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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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Na+ ions gate GIRK channels after activation by G protein βγ subunits. (A) Single-channel activity (NPo, bin = 5 s) plotted as a function of time. The data were obtained from an inside-out patch excised from an oocyte expressing the recombinant channel GIRK1/GIRK4. 20 mM Na+ and 10 μM GTPγS were applied as indicated by the bars. The membrane was clamped at −80 mV and 5 μM acetylcholine was in the pipette solution. (B) The mean NPo for seven patches is plotted for different conditions. Steady state channel activity after activation by GTPγS was taken as reference (GTPγS) and NPo were normalized to it. Na+ concentration was 20 mM and GTPγS was 10 μM. GTPγS+Na+ corresponds to the application of 20 mM Na+ after the washout of the GTP analogue. SEM are indicated by the vertical bars. The normalized mean NPo was 0.057 ± 0.018 (mean ± SEM) in control solution, 0.277 ± 0.095 in the presence of 20 mM Na+ ions, 1 after the application of 10 μM GTPγS, and 4.21 ± 0.59 in the presence of 20 mM Na+ ions after channel activation by GTPγS. (C) NPo vs. time plot for the channel activity recorded in an inside-out patch from an oocyte expressing GIRK1/GIRK4. 20 mM Na+ and 20 nM β1γ7 purified subunits were applied via the bath as indicated by the bars. Vm = −80 mV. 5 μM acetylcholine was present in the pipette. (D) The mean NPo for nine patches are plotted for different conditions. Steady state channel activity after β1γ7 activation (after β1γ7 washout) was taken as reference and NPo was normalized to it. Na+ concentration was 20 mM and β1γ7 was 20 nM. βγ+Na+ refers to the application of 20 mM Na+ after the washout of βγ. The vertical bars represent SEM. The normalized mean NPo was 0.084 ± 0.039 (mean ± SEM) in control solution, 0.206 ± 0.12 in the presence of 20 mM Na+ ions, 1 after the application of 20 nM β1γ7, and 3.1 ± 0.84 in the presence of 20 mM Na+ ions after activation of the channel by the G protein subunits.
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Figure 2: Na+ ions gate GIRK channels after activation by G protein βγ subunits. (A) Single-channel activity (NPo, bin = 5 s) plotted as a function of time. The data were obtained from an inside-out patch excised from an oocyte expressing the recombinant channel GIRK1/GIRK4. 20 mM Na+ and 10 μM GTPγS were applied as indicated by the bars. The membrane was clamped at −80 mV and 5 μM acetylcholine was in the pipette solution. (B) The mean NPo for seven patches is plotted for different conditions. Steady state channel activity after activation by GTPγS was taken as reference (GTPγS) and NPo were normalized to it. Na+ concentration was 20 mM and GTPγS was 10 μM. GTPγS+Na+ corresponds to the application of 20 mM Na+ after the washout of the GTP analogue. SEM are indicated by the vertical bars. The normalized mean NPo was 0.057 ± 0.018 (mean ± SEM) in control solution, 0.277 ± 0.095 in the presence of 20 mM Na+ ions, 1 after the application of 10 μM GTPγS, and 4.21 ± 0.59 in the presence of 20 mM Na+ ions after channel activation by GTPγS. (C) NPo vs. time plot for the channel activity recorded in an inside-out patch from an oocyte expressing GIRK1/GIRK4. 20 mM Na+ and 20 nM β1γ7 purified subunits were applied via the bath as indicated by the bars. Vm = −80 mV. 5 μM acetylcholine was present in the pipette. (D) The mean NPo for nine patches are plotted for different conditions. Steady state channel activity after β1γ7 activation (after β1γ7 washout) was taken as reference and NPo was normalized to it. Na+ concentration was 20 mM and β1γ7 was 20 nM. βγ+Na+ refers to the application of 20 mM Na+ after the washout of βγ. The vertical bars represent SEM. The normalized mean NPo was 0.084 ± 0.039 (mean ± SEM) in control solution, 0.206 ± 0.12 in the presence of 20 mM Na+ ions, 1 after the application of 20 nM β1γ7, and 3.1 ± 0.84 in the presence of 20 mM Na+ ions after activation of the channel by the G protein subunits.

Mentions: Fig. 2A and Fig. B, show representative and summary data from experiments where Na+ ions gated GIRK1/GIRK4 channels after activation by G proteins. Inside-out patches from oocytes expressing these channels showed no channel activity upon application of 20 mM Na+. This result suggested a low presence of PIP2 in the membrane. However, this PIP2 concentration was sufficient to allow persistent channel activation by a brief exposure to 10 μM GTPγS. Reapplication of Na+ ions produced a more than fourfold increase in the channel activity above the level obtained with GTPγS. It should be noted that the effect of Na+ ions on the basal channel activity was variable from patch to patch, presumably reflecting different levels of endogenous PIP2 at the time of Na+ application.


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)

Na+ ions gate GIRK channels after activation by G protein βγ subunits. (A) Single-channel activity (NPo, bin = 5 s) plotted as a function of time. The data were obtained from an inside-out patch excised from an oocyte expressing the recombinant channel GIRK1/GIRK4. 20 mM Na+ and 10 μM GTPγS were applied as indicated by the bars. The membrane was clamped at −80 mV and 5 μM acetylcholine was in the pipette solution. (B) The mean NPo for seven patches is plotted for different conditions. Steady state channel activity after activation by GTPγS was taken as reference (GTPγS) and NPo were normalized to it. Na+ concentration was 20 mM and GTPγS was 10 μM. GTPγS+Na+ corresponds to the application of 20 mM Na+ after the washout of the GTP analogue. SEM are indicated by the vertical bars. The normalized mean NPo was 0.057 ± 0.018 (mean ± SEM) in control solution, 0.277 ± 0.095 in the presence of 20 mM Na+ ions, 1 after the application of 10 μM GTPγS, and 4.21 ± 0.59 in the presence of 20 mM Na+ ions after channel activation by GTPγS. (C) NPo vs. time plot for the channel activity recorded in an inside-out patch from an oocyte expressing GIRK1/GIRK4. 20 mM Na+ and 20 nM β1γ7 purified subunits were applied via the bath as indicated by the bars. Vm = −80 mV. 5 μM acetylcholine was present in the pipette. (D) The mean NPo for nine patches are plotted for different conditions. Steady state channel activity after β1γ7 activation (after β1γ7 washout) was taken as reference and NPo was normalized to it. Na+ concentration was 20 mM and β1γ7 was 20 nM. βγ+Na+ refers to the application of 20 mM Na+ after the washout of βγ. The vertical bars represent SEM. The normalized mean NPo was 0.084 ± 0.039 (mean ± SEM) in control solution, 0.206 ± 0.12 in the presence of 20 mM Na+ ions, 1 after the application of 20 nM β1γ7, and 3.1 ± 0.84 in the presence of 20 mM Na+ ions after activation of the channel by the G protein subunits.
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Related In: Results  -  Collection

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Figure 2: Na+ ions gate GIRK channels after activation by G protein βγ subunits. (A) Single-channel activity (NPo, bin = 5 s) plotted as a function of time. The data were obtained from an inside-out patch excised from an oocyte expressing the recombinant channel GIRK1/GIRK4. 20 mM Na+ and 10 μM GTPγS were applied as indicated by the bars. The membrane was clamped at −80 mV and 5 μM acetylcholine was in the pipette solution. (B) The mean NPo for seven patches is plotted for different conditions. Steady state channel activity after activation by GTPγS was taken as reference (GTPγS) and NPo were normalized to it. Na+ concentration was 20 mM and GTPγS was 10 μM. GTPγS+Na+ corresponds to the application of 20 mM Na+ after the washout of the GTP analogue. SEM are indicated by the vertical bars. The normalized mean NPo was 0.057 ± 0.018 (mean ± SEM) in control solution, 0.277 ± 0.095 in the presence of 20 mM Na+ ions, 1 after the application of 10 μM GTPγS, and 4.21 ± 0.59 in the presence of 20 mM Na+ ions after channel activation by GTPγS. (C) NPo vs. time plot for the channel activity recorded in an inside-out patch from an oocyte expressing GIRK1/GIRK4. 20 mM Na+ and 20 nM β1γ7 purified subunits were applied via the bath as indicated by the bars. Vm = −80 mV. 5 μM acetylcholine was present in the pipette. (D) The mean NPo for nine patches are plotted for different conditions. Steady state channel activity after β1γ7 activation (after β1γ7 washout) was taken as reference and NPo was normalized to it. Na+ concentration was 20 mM and β1γ7 was 20 nM. βγ+Na+ refers to the application of 20 mM Na+ after the washout of βγ. The vertical bars represent SEM. The normalized mean NPo was 0.084 ± 0.039 (mean ± SEM) in control solution, 0.206 ± 0.12 in the presence of 20 mM Na+ ions, 1 after the application of 20 nM β1γ7, and 3.1 ± 0.84 in the presence of 20 mM Na+ ions after activation of the channel by the G protein subunits.
Mentions: Fig. 2A and Fig. B, show representative and summary data from experiments where Na+ ions gated GIRK1/GIRK4 channels after activation by G proteins. Inside-out patches from oocytes expressing these channels showed no channel activity upon application of 20 mM Na+. This result suggested a low presence of PIP2 in the membrane. However, this PIP2 concentration was sufficient to allow persistent channel activation by a brief exposure to 10 μM GTPγS. Reapplication of Na+ ions produced a more than fourfold increase in the channel activity above the level obtained with GTPγS. It should be noted that the effect of Na+ ions on the basal channel activity was variable from patch to patch, presumably reflecting different levels of endogenous PIP2 at the time of Na+ application.

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