit that mediates the Gk effect.

Different properties of the atrial G protein-gated K+ channels activated by extracellular ATP and adenosine

1995 ◽  
Vol 269 (4) ◽  
pp. H1349-H1358 ◽  
Author(s):  
C. Fu ◽  
A. Pleumsamran ◽  
U. Oh ◽  
D. Kim
Keyword(s):  
G Protein ◽  
Single Channel ◽  
Extracellular Atp ◽  
K Channel ◽  
Affinity Constant ◽  
Channel Activity ◽  
K Channels ◽  
Atrial Cells ◽  
K Current ◽  
Inside Out

Extracellular ATP (ATPo) and adenosine activate G protein-gated inwardly rectifying K+ currents in atrial cells. Earlier studies have suggested that the two agonists may use separate pathways to activate the K+ current. Therefore, we examined whether the K+ channels activated by the two agonists have different properties under identical ionic conditions. In cell-attached patches, K+ channels activated by 100 microM ATP in the pipette had a single-channel conductance and mean open time of 32.0 +/- 0.2 pS and 0.5 +/- 0.1 ms, respectively, compared with 31.3 +/- 0.3 pS and 0.9 +/- 0.1 ms for the K+ channels activated by adenosine (140 mM KCl). With ATPo as the agonist, the K+ channel activity in cell-attached patches was approximately threefold lower than that in inside-out patches with 100 microM GTP in the bath. Applying ATP to the cytoplasmic side of the membrane (ATPi) produced a biphasic concentration-dependent effect on channel activity: an increase at low [mean affinity constant (K0.5) = 190 microM] and a decrease at high (K0.5 = 1.3 mM) concentrations. In contrast, with adenosine as the agonist, K+ channel activity in cell-attached patches was approximately fourfold greater than that in inside-out patches with 100 microM GTP in the bath. In inside-out patches, ATPi only augmented the K+ channel activity (K0.5 = 32 microM). These results show that although both ATPo and adenosine activate kinetically similar K+ channels in atrial cells, the channels are regulated differently by intracellular nucleotides.

Abstract 485: Enhanced Large Conductance Ca+2 -sensitive K+ Channels (bk) Activity Impairs the Myogenic Response in the Cerebral Vasculature of Fawn Hooded Hypertensive (fhh) Rat

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Mallikarjuna R Pabbidi ◽  
Jerry Farley ◽  
Debebe Gebremedhin ◽  
David R. Harder ◽  
Richard J. Roman
Keyword(s):  
Congenic Strain ◽  
Single Channel ◽  
Cerebral Arteries ◽  
Fold Increase ◽  
Bk Channel ◽  
Chromosome 1 ◽  
K Channel ◽  
Myogenic Response ◽  
Whole Cell Patch Clamp ◽  
Current Densities

Our recent studies have revealed that the myogenic response in cerebral arteries and autoregulation of cerebral blood flow is impaired in FHH and that transfer of a 2.4 Mb region of chromosome 1 from BN into FHH.1 BN congenic strain restores these responses. The present study examined the role of large conductance calcium activated potassium (BK) channel in altering the myogenic response in FHH rats. Whole-cell patch-clamp of cerebral vascular smooth muscle cells (VSMC) revealed a 4.6 fold increase in outward potassium (K) channel current densities (pA/pF) in FHH rats compared to FHH.1 BN congenic strain. Iberiotoxin (IBTX -a selective BK channel inhibitor) sensitive current densities are significantly greater in FHH rats compared with the FHH.1 BN congenic strain (FHH rats: +40mV; pre IBTX 43.1 ± 7.2, after IBTX 11.8 ± 2.2 pA/pF versus pre IBTX 5.6 ± 1 pA/pF and after IBTX 4.1 ± 0.6 pA/pF in the FHH.1 BN congenic strain). In excised patches, the BK channel exhibited similar single-channel slope conductance for FHH and the FHH.1 BN rats (208.9 pS versus 208.7 pS). However, the open channel probability (NP o ) was ~10 fold higher in FHH rats than in FHH.1 BN rats (1μM free (Ca +2 ) i : +40mV: FHH; 0.8 ± 0.04 versus 0.08 ± 0.004 in FHH.1 BN rats). Voltage and Ca 2+ sensitivity of the BK channel is similar in cerebral VSMC isolated from FHH and FHH.1 BN rats. Middle cerebral arterioles (MCA) isolated from FHH rats increased in diameter from 142 ± 16 to 157 ± 19 μm when pressure was increased from 40 to 140 mmHg. In contrast, the diameter of the MCA decreased by 49% in the FHH.1 BN congenic strain from 127 ± 16 to 65 ± 13 μm. Pharmacological block of BK channel by IBTX (100nM) restored myogenic response in FHH rats but had no effect in FHH.1 BN rats. These results indicate that the impaired myogenic response of the cerebral vessels in FHH rats is mediated via a gene and mechanism that enhances BK channel activity.

scholarly journals Orexin (Hypocretin) Effects on Constitutively Active Inward Rectifier K+ Channels in Cultured Nucleus Basalis Neurons

2004 ◽  
Vol 92 (6) ◽  
pp. 3183-3191 ◽  
Author(s):  
Q. V. Hoang ◽  
P. Zhao ◽  
S. Nakajima ◽  
Y. Nakajima
Keyword(s):  
G Protein ◽  
Single Channel ◽  
Cholinergic Neurons ◽  
Reversal Potential ◽  
Inward Rectifier ◽  
Nucleus Basalis ◽  
Acid Pretreatment ◽  
Girk Channels ◽  
Neuronal Excitation ◽  
Constitutively Active

Orexins are excitatory transmitters implicated in sleep disorders. Because orexins were discovered only recently, their ionic and signal transduction mechanisms have not been well clarified. We recently reported that orexin A (OXA) inhibits G protein–coupled inward rectifier K+ (GIRK) channels in cultured locus coeruleus and nucleus tuberomammillaris neurons. Other work in our laboratory revealed the existence of a novel inward rectifier K+ channel (KirNB), which is located in cholinergic neurons of the nucleus basalis (NB) and possesses unique single-channel characteristics. The mean open time is considerably shorter in KirNB than in Kir2.0 channels. Constitutive activity and a smaller unitary conductance set KirNB apart from cloned Kir3.0 channels. Previously, we found that substance P excites NB neurons by inhibiting KirNB channels. Here we show that orexins suppress KirNB channel activity, likely leading to neuronal excitation. Electrophysiological studies were performed on cultured NB neurons from the basal forebrain. OXA application decreased whole cell conductance through a pertussis toxin (PTX)-insensitive G protein. The OXA-suppressed current was inwardly rectifying with a reversal potential around EK. Single-channel recordings of NB neurons revealed that constitutively active KirNB channels were transiently inhibited by OXA. Okadaic acid pretreatment abolished the recovery. The results suggest that OXA inhibition of KirNB is mediated by a PTX-insensitive G protein (i.e., Gq/11), which eventually results in channel phosphorylation. Recovery from this inhibition is by dephosphorylation. These results, taken together with our previous study, suggest that orexin receptors can elicit neuronal excitation through at least two families of inward rectifier K+ channels: GIRK and KirNB channels.

Protein kinase-independent inhibition of muscarinic K+ channels by staurosporine

1994 ◽  
Vol 266 (4) ◽  
pp. C1128-C1132 ◽  
Author(s):  
C. F. Lo ◽  
G. E. Breitwieser
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Kinase Inhibitors ◽  
Single Channel ◽  
Protein Kinase Inhibitors ◽  
K Channel ◽  
K Current ◽  
Excised Patches ◽  
Dose Dependent Decrease ◽  
Gamma S

Acetylcholine (ACh) binding to atrial muscarinic receptors activates an inwardly rectifying K+ current (IK[ACh]) via a pertussis toxin-sensitive GTP-binding protein (GK). The muscarinic K+ channel (termed GIRK1) has been cloned, and the nucleotide sequence contains nine consensus sites for protein kinase C (PKC) phosphorylation (16). Dephosphorylation of the muscarinic K+ channel has been implicated in rapid IK[ACh] desensitization in the presence of agonist (13). Staurosporine is a widely used membrane-permeant inhibitor of PKC and other protein kinases (7), including G protein-coupled receptor kinases. We investigated the role of phosphorylation in the regulation of IK[ACh] by examining the effect of a variety of protein kinase inhibitors. Staurosporine produced a rapid and reversible dose-dependent decrease in IK[ACh], activated by either GTP or guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). Other PKC inhibitors, including calphostin C and K-252b, were without effect on GTP gamma S-activated IK[ACh]. In excised patches of atrial membrane under nonphosphorylating conditions (0 ATP, 1 mM 5'-adenylylimidodiphosphate), staurosporine reversibly reduced muscarinic K+ channel activity without altering single-channel current amplitude. These results suggest that staurosporine inhibits IK[ACh] by a mechanism independent of intracellular protein kinases.

Development of inwardly rectifying K+ channel family in rat ventricular myocytes

1997 ◽  
Vol 272 (4) ◽  
pp. H1741-H1750 ◽  
Author(s):  
L. H. Xie ◽  
M. Takano ◽  
A. Noma
Keyword(s):  
Single Channel ◽  
Ventricular Myocytes ◽  
K Channel ◽  
Resting Membrane Potential ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Rat Ventricular Myocytes ◽  
Adult Rat ◽  
K Current ◽  
Inwardly Rectifying

The ATP-sensitive K+ current (I(K,ATP)), the inward rectifier K+ current (I(K1)), and the acetylcholine-activated K+ current (I(K,ACh)) were recorded in fetal, neonatal, and adult rat ventricular myocytes using the patch-clamp technique. The density (pA/pF) of I(K1) increased from gestation day 10 through neonatal day 1 and then decreased after neonatal day 30. The density of I(K,ATP) activated maximally by metabolic inhibition changed in parallel with the I(K1) density, and the density of I(K,ATP) channel distribution was 1.3 times higher than that of I(K1) throughout the development. We failed to observe developmental changes in the single-channel conductance and the mean open time of I(K1) and I(K,ATP) channels. However, the open probability of the I(K,ATP) channel was lower in fetuses, and the sensitivity to ATP was highest in 1-day neonates. I(K,ACh) were present in the ventricle at all stages of development but at a much lower density than in atrium. The relationship between the resting membrane potential and the development of the inwardly rectifying K-channel family is discussed.

ATP-dependent regulation of a G protein-coupled K+ channel (GIRK1/GIRK4) expressed in oocytes

1997 ◽  
Vol 272 (1) ◽  
pp. H195-H206
Author(s):  
D. Kim ◽  
M. Watson ◽  
V. Indyk
Keyword(s):  
G Protein ◽  
Single Channel ◽  
K Channel ◽  
Time Duration ◽  
Cytosolic Protein ◽  
Open Time ◽  
Cytosolic Extract ◽  
K Current ◽  
G Protein Coupled ◽  
Inside Out

Recent studies suggest that activation of the atrial muscarinic K+ current by acetylcholine (ACh) involves an ATP-dependent process that is then inhibited by a cytosolic protein to result in the rapid desensitization. To obtain further evidence in support of such a dually regulated process, we studied the properties of GIRK1 and GIRK4, which, when coexpressed in oocytes, form a heteromultimer that closely resembles the muscarinic K+ channel. ACh activated an inwardly rectifying K+ current that desensitized slowly. In cell-attached patches with ACh in the pipette, the mean open times (tau zero) of GIRK1/GIRK4 were 1.2 +/- 0.1 (28%) and 6.7 +/- 0.8 ms (72%) and did not change significantly with time. However, in inside-out patches, the tau zero of GIRK1/GIRK4 activated with guanosine 5'-O-(3-thiotriphosphate) was 1.3 +/- 0.1 ms (100%), and the channel activity (NP0) was almost fivefold lower. These changes in channel kinetics did not occur in the presence of sodium orthovanadate (3 mM), an inhibitor of phosphatase. Addition of 1 mM ATP, but not adenylimidodiphosphate, to inside-out patches resulted in increases in NP zero (4.8-fold) and the open-time duration of GIRK1/GIRK4, such that tau zero were 1.2 +/- 0.2 (32%) and 6.2 +/- 0.6 ms (68%). Single channel conductances were unchanged (34 +/- 1 pS). Cytosolic extract from atria, but not oocytes, could reverse these effects of ATP. These results provide further evidence that the antagonistic modulation of G protein-gated K+ channels by ATP and the atrial cytosolic protein produces the early rapid desensitization in atrial cells. In oocytes the ATP-dependent step is dominant and thus provides a major component of the total GIRK1/GIRK4 current activated by ACh.

Voltage dependence of the Ca2+-activated K+channel KCa3.1 in human erythroleukemia cells

2013 ◽  
Vol 304 (9) ◽  
pp. C858-C872 ◽  
Author(s):  
Colin J. Stoneking ◽  
Oshini Shivakumar ◽  
David Nicholson Thomas ◽  
William H. Colledge ◽  
Michael J. Mason
Keyword(s):  
Voltage Dependence ◽  
Single Channel ◽  
K Channel ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Erythroleukemia Cells ◽  
Voltage Dependent ◽  
Hel Cells ◽  
Cell Current ◽  
Whole Cell Current

We have isolated a K+-selective, Ca2+-dependent whole cell current and single-channel correlate in the human erythroleukemia (HEL) cell line. The whole cell current was inhibited by the intermediate-conductance KCa3.1 inhibitors clotrimazole, TRAM-34, and charybdotoxin, unaffected by the small-conductance KCa2 family inhibitor apamin and the large-conductance KCa1.1 inhibitors paxilline and iberiotoxin, and augmented by NS309. The single-channel correlate of the whole cell current was blocked by TRAM-34 and clotrimazole, insensitive to paxilline, and augmented by NS309 and had a single-channel conductance in physiological K+gradients of ∼9 pS. RT-PCR revealed that the KCa3.1 gene, but not the KCa1.1 gene, was expressed in HEL cells. The KCa3.1 current, isolated in HEL cells under whole cell patch-clamp conditions, displayed an activated current component during depolarizing voltage steps from hyperpolarized holding potentials and tail currents upon repolarization, consistent with voltage-dependent modulation. This activated current increased with increasing voltage steps above −40 mV and was sensitive to inhibition by clotrimazole, TRAM-34, and charybdotoxin and insensitive to apamin, paxilline, and iberiotoxin. In single-channel experiments, depolarization resulted in an increase in open channel probability ( Po) of KCa3.1, with no increase in channel number. The voltage modulation of Powas an increasing monotonic function of voltage. In the absence of elevated Ca2+, voltage was ineffective at inducing channel activity in whole cell and single-channel experiments. These data indicate that KCa3.1 in HEL cells displays a unique form of voltage dependence modulating Po.

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