G protein-mediated inhibition of inwardly rectifying K+ channels in guinea pig chromaffin cells

Properties of inwardly directed rectification and its G protein-mediated inhibition in guinea pig chromaffin cells were studied using the whole cell version of the patch-clamp technique. The current-voltage (I-V) relationship for plateau currents in response to a 50-ms pulse showed an inwardly directed rectification between -80 and -140 mV and a negative slope at more negative potentials in normal solution. Replacement of Na+ with N-methyl-D-glucamine (NMDG) in the perfusate did not alter the plateau I-V relationship between -110 and -130 mV but did abolish the negative slope below -140 mV. The zero current or resting membrane potential in the NMDG solution was in fair agreement with the equilibrium potential for K+. The chord conductance-voltage relationship showed a good fit with the Boltzmann equation and shifted along the voltage axis by an approximate change in driving force on K+ when K+ concentration was increased. External Cs+ and Ba2+ produced a voltage-dependent inhibition of the inwardly directed rectification. These results indicate that inwardly rectifying (IR) K+ channels are mediating an inwardly directed rectification. Intracellular dialysis with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) produced a complete suppression of this IR K+ channel, irrespective of treatment with pertussis toxin. Adding GTP or guanosine 5'-O-(2-thiodiphosphate) to the patch solution resulted in a decrease in GTP gamma S inhibition of the K+ current. Internal application of vanadate was without effect. Time course of the inhibition of the IR K+ current coincided in part with that of inactivation of a nonselective cation current. In conclusion, IR K+ channels in the chromaffin cell are subject to G protein-mediated inhibition.

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Phosphatase is responsible for run down, and probably G protein-mediated inhibition of inwardly rectifying K+ currents in guinea pig chromaffin cells.

The Journal of General Physiology ◽

10.1085/jgp.105.2.249 ◽

1995 ◽

Vol 105 (2) ◽

pp. 249-266 ◽

Cited By ~ 17

Author(s):

M Inoue ◽

I Imanaga

Keyword(s):

G Protein ◽

Chromaffin Cells ◽

Kinase Inhibitor ◽

Specific Inhibitor ◽

Muscarinic Agonist ◽

Calyculin A ◽

Patch Clamp Technique ◽

Inward Current ◽

Gamma S ◽

Inwardly Rectifying

The mechanism of G protein-mediated inhibition of an inwardly rectifying K+ current (IIR) in adrenal chromaffin cells was investigated using the whole-cell version of the patch clamp technique. In case of recording with use of ATP-containing patch solution, the IIR was well maintained; otherwise, it ran down within 15 min. This run down was not prevented by replacement with adenylyl-imidodiphosphate, a nonhydrolysable analogue of ATP, but was markedly reduced by the addition to the ATP-free solution of 1 microM calyculin A, a specific inhibitor of serine/threonine phosphatase 1 (PP1) and 2A (PP2A). The addition of alkaline phosphatase to the ATP-containing solution facilitated run down of the current, and application of 100 microM H-7, a general kinase inhibitor, reversibly suppressed IIR. These results taken together suggest that inwardly rectifying K+ channels are under the influence of kinase and phosphatase without external signals. Infusion of nonhydrolysable analogues of GTP, guanosine-5'-O-(3-thiophosphate) (GTP gamma S) or guanylyl-imidodiphosphate, through the pipette produced little inward current at -55 mV, but completely inhibited IIR within approximately 5 or 6 min in all cells tested in the presence of 12 microM Mg2+ inside the cell. In contrast, infusion of aluminum fluoride (AlF) complex, another GTP binding (G) protein activator, consistently produced large inward currents, but did not alter IIR noticeably for 15 min in 17% of the cells tested. In the other cells, the inhibition of IIR developed slowly after long latent periods. This inhibitory potency of AlF was not enhanced by an increase in Mg2+ concentrations. Subtraction of the current-voltage relationship before from that noted during the generation of inward current by AlF complex revealed that the inward current diminished progressively with hyperpolarizations, as is the case with a nonselective cation current (INS) induced by a muscarinic agonist. Thus, AlF complex seems to be potent with the generation of INS, but not with IIR inhibition. The addition of 3 microM calyculin A significantly retarded the IIR inhibition by GTP gamma S, whereas that of 1 microM okadaic acid, another inhibitor of PPI and PP2A, markedly prevented the decline of IIR by AIF complex. Our observations suggest that the low potency of AlF complex in inhibiting IIR may be due to interference with phosphatase activity and that the activation of G protein suppresses IIR, probably by enhancing the apparent activity of phosphatase, which may explain run down of the current.

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Involvement of A pertussis Toxin Sensitive G-Protein in the Inhibition of Inwardly Rectifying K+Currents by Platelet-Activating Factor in Guinea-Pig Atrial Cardiomyocytes

Mediators of Inflammation ◽

10.1155/s0962935194000086 ◽

1994 ◽

Vol 3 (1) ◽

pp. 45-51

Author(s):

M. Gollasch ◽

T. Kleppisch ◽

D. Krautwurst ◽

D. Lewinsohn ◽

J. Hescheler

Keyword(s):

Cyclic Amp ◽

Guinea Pig ◽

G Protein ◽

Pertussis Toxin ◽

Platelet Activating Factor ◽

Resting Membrane Potential ◽

Patch Clamp Technique ◽

Guinea Pig Heart ◽

Ventricular Cells ◽

Inwardly Rectifying

Platelet-activating factor (PAF) inhibits single inwardly rectifying K+channels in guinea-pig ventricular cells. There is currently little information as to the mechanism by which these channels are modulated. The effect of PAF on quasi steady-state inwardly rectifying K+currents (presumably of the IK1type) of auricular, atrial and ventricular cardiomyocytes from guinea-pig were studied. Applying the patch-clamp technique in the whole-cell configuration, PAF (10 nM) reduced the K+currents in all three cell types. The inhibitory effect of PAF occurred within seconds and was reversible upon wash-out. It was almost completely abolished by the PAF receptor antagonist BN 50730. Intracellular infusion of atrial cells with guanine 5′-(β-thio)diphosphate (GDPS) or pretreatment of cells with pertussis toxin abolished the PAF dependent reduction of the currents. Neither extracellularly applied isoproterenol nor intracellularly applied adenosine 3′,5′-cyclic monophosphate (cyclic AMP) attenuated the PAF effect. In multicellular preparations of auricles, PAF (10 nM) induced arrhythmias. The arrhythmogenic activity was also reduced by BN 50730. The data indicate that activated PAF receptors inhibit inwardly rectifying K+currents via a pertussis toxin sensitive G-protein without involvement of a cyclic AMP-dependent step. Since IK1is a major component in stabilizing the resting membrane potential, the observed inhibition of this type of channel could play an important role in PAF dependent arrhythmogenesis in guinea-pig heart.

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Lamellipod extension and K+ current in osteoclasts are regulated by different types of G proteins

Journal of Cell Science ◽

10.1242/jcs.107.2.517 ◽

1994 ◽

Vol 107 (2) ◽

pp. 517-526 ◽

Cited By ~ 1

Author(s):

S.A. Arkett ◽

S.J. Dixon ◽

S.M. Sims

Keyword(s):

Molecular Mass ◽

G Protein ◽

G Proteins ◽

Actin Polymerization ◽

Complete Suppression ◽

Patch Clamp Technique ◽

Membrane Area ◽

Whole Cell ◽

K Current ◽

Inwardly Rectifying

Osteoclasts are the cells responsible for the resorption of bone and other mineralized tissues. GTP-binding proteins (G proteins) play important roles in regulating the activity of many cell types; however, there is limited knowledge of their functions in osteoclasts. We used the patch-clamp technique in the whole-cell configuration to introduce either hydrolysis-resistant guanosine triphosphate analogues or fluoroaluminate into single rat osteoclasts, and examined the effects of G protein activation on cell morphology and ionic conductances. Guanosine 5′-O-(3-thiotriphosphate) or 5′-guanylyl-imidodiphosphate, but not the control compounds adenosine 5′-O-(3-thiotriphosphate) or guanosine 5′-O-(2-thiodiphosphate), induced: (1) prompt spreading due to extension of lamellipodia; and (2) after a latency of several minutes, complete suppression of the inwardly rectifying K+ current. Pertussis toxin did not alter either spreading or suppression of K+ current induced by guanosine 5′-O-(3-thiotriphosphate). Cytochalasin D, but not colchicine, prevented guanosine 5′-O-(3-thiotriphosphate)-induced spreading, consistent with actin polymerization underlying lamellipod extension. Whole-cell capacitance did not change during guanosine 5′-O-(3-thiotriphosphate)-induced spreading, which is consistent with a lack of change in total plasma membrane area. Fluoroaluminate did not induce spreading, but it did suppress the K+ current. The differential effects of fluoroaluminate and guanosine 5′-O-(3-thiotriphosphate) suggest that lamellipod extension is regulated by a small molecular mass, monomeric G protein, whereas the inwardly rectifying K+ current is regulated by a large molecular mass, heterotrimeric G protein. Thus, osteoclast motility and ion transport are regulated by separate G protein-coupled pathways.

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On the mechanism of basal and agonist-induced activation of the G protein-gated muscarinic K+ channel in atrial myocytes of guinea pig heart.

The Journal of General Physiology ◽

10.1085/jgp.98.3.517 ◽

1991 ◽

Vol 98 (3) ◽

pp. 517-533 ◽

Cited By ~ 53

Author(s):

H Ito ◽

T Sugimoto ◽

I Kobayashi ◽

K Takahashi ◽

T Katada ◽

...

Keyword(s):

Guinea Pig ◽

G Protein ◽

K Channel ◽

Dependent Manner ◽

Atrial Myocytes ◽

Patch Clamp Technique ◽

Open Probability ◽

Concentration Dependent Manner ◽

Guinea Pig Heart ◽

Gamma S

Using the patch clamp technique, we examined the agonist-free, basal interaction between the muscarinic acetylcholine (m-ACh) receptor and the G protein (GK)-gated muscarinic K+ channel (IK.ACh), and the modification of this interaction by ACh binding to the receptor in single atrial myocytes of guinea pig heart. In the whole cell clamp mode, guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma S) gradually increased the IK.ACh current in the absence of agonists (e.g., acetylcholine). This increase was inhibited in cells that were pretreated with islet-activating protein (IAP, pertussis toxin) or N-ethylmaleimide (NEM). In inside-out patches, even in the absence of agonists, intracellular GTP caused openings of IK.ACh in a concentration-dependent manner in approximately 80% of the patches. Channel activation by GTP in the absence of agonist was much less than that caused by GTP-gamma S. The agonist-independent, GTP-induced activation of IK.ACh was inhibited by the A promoter of IAP (with nicotinamide adenine dinucleotide) or NEM. As the ACh concentration was increased, the GTP-induced maximal open probability of IK.ACh was increased and the GTP concentration for the half-maximal activation of IK.ACh was decreased. Intracellular GDP inhibited the GTP-induced openings of IK.ACh in a concentration-dependent fashion. The half-inhibition of IK.ACh openings occurred at a much lower concentration of GDP in the absence of agonists than in the presence of ACh. From these results, we concluded (a) that the interaction between the m-ACh receptor and GK is essential for basal stimulation of IK.ACh, and (b) that ACh binding to the receptor accelerates the turnover of GK and increases GK's affinity to GTP analogues over GDP.

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G protein control of potassium channel activity in a mast cell line.

The Journal of General Physiology ◽

10.1085/jgp.95.2.205 ◽

1990 ◽

Vol 95 (2) ◽

pp. 205-227 ◽

Cited By ~ 50

Author(s):

M A McCloskey ◽

M D Cahalan

Keyword(s):

Cell Line ◽

G Protein ◽

Pertussis Toxin ◽

Inward Rectifier ◽

K Channel ◽

Half Time ◽

Patch Clamp Technique ◽

Whole Cell ◽

K Channels ◽

Gamma S

Using the patch-clamp technique, we studied regulation of potassium channels by G protein activators in the histamine-secreting rat basophilic leukemia (RBL-2H3) cell line. These cells normally express inward rectifier K+ channels, with a macroscopic whole-cell conductance in normal Ringer ranging from 1 to 16 nS/cell. This conductance is stabilized by including ATP or GTP in the pipette solution. Intracellular dialysis with any of three different activators of G proteins (GTP gamma S, GppNHp, or AlF-4) completely inhibited the inward rectifier K+ conductance with a half-time for decline averaging approximately 300 s after "break-in" to achieve whole-cell recording. In addition, with a half-time averaging approximately 200 s, G protein activators induced the appearance of a novel time-independent outwardly rectifying K+ conductance, which reached a maximum of 1-14 nS. The induced K+ channels are distinct from inward rectifier channels, having a smaller single-channel conductance of approximately 8 pS in symmetrical 160 mM K+, and being more sensitive to block by quinidine, but less sensitive to block by Ba2+. The induced K+ channels were also highly permeable to Rb+ but not to Na+ or Cs+. The current was not activated by the second messengers Ca2+, inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, or by cyclic AMP-dependent phosphorylation. Pretreatment of cells with pertussis toxin (0.1 microgram/ml for 12-13 h) prevented this current's induction both by guanine nucleotides and aluminum fluoride, but had no effect on the decrease in inward rectifier conductance. Since GTP gamma S is known to stimulate secretion from patch-clamped rat peritoneal mast cells, it is conceivable that K+ channels become inserted into the plasma membrane from secretory granules. However, total membrane capacitance remained nearly constant during appearance of the K+ channels, suggesting that secretion induced by GTP gamma S was minimal. Furthermore, pertussis toxin had no effect on secretion triggered by antigen, and triggering of secretion before electrical recording failed to induce the outward K+ current. Finally, GTP gamma S activated the K+ channel in excised inside-out patches of membrane. We conclude that two different GTP-binding proteins differentially regulate two subsets of K+ channels, causing the inward rectifier to close and a novel K+ channel to open when activated.

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K+ and Cl- contribute to resting membrane conductance of cultured porcine endocardial endothelial cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1997.272.4.h1770 ◽

1997 ◽

Vol 272 (4) ◽

pp. H1770-H1779 ◽

Cited By ~ 1

Author(s):

P. Fransen ◽

S. U. Sys

Keyword(s):

Endothelial Cells ◽

Membrane Conductance ◽

Flufenamic Acid ◽

Equilibrium Potential ◽

Channel Blockers ◽

Patch Clamp Technique ◽

K Channels ◽

Endocardial Endothelial Cells ◽

Inwardly Rectifying ◽

In Cells

The conventional whole cell patch-clamp technique was used to measure the resting membrane conductance and membrane currents of single, nonstimulated, cultured endocardial endothelial cells of the porcine right ventricle in different ionic conditions. All cells displayed the barium-sensitive, inwardly rectifying potassium (K+) current (I(Ki)). In 65% of the cells, I(Ki) was the predominant membrane current. The mean zero-current potential (V0) was -61.0 +/- 12.5 mV (+/- SD, n = 45). In 35% of the cells, I(Ki) was superposed on an outwardly rectifying (OR) current. V0 of these cells was more depolarized (-33.5 +/- 22.0 mV, n = 26). High intracellular Cl- (122 instead of 52 mmol/l) activated or increased the OR current and shifted V0 in the direction of the equilibrium potential for Cl-. In cells displaying the OR current, V0 was dependent on extracellular Cl-, indicating the contribution of an OR Cl- current in setting V0. At low intracellular Cl- (6 instead of 52 mmol/l), the OR current was decreased and V0 shifted in the direction of the equilibrium potential for K+. In cells not displaying the OR current, V0 was dependent on extracellular K+ but not on Cl-, indicating major permeability to K+ in these conditions. Block of the OR current by the Cl(-)-channel blockers anthracene-9-carboxylic acid (1 mmol/l), flufenamic acid (100-500 micromol/l), and Zn2+ (100-200 micromol/l) provided further evidence for the anionic nature of the OR current. After inhibition of I(Ki) and the OR Cl- current, a third current component was observed in 50% of the cells. The pharmacology and voltage dependence of this current suggested the presence of Ca2+-activated K+ channels in endocardial endothelial cells. We concluded that the resting membrane conductance of nonstimulated endocardial endothelial cells is mainly determined by the combined activity of inwardly rectifying K+, OR Cl-, and Ca2+-activated K+ channels.

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Regulation of Ca2+ current in frog ventricular cardiomyocytes by guanosine 5'-triphosphate analogues and isoproterenol.

The Journal of General Physiology ◽

10.1085/jgp.102.3.525 ◽

1993 ◽

Vol 102 (3) ◽

pp. 525-549 ◽

Cited By ~ 10

Author(s):

T D Parsons ◽

H C Hartzell

Keyword(s):

G Protein ◽

G Proteins ◽

Ventricular Myocytes ◽

Calcium Currents ◽

Internal Perfusion ◽

High Concentration ◽

Patch Clamp Technique ◽

Intact Cells ◽

Molecule Interactions ◽

Gamma S

Calcium currents (ICa) were measured in frog ventricular myocytes using the whole-cell patch clamp technique and a perfused pipette. To gain insight into the role of G proteins in the regulation of ICa in intact cells, the effect of internal perfusion with hydrolysis-resistant GTP analogues, guanylyl 5'-imidodiphosphate (GppNHp) or guanosine 5'-thiotriphosphate (GTP gamma S), on ICa stimulated by isoproterenol (Iso) or forskolin (Forsk) was examined. Significant differences were observed between the effects of the two GTP analogues. Internal perfusion of GppNHp resulted in a near-complete (approximately 80%) and irreversible inhibition of Iso-stimulated ICa. In contrast, internal perfusion with GTP gamma S resulted in only a partial (approximately 40%) inhibition of Iso- or Forsk-stimulated ICa. The fraction of the current not inhibited by GTP gamma S remained persistently elevated after the washout of Iso but declined to basal levels upon washout of Forsk. Excess internal GTP or GppNHp did not reduce the persistent ICa. Internal adenosine 5'-thiotriphosphate (ATP gamma S) mimicked the GTP gamma S-induced, persistent ICa. GppNHp sometimes induced a persistent ICa, but only if GppNHp was present at high concentration before Iso exposure. Inhibitors of protein kinase A inhibited both the GTP gamma S- and ATP gamma S-induced, persistent ICa. We conclude that: (a) GTP gamma S is less effective than GppNHp in inhibiting adenylyl cyclase (AC) via the inhibitory G protein, Gi; and (b) the persistent ICa results from a long-lived Gs-GTP gamma S complex that can activate AC in the absence of Iso. These results suggest that different hydrolysis-resistant nucleotide analogues may behave differently in activating G proteins and imply that the efficacy of G protein-effector molecule interactions can depend on the GTP analogue with which the G protein is activated.

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G proteins activate ionic conductances at multiple sites in T84 cells

AJP Cell Physiology ◽

10.1152/ajpcell.1997.272.4.c1222 ◽

1997 ◽

Vol 272 (4) ◽

pp. C1222-C1231

Author(s):

L. Izu ◽

M. Li ◽

R. DeMuro ◽

M. E. Duffey

Keyword(s):

G Protein ◽

G Proteins ◽

Cell Voltage ◽

Equilibrium Potential ◽

T84 Cells ◽

Ionic Conductances ◽

Inward Currents ◽

Cl Channels ◽

K Current

We examined the role of G proteins in activation of ionic conductances in isolated T84 cells during cholinergic stimulation. When cells were whole cell voltage clamped to the K+ equilibrium potential (E(K)) or Cl- equilibrium potential (E(Cl)) under standard conditions, the cholinergic agonist, carbachol, induced a large oscillating K+ current but only a small inward current. Addition of the GDP analogue, guanosine 5'-O-(2-thiodiphosphate), to pipettes blocked the ability of carbachol to activate the K+ current. Addition of the nonhydrolyzable GTP analogue, guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), to pipettes stimulated large oscillating K+ and inward currents. This occurred even when Ca2+ was absent from the bath but not when the Ca2+ chelator, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, was added to pipettes. When all pipette and bath K+ was replaced with Na+ and cells were voltage clamped between E(Na) and E(Cl), GTPgammaS activated oscillating Na+ and Cl- currents. Finally, addition of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] to pipettes activated large oscillating K+ currents but only small inward currents. These results suggest that a carbachol-induced release of Ca2+ from intracellular stores is activated by a G protein through the phospholipase C-Ins(1,4,5)P3 signaling pathway. In addition, this or another G protein activates Cl- current by directly gating Cl- channels to increase their sensitivity to Ca2+.

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