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Synergistic activation of G protein-gated inwardly rectifying potassium channels by the betagamma subunits of G proteins and Na(+) and Mg(2+) ions.

Petit-Jacques J, Sui JL, Logothetis DE - J. Gen. Physiol. (1999)

Bottom Line: Native and recombinant G protein-gated inwardly rectifying potassium (GIRK) channels are directly activated by the betagamma subunits of GTP-binding (G) proteins.The presence of phosphatidylinositol-bis-phosphate (PIP(2)) is required for G protein activation.At high levels of PIP(2), synergistic interactions among Na(+), Mg(2+), and G(betagamma) subunits resulted in severalfold stimulated levels of channel activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Mount Sinai School of Medicine of the New York University, New York, New York 10029, USA.

ABSTRACT
Native and recombinant G protein-gated inwardly rectifying potassium (GIRK) channels are directly activated by the betagamma subunits of GTP-binding (G) proteins. The presence of phosphatidylinositol-bis-phosphate (PIP(2)) is required for G protein activation. Formation (via hydrolysis of ATP) of endogenous PIP(2) or application of exogenous PIP(2) increases the mean open time of GIRK channels and sensitizes them to gating by internal Na(+) ions. In the present study, we show that the activity of ATP- or PIP(2)-modified channels could also be stimulated by intracellular Mg(2+) ions. In addition, Mg(2+) ions reduced the single-channel conductance of GIRK channels, independently of their gating ability. Both Na(+) and Mg(2+) ions exert their gating effects independently of each other or of the activation by the G(betagamma) subunits. At high levels of PIP(2), synergistic interactions among Na(+), Mg(2+), and G(betagamma) subunits resulted in severalfold stimulated levels of channel activity. Changes in ionic concentrations and/or G protein subunits in the local environment of these K(+) channels could provide a rapid amplification mechanism for generation of graded activity, thereby adjusting the level of excitability of the cells.

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Impairment of G protein signaling does not affect the activation of KACh by MgATP and Na+. (A) Single channel activity (top; NPo, bin = 5 s) plotted as a function of time. The data were obtained from an inside-out patch excised from an atrial cell. The KACh channel was stimulated by maintaining the membrane at −80 mV and by the presence of 5 μM acetylcholine in the pipette. 10 μM GTPγS, 50 μM QEHA, and 5/20 mM MgATP/Na+ were applied for the duration indicated by the bars. Sample single-channel currents in each condition at the time marked by the arrows are shown under the plot (bottom). (B) NPo plot of KACh channel activity (top, bin = 5 s) in an inside-out patch from an oocyte expressing the human GIRK1/GIRK4 and the construct βARK-PH. The membrane was clamped at −80 mV and 5 μM acetylcholine was present in the pipette. Application of 10 μM GTPγS and 5/20 mM MgATP/Na+ are illustrated by the bars. Labeled arrows correspond to the sample single-channel currents shown under the plot (bottom).
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Figure 1: Impairment of G protein signaling does not affect the activation of KACh by MgATP and Na+. (A) Single channel activity (top; NPo, bin = 5 s) plotted as a function of time. The data were obtained from an inside-out patch excised from an atrial cell. The KACh channel was stimulated by maintaining the membrane at −80 mV and by the presence of 5 μM acetylcholine in the pipette. 10 μM GTPγS, 50 μM QEHA, and 5/20 mM MgATP/Na+ were applied for the duration indicated by the bars. Sample single-channel currents in each condition at the time marked by the arrows are shown under the plot (bottom). (B) NPo plot of KACh channel activity (top, bin = 5 s) in an inside-out patch from an oocyte expressing the human GIRK1/GIRK4 and the construct βARK-PH. The membrane was clamped at −80 mV and 5 μM acetylcholine was present in the pipette. Application of 10 μM GTPγS and 5/20 mM MgATP/Na+ are illustrated by the bars. Labeled arrows correspond to the sample single-channel currents shown under the plot (bottom).

Mentions: To further test for a dependence of the MgATP/Na+ activation on G protein gating of KAch, we designed experiments where G protein–dependent activation of the channel was impaired. As shown in Fig. 1 A, the KACh channel in an inside-out atrial myocyte patch was activated persistently by 10 μM GTPγS, a nonhydrolyzable analogue of GTP. Activation of the channel by GTPγS was blocked upon perfusion of the QEHA peptide. QEHA is a 27 amino acid long peptide derived from the COOH terminus of the Gβγ-sensitive adenylate cyclase 2 isoform. It has been shown to block Gβγ activation of several different effectors, including the KACh channel (Chen et al. 1995). QEHA (50 μM) application abolished the GTPγS activation of KACh in <2 min (n = 3). After washout, the channel activity remained very low, suggesting the persistence of the QEHA-blocking effect. However, under these conditions, the KACh channel could be activated by MgATP/Na+ (5/20 mM). QEHA coapplication with MgATP/Na+ failed to block channel activation, whereas QEHA did block GTPγS-induced activation in the same oocyte patches (n = 3) (data not shown).


Synergistic activation of G protein-gated inwardly rectifying potassium channels by the betagamma subunits of G proteins and Na(+) and Mg(2+) ions.

Petit-Jacques J, Sui JL, Logothetis DE - J. Gen. Physiol. (1999)

Impairment of G protein signaling does not affect the activation of KACh by MgATP and Na+. (A) Single channel activity (top; NPo, bin = 5 s) plotted as a function of time. The data were obtained from an inside-out patch excised from an atrial cell. The KACh channel was stimulated by maintaining the membrane at −80 mV and by the presence of 5 μM acetylcholine in the pipette. 10 μM GTPγS, 50 μM QEHA, and 5/20 mM MgATP/Na+ were applied for the duration indicated by the bars. Sample single-channel currents in each condition at the time marked by the arrows are shown under the plot (bottom). (B) NPo plot of KACh channel activity (top, bin = 5 s) in an inside-out patch from an oocyte expressing the human GIRK1/GIRK4 and the construct βARK-PH. The membrane was clamped at −80 mV and 5 μM acetylcholine was present in the pipette. Application of 10 μM GTPγS and 5/20 mM MgATP/Na+ are illustrated by the bars. Labeled arrows correspond to the sample single-channel currents shown under the plot (bottom).
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Related In: Results  -  Collection

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Figure 1: Impairment of G protein signaling does not affect the activation of KACh by MgATP and Na+. (A) Single channel activity (top; NPo, bin = 5 s) plotted as a function of time. The data were obtained from an inside-out patch excised from an atrial cell. The KACh channel was stimulated by maintaining the membrane at −80 mV and by the presence of 5 μM acetylcholine in the pipette. 10 μM GTPγS, 50 μM QEHA, and 5/20 mM MgATP/Na+ were applied for the duration indicated by the bars. Sample single-channel currents in each condition at the time marked by the arrows are shown under the plot (bottom). (B) NPo plot of KACh channel activity (top, bin = 5 s) in an inside-out patch from an oocyte expressing the human GIRK1/GIRK4 and the construct βARK-PH. The membrane was clamped at −80 mV and 5 μM acetylcholine was present in the pipette. Application of 10 μM GTPγS and 5/20 mM MgATP/Na+ are illustrated by the bars. Labeled arrows correspond to the sample single-channel currents shown under the plot (bottom).
Mentions: To further test for a dependence of the MgATP/Na+ activation on G protein gating of KAch, we designed experiments where G protein–dependent activation of the channel was impaired. As shown in Fig. 1 A, the KACh channel in an inside-out atrial myocyte patch was activated persistently by 10 μM GTPγS, a nonhydrolyzable analogue of GTP. Activation of the channel by GTPγS was blocked upon perfusion of the QEHA peptide. QEHA is a 27 amino acid long peptide derived from the COOH terminus of the Gβγ-sensitive adenylate cyclase 2 isoform. It has been shown to block Gβγ activation of several different effectors, including the KACh channel (Chen et al. 1995). QEHA (50 μM) application abolished the GTPγS activation of KACh in <2 min (n = 3). After washout, the channel activity remained very low, suggesting the persistence of the QEHA-blocking effect. However, under these conditions, the KACh channel could be activated by MgATP/Na+ (5/20 mM). QEHA coapplication with MgATP/Na+ failed to block channel activation, whereas QEHA did block GTPγS-induced activation in the same oocyte patches (n = 3) (data not shown).

Bottom Line: Native and recombinant G protein-gated inwardly rectifying potassium (GIRK) channels are directly activated by the betagamma subunits of GTP-binding (G) proteins.The presence of phosphatidylinositol-bis-phosphate (PIP(2)) is required for G protein activation.At high levels of PIP(2), synergistic interactions among Na(+), Mg(2+), and G(betagamma) subunits resulted in severalfold stimulated levels of channel activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Mount Sinai School of Medicine of the New York University, New York, New York 10029, USA.

ABSTRACT
Native and recombinant G protein-gated inwardly rectifying potassium (GIRK) channels are directly activated by the betagamma subunits of GTP-binding (G) proteins. The presence of phosphatidylinositol-bis-phosphate (PIP(2)) is required for G protein activation. Formation (via hydrolysis of ATP) of endogenous PIP(2) or application of exogenous PIP(2) increases the mean open time of GIRK channels and sensitizes them to gating by internal Na(+) ions. In the present study, we show that the activity of ATP- or PIP(2)-modified channels could also be stimulated by intracellular Mg(2+) ions. In addition, Mg(2+) ions reduced the single-channel conductance of GIRK channels, independently of their gating ability. Both Na(+) and Mg(2+) ions exert their gating effects independently of each other or of the activation by the G(betagamma) subunits. At high levels of PIP(2), synergistic interactions among Na(+), Mg(2+), and G(betagamma) subunits resulted in severalfold stimulated levels of channel activity. Changes in ionic concentrations and/or G protein subunits in the local environment of these K(+) channels could provide a rapid amplification mechanism for generation of graded activity, thereby adjusting the level of excitability of the cells.

Show MeSH
Related in: MedlinePlus