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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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Related in: MedlinePlus

Synergistic effects of Gβγ, Na+, and Mg2+ ions in activating GIRK channels. (A) The mean NPo for seven patches are plotted for different conditions. The data were obtained from inside-out patches excised from oocytes expressing the recombinant channel GIRK1/GIRK4. The membrane was held at −80 mV and 5 μM acetylcholine was present in the pipette. PIP2 was 2.5 μM, Mg2+ was 10 mM, and Na+ was 10 mM. SEM are indicated by the vertical bars. The mean NPo for the channel activity was 0.011 ± 0.003 in control conditions, and 0.01 ± 0.006 during the application of 2.5 μM PIP2. When 10 mM Mg2+ ions were applied with PIP2, the mean NPo was 0.43 ± 0.14. 10 mM Na+ ions gave a mean NPo of 0.40 ± 0.12. When applied together, in the presence of PIP2, Mg2+ and Na+ ions (10 mM each) yielded a mean NPo of 2.08 ± 0.52. (B) Mean NPo plots for six inside-out patches from oocytes expressing GIRK1/GIRK4. Vm was −80 mV. 5 μM acetylcholine was in the pipette. Mg2+ was 10 mM, Na+ was 10 mM, and GTPγS was 10 μM. The columns GTPγS and GTPγS+x depict the channel activity measured after the GTP analogue was washed out and substance(s) x were added. SEM are indicated by vertical bars. The mean NPo of the channel was 0.023 ± 0.012 in control conditions and 0.035 ± 0.02 in the presence of 10 mM Mg2+ ions. 10 μM GTPγS gave a mean NPo of 0.12 ± 0.045. After GTPγS washout, Mg2+ ions gave a mean NPo of 0.12 ± 0.03. 10 mM Na+ ions gave a mean NPo of 0.23 ± 0.06. Coapplication of Mg2+ and Na+ ions resulted in a mean NPo of 0.26 ± 0.08. (C) Mean NPo plots for six patches. The inside-out patches were excised from oocytes expressing GIRK1/GIRK4. Vm was −80 mV. 5 μM ACh present in the pipette. PIP2 was 2.5 μM, Mg2+ was 10 mM, Na+ was 10 mM, and GTPγS was 10 μM. PIP2+GTPγS refers to the channel activity (at steady state) during the application of the GTP analogue. PIP2+GTPγS+x columns depict the channel activity measured after the washout of the GTP analogue and addition of substance(s) x. In absence of 10 mM Mg2+, all solutions contained 50 μM Mg2+. This low concentration of Mg2+ was necessary to render GTPγS effective. Vertical bars represent SEM. The mean NPo for the channel activity was 0.004 ± 0.002 in control conditions and 0.0007 ± 0.0002 in the presence of 2.5 μM PIP2. When 10 mM Mg2+ ions were applied to the patches in the presence of PIP2, a mean NPo of 0.034 ± 0.013 was obtained. Although this activity appeared small, it was significantly higher than that in PIP2 alone (P < 0.005, paired t test, log scale). 10 mM each of Mg2+ and Na+ ions in combination yielded a mean NPo of 0.2 ± 0.09. GTPγS gave a mean NPo of 0.058 ± 0.03. Again, although this activity appeared relatively small, it was significantly higher than that in PIP2 alone (P < 0.005, paired t test, log scale). When 10 mM Mg2+ ions were applied to the patches after the GTPγS treatment a mean NPo of 0.42 ± 0.19 was obtained. Mg2+ and Na+ ions applied together resulted in NPo of 1.6 ± 0.49.
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Figure 7: Synergistic effects of Gβγ, Na+, and Mg2+ ions in activating GIRK channels. (A) The mean NPo for seven patches are plotted for different conditions. The data were obtained from inside-out patches excised from oocytes expressing the recombinant channel GIRK1/GIRK4. The membrane was held at −80 mV and 5 μM acetylcholine was present in the pipette. PIP2 was 2.5 μM, Mg2+ was 10 mM, and Na+ was 10 mM. SEM are indicated by the vertical bars. The mean NPo for the channel activity was 0.011 ± 0.003 in control conditions, and 0.01 ± 0.006 during the application of 2.5 μM PIP2. When 10 mM Mg2+ ions were applied with PIP2, the mean NPo was 0.43 ± 0.14. 10 mM Na+ ions gave a mean NPo of 0.40 ± 0.12. When applied together, in the presence of PIP2, Mg2+ and Na+ ions (10 mM each) yielded a mean NPo of 2.08 ± 0.52. (B) Mean NPo plots for six inside-out patches from oocytes expressing GIRK1/GIRK4. Vm was −80 mV. 5 μM acetylcholine was in the pipette. Mg2+ was 10 mM, Na+ was 10 mM, and GTPγS was 10 μM. The columns GTPγS and GTPγS+x depict the channel activity measured after the GTP analogue was washed out and substance(s) x were added. SEM are indicated by vertical bars. The mean NPo of the channel was 0.023 ± 0.012 in control conditions and 0.035 ± 0.02 in the presence of 10 mM Mg2+ ions. 10 μM GTPγS gave a mean NPo of 0.12 ± 0.045. After GTPγS washout, Mg2+ ions gave a mean NPo of 0.12 ± 0.03. 10 mM Na+ ions gave a mean NPo of 0.23 ± 0.06. Coapplication of Mg2+ and Na+ ions resulted in a mean NPo of 0.26 ± 0.08. (C) Mean NPo plots for six patches. The inside-out patches were excised from oocytes expressing GIRK1/GIRK4. Vm was −80 mV. 5 μM ACh present in the pipette. PIP2 was 2.5 μM, Mg2+ was 10 mM, Na+ was 10 mM, and GTPγS was 10 μM. PIP2+GTPγS refers to the channel activity (at steady state) during the application of the GTP analogue. PIP2+GTPγS+x columns depict the channel activity measured after the washout of the GTP analogue and addition of substance(s) x. In absence of 10 mM Mg2+, all solutions contained 50 μM Mg2+. This low concentration of Mg2+ was necessary to render GTPγS effective. Vertical bars represent SEM. The mean NPo for the channel activity was 0.004 ± 0.002 in control conditions and 0.0007 ± 0.0002 in the presence of 2.5 μM PIP2. When 10 mM Mg2+ ions were applied to the patches in the presence of PIP2, a mean NPo of 0.034 ± 0.013 was obtained. Although this activity appeared small, it was significantly higher than that in PIP2 alone (P < 0.005, paired t test, log scale). 10 mM each of Mg2+ and Na+ ions in combination yielded a mean NPo of 0.2 ± 0.09. GTPγS gave a mean NPo of 0.058 ± 0.03. Again, although this activity appeared relatively small, it was significantly higher than that in PIP2 alone (P < 0.005, paired t test, log scale). When 10 mM Mg2+ ions were applied to the patches after the GTPγS treatment a mean NPo of 0.42 ± 0.19 was obtained. Mg2+ and Na+ ions applied together resulted in NPo of 1.6 ± 0.49.

Mentions: In experiments shown in Fig. 7, where exogenous PIP2 was applied throughout the experiment (i.e., Fig. 7A and Fig. C), occasional applications of the same ion as a function of time in the experiment were used as control to ascertain that the synergistic effects described were not due to a time-dependent accumulation of PIP2 in the membrane patch. Similar precautions were taken in the experiments shown in Fig. 4. Experiments used to generate the data shown in these two figures were never longer than 14 min (usually 10–13 min). Na+ and/or Mg2+ ions were applied for 30 s.


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)

Synergistic effects of Gβγ, Na+, and Mg2+ ions in activating GIRK channels. (A) The mean NPo for seven patches are plotted for different conditions. The data were obtained from inside-out patches excised from oocytes expressing the recombinant channel GIRK1/GIRK4. The membrane was held at −80 mV and 5 μM acetylcholine was present in the pipette. PIP2 was 2.5 μM, Mg2+ was 10 mM, and Na+ was 10 mM. SEM are indicated by the vertical bars. The mean NPo for the channel activity was 0.011 ± 0.003 in control conditions, and 0.01 ± 0.006 during the application of 2.5 μM PIP2. When 10 mM Mg2+ ions were applied with PIP2, the mean NPo was 0.43 ± 0.14. 10 mM Na+ ions gave a mean NPo of 0.40 ± 0.12. When applied together, in the presence of PIP2, Mg2+ and Na+ ions (10 mM each) yielded a mean NPo of 2.08 ± 0.52. (B) Mean NPo plots for six inside-out patches from oocytes expressing GIRK1/GIRK4. Vm was −80 mV. 5 μM acetylcholine was in the pipette. Mg2+ was 10 mM, Na+ was 10 mM, and GTPγS was 10 μM. The columns GTPγS and GTPγS+x depict the channel activity measured after the GTP analogue was washed out and substance(s) x were added. SEM are indicated by vertical bars. The mean NPo of the channel was 0.023 ± 0.012 in control conditions and 0.035 ± 0.02 in the presence of 10 mM Mg2+ ions. 10 μM GTPγS gave a mean NPo of 0.12 ± 0.045. After GTPγS washout, Mg2+ ions gave a mean NPo of 0.12 ± 0.03. 10 mM Na+ ions gave a mean NPo of 0.23 ± 0.06. Coapplication of Mg2+ and Na+ ions resulted in a mean NPo of 0.26 ± 0.08. (C) Mean NPo plots for six patches. The inside-out patches were excised from oocytes expressing GIRK1/GIRK4. Vm was −80 mV. 5 μM ACh present in the pipette. PIP2 was 2.5 μM, Mg2+ was 10 mM, Na+ was 10 mM, and GTPγS was 10 μM. PIP2+GTPγS refers to the channel activity (at steady state) during the application of the GTP analogue. PIP2+GTPγS+x columns depict the channel activity measured after the washout of the GTP analogue and addition of substance(s) x. In absence of 10 mM Mg2+, all solutions contained 50 μM Mg2+. This low concentration of Mg2+ was necessary to render GTPγS effective. Vertical bars represent SEM. The mean NPo for the channel activity was 0.004 ± 0.002 in control conditions and 0.0007 ± 0.0002 in the presence of 2.5 μM PIP2. When 10 mM Mg2+ ions were applied to the patches in the presence of PIP2, a mean NPo of 0.034 ± 0.013 was obtained. Although this activity appeared small, it was significantly higher than that in PIP2 alone (P < 0.005, paired t test, log scale). 10 mM each of Mg2+ and Na+ ions in combination yielded a mean NPo of 0.2 ± 0.09. GTPγS gave a mean NPo of 0.058 ± 0.03. Again, although this activity appeared relatively small, it was significantly higher than that in PIP2 alone (P < 0.005, paired t test, log scale). When 10 mM Mg2+ ions were applied to the patches after the GTPγS treatment a mean NPo of 0.42 ± 0.19 was obtained. Mg2+ and Na+ ions applied together resulted in NPo of 1.6 ± 0.49.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230539&req=5

Figure 7: Synergistic effects of Gβγ, Na+, and Mg2+ ions in activating GIRK channels. (A) The mean NPo for seven patches are plotted for different conditions. The data were obtained from inside-out patches excised from oocytes expressing the recombinant channel GIRK1/GIRK4. The membrane was held at −80 mV and 5 μM acetylcholine was present in the pipette. PIP2 was 2.5 μM, Mg2+ was 10 mM, and Na+ was 10 mM. SEM are indicated by the vertical bars. The mean NPo for the channel activity was 0.011 ± 0.003 in control conditions, and 0.01 ± 0.006 during the application of 2.5 μM PIP2. When 10 mM Mg2+ ions were applied with PIP2, the mean NPo was 0.43 ± 0.14. 10 mM Na+ ions gave a mean NPo of 0.40 ± 0.12. When applied together, in the presence of PIP2, Mg2+ and Na+ ions (10 mM each) yielded a mean NPo of 2.08 ± 0.52. (B) Mean NPo plots for six inside-out patches from oocytes expressing GIRK1/GIRK4. Vm was −80 mV. 5 μM acetylcholine was in the pipette. Mg2+ was 10 mM, Na+ was 10 mM, and GTPγS was 10 μM. The columns GTPγS and GTPγS+x depict the channel activity measured after the GTP analogue was washed out and substance(s) x were added. SEM are indicated by vertical bars. The mean NPo of the channel was 0.023 ± 0.012 in control conditions and 0.035 ± 0.02 in the presence of 10 mM Mg2+ ions. 10 μM GTPγS gave a mean NPo of 0.12 ± 0.045. After GTPγS washout, Mg2+ ions gave a mean NPo of 0.12 ± 0.03. 10 mM Na+ ions gave a mean NPo of 0.23 ± 0.06. Coapplication of Mg2+ and Na+ ions resulted in a mean NPo of 0.26 ± 0.08. (C) Mean NPo plots for six patches. The inside-out patches were excised from oocytes expressing GIRK1/GIRK4. Vm was −80 mV. 5 μM ACh present in the pipette. PIP2 was 2.5 μM, Mg2+ was 10 mM, Na+ was 10 mM, and GTPγS was 10 μM. PIP2+GTPγS refers to the channel activity (at steady state) during the application of the GTP analogue. PIP2+GTPγS+x columns depict the channel activity measured after the washout of the GTP analogue and addition of substance(s) x. In absence of 10 mM Mg2+, all solutions contained 50 μM Mg2+. This low concentration of Mg2+ was necessary to render GTPγS effective. Vertical bars represent SEM. The mean NPo for the channel activity was 0.004 ± 0.002 in control conditions and 0.0007 ± 0.0002 in the presence of 2.5 μM PIP2. When 10 mM Mg2+ ions were applied to the patches in the presence of PIP2, a mean NPo of 0.034 ± 0.013 was obtained. Although this activity appeared small, it was significantly higher than that in PIP2 alone (P < 0.005, paired t test, log scale). 10 mM each of Mg2+ and Na+ ions in combination yielded a mean NPo of 0.2 ± 0.09. GTPγS gave a mean NPo of 0.058 ± 0.03. Again, although this activity appeared relatively small, it was significantly higher than that in PIP2 alone (P < 0.005, paired t test, log scale). When 10 mM Mg2+ ions were applied to the patches after the GTPγS treatment a mean NPo of 0.42 ± 0.19 was obtained. Mg2+ and Na+ ions applied together resulted in NPo of 1.6 ± 0.49.
Mentions: In experiments shown in Fig. 7, where exogenous PIP2 was applied throughout the experiment (i.e., Fig. 7A and Fig. C), occasional applications of the same ion as a function of time in the experiment were used as control to ascertain that the synergistic effects described were not due to a time-dependent accumulation of PIP2 in the membrane patch. Similar precautions were taken in the experiments shown in Fig. 4. Experiments used to generate the data shown in these two figures were never longer than 14 min (usually 10–13 min). Na+ and/or Mg2+ ions were applied for 30 s.

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