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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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Mg2+ ions act at a site distinct of that used by Na+ ions. (A) Single channel activity (NPo, bin = 5 s) plotted as a function of time. An inside-out patch from an oocyte expressing GIRK4*(D223N) (see text) and human muscarinic receptor type 2 receptor was exposed to 2.5 μM PIP2 and 20 mM Na+ or 1 mM Mg2+ ions, and channel activity was recorded. The membrane potential was kept at −80 mV. The pipette solution contained 5 μM ACh. (B) Summary data plotting mean NPo of four experiments such as that shown in A. The mean NPo values were 0.14 ± 0.1 (mean ± SEM) in control conditions, 0.21 ± 0.06 in the presence of PIP2, 0.21 ± 0.08 in the presence of PIP2 and Na+ ions, and 2.97 ± 0.31 in the presence of PIP2 and Mg2+ ions.
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Figure 5: Mg2+ ions act at a site distinct of that used by Na+ ions. (A) Single channel activity (NPo, bin = 5 s) plotted as a function of time. An inside-out patch from an oocyte expressing GIRK4*(D223N) (see text) and human muscarinic receptor type 2 receptor was exposed to 2.5 μM PIP2 and 20 mM Na+ or 1 mM Mg2+ ions, and channel activity was recorded. The membrane potential was kept at −80 mV. The pipette solution contained 5 μM ACh. (B) Summary data plotting mean NPo of four experiments such as that shown in A. The mean NPo values were 0.14 ± 0.1 (mean ± SEM) in control conditions, 0.21 ± 0.06 in the presence of PIP2, 0.21 ± 0.08 in the presence of PIP2 and Na+ ions, and 2.97 ± 0.31 in the presence of PIP2 and Mg2+ ions.

Mentions: Recent work has identified an aspartate amino acid residue as the site of action of Na+ ions on GIRK channels, GIRK2 (D228) and GIRK4 (D223) (Ho and Murrell-Lagnado 1999; Zhang et al. 1999). Moreover, it was shown that Na+ sensitivity lies entirely with the heteromeric partners of GIRK1, as this channel possesses an asparagine instead of an aspartate residue at the equivalent position. We used the point mutant GIRK4(S143T) (referred to as GIRK4*) that allows for high levels of activity of homotetrameric GIRK4 channels (Vivaudou et al. 1991) to test for Na+ and Mg2+ sensitivity. GIRK4* channel activity shows high sensitivity to both Na+ and Mg2+. Fig. 5 shows that indeed GIRK4*(D223N) loses its sensitivity to Na+ ions (20 mM). However Mg2+ ion (1 mM) sensitivity was intact (Fig. 5 A). Summary data are shown in Fig. 5 B. These data indicate that Na+ and Mg2+ ions act at distinct sites to activate GIRK channels.


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)

Mg2+ ions act at a site distinct of that used by Na+ ions. (A) Single channel activity (NPo, bin = 5 s) plotted as a function of time. An inside-out patch from an oocyte expressing GIRK4*(D223N) (see text) and human muscarinic receptor type 2 receptor was exposed to 2.5 μM PIP2 and 20 mM Na+ or 1 mM Mg2+ ions, and channel activity was recorded. The membrane potential was kept at −80 mV. The pipette solution contained 5 μM ACh. (B) Summary data plotting mean NPo of four experiments such as that shown in A. The mean NPo values were 0.14 ± 0.1 (mean ± SEM) in control conditions, 0.21 ± 0.06 in the presence of PIP2, 0.21 ± 0.08 in the presence of PIP2 and Na+ ions, and 2.97 ± 0.31 in the presence of PIP2 and Mg2+ ions.
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Related In: Results  -  Collection

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

Figure 5: Mg2+ ions act at a site distinct of that used by Na+ ions. (A) Single channel activity (NPo, bin = 5 s) plotted as a function of time. An inside-out patch from an oocyte expressing GIRK4*(D223N) (see text) and human muscarinic receptor type 2 receptor was exposed to 2.5 μM PIP2 and 20 mM Na+ or 1 mM Mg2+ ions, and channel activity was recorded. The membrane potential was kept at −80 mV. The pipette solution contained 5 μM ACh. (B) Summary data plotting mean NPo of four experiments such as that shown in A. The mean NPo values were 0.14 ± 0.1 (mean ± SEM) in control conditions, 0.21 ± 0.06 in the presence of PIP2, 0.21 ± 0.08 in the presence of PIP2 and Na+ ions, and 2.97 ± 0.31 in the presence of PIP2 and Mg2+ ions.
Mentions: Recent work has identified an aspartate amino acid residue as the site of action of Na+ ions on GIRK channels, GIRK2 (D228) and GIRK4 (D223) (Ho and Murrell-Lagnado 1999; Zhang et al. 1999). Moreover, it was shown that Na+ sensitivity lies entirely with the heteromeric partners of GIRK1, as this channel possesses an asparagine instead of an aspartate residue at the equivalent position. We used the point mutant GIRK4(S143T) (referred to as GIRK4*) that allows for high levels of activity of homotetrameric GIRK4 channels (Vivaudou et al. 1991) to test for Na+ and Mg2+ sensitivity. GIRK4* channel activity shows high sensitivity to both Na+ and Mg2+. Fig. 5 shows that indeed GIRK4*(D223N) loses its sensitivity to Na+ ions (20 mM). However Mg2+ ion (1 mM) sensitivity was intact (Fig. 5 A). Summary data are shown in Fig. 5 B. These data indicate that Na+ and Mg2+ ions act at distinct sites to activate GIRK channels.

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