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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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High concentrations of Mg2+ ions reduce the GIRK single-channel currents. (A) Single-channel records of GIRK channels in an inside-out patch excised from an oocyte expressing GIRK1/GIRK4. The membrane was clamped at −120 mV and 5 μM acetylcholine was present in the pipette. The channel was preactivated by 10 μM GTPγS and the current traces shown were recorded after washout of the GTP analogue. The switch between bath solutions containing 1 and 20 mM Mg2+ is visualized by the arrow (and by the corresponding electrical artifact on the record) on top of the second current trace. Associated all-point histogram plots indicate the amplitudes resulting from the various activity levels ranging from closed to multiple open levels and are shown for the first (1 mM Mg2+) and third (20 mM Mg2+) current traces. Data points are shown on a logarithmic scale ranging from 5 to 50,000. (B) KACh channel activity in an inside-out patch from a cardiac cell. The membrane was held at −90 mV and the pipette contained 5 μM acetylcholine. The patch was preincubated with 5 μM PIP2 and the current traces shown were recorded after the washout of PIP2. The arrow on top of the second current trace visualizes the switch between bath solutions containing 20 mM Mg2+ and 20 mM Na+ + 1 mM Mg2+. Associated all-point histogram plots are shown for the first (20 mM Mg2+) and third (20 mM Na+ + 1 mM Mg2+) current traces. Data points are shown on a logarithmic scale ranging from 20 to 50,000.
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Figure 6: High concentrations of Mg2+ ions reduce the GIRK single-channel currents. (A) Single-channel records of GIRK channels in an inside-out patch excised from an oocyte expressing GIRK1/GIRK4. The membrane was clamped at −120 mV and 5 μM acetylcholine was present in the pipette. The channel was preactivated by 10 μM GTPγS and the current traces shown were recorded after washout of the GTP analogue. The switch between bath solutions containing 1 and 20 mM Mg2+ is visualized by the arrow (and by the corresponding electrical artifact on the record) on top of the second current trace. Associated all-point histogram plots indicate the amplitudes resulting from the various activity levels ranging from closed to multiple open levels and are shown for the first (1 mM Mg2+) and third (20 mM Mg2+) current traces. Data points are shown on a logarithmic scale ranging from 5 to 50,000. (B) KACh channel activity in an inside-out patch from a cardiac cell. The membrane was held at −90 mV and the pipette contained 5 μM acetylcholine. The patch was preincubated with 5 μM PIP2 and the current traces shown were recorded after the washout of PIP2. The arrow on top of the second current trace visualizes the switch between bath solutions containing 20 mM Mg2+ and 20 mM Na+ + 1 mM Mg2+. Associated all-point histogram plots are shown for the first (20 mM Mg2+) and third (20 mM Na+ + 1 mM Mg2+) current traces. Data points are shown on a logarithmic scale ranging from 20 to 50,000.

Mentions: We observed, particularly at high concentrations (>5 mM), that internal Mg2+ ions reduced the amplitude of single GIRK channel currents. In Fig. 6 A, the activity of the coexpressed channel subunits GIRK1/GIRK4 from an inside-out patch was recorded at −120 mV. After activation by 10 μM GTPγS, channel activity was recorded in a solution containing 1 mM Mg2+ ions, showing an approximate amplitude of −3.2 pA. When the solution applied to the patch was switched to one containing 20 mM Mg2+ ions, the amplitude of the single openings was rapidly reduced to a lower value, approximately −2.5 pA (n = 5). In Fig. 6 B, the activity of native KACh channels in an inside-out patch from an atrial cell was recorded at −90 mV. After exposure to 5 μM PIP2, the patch was perfused with a solution containing 20 mM Mg2+ ions, giving an amplitude of approximately −2.2 pA. When the solution applied to the patch was switched to one containing 20 mM Na+ and 1 mM Mg2+ ions, the channel amplitude immediately increased to a value of approximately −3.5 pA. This amplitude was also obtained in control conditions, where 1 mM Mg2+ ions were present (n = 5). The reduction in the single-channel amplitude was observed at various voltages. Since it was present at negative potentials (i.e., −80, −90, and −120 mV) where no rectification occurs, it is likely to proceed by a mechanism distinct from that of the rectification phenomenon. Mg2+ ions at high concentrations also decreased the amplitude of GIRK single channels when applied together with Na+ ions (data not shown). Thus, regardless of their ability to gate GIRK channels (see Fig. 3 and Fig. 7), Mg2+ ions at high concentrations (>5 mM) show a clear inhibition on single-channel current amplitudes. These data suggest that the inhibitory effect of Mg2+ ions on the single-channel amplitude was not dependent on their ability to gate the channel.


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)

High concentrations of Mg2+ ions reduce the GIRK single-channel currents. (A) Single-channel records of GIRK channels in an inside-out patch excised from an oocyte expressing GIRK1/GIRK4. The membrane was clamped at −120 mV and 5 μM acetylcholine was present in the pipette. The channel was preactivated by 10 μM GTPγS and the current traces shown were recorded after washout of the GTP analogue. The switch between bath solutions containing 1 and 20 mM Mg2+ is visualized by the arrow (and by the corresponding electrical artifact on the record) on top of the second current trace. Associated all-point histogram plots indicate the amplitudes resulting from the various activity levels ranging from closed to multiple open levels and are shown for the first (1 mM Mg2+) and third (20 mM Mg2+) current traces. Data points are shown on a logarithmic scale ranging from 5 to 50,000. (B) KACh channel activity in an inside-out patch from a cardiac cell. The membrane was held at −90 mV and the pipette contained 5 μM acetylcholine. The patch was preincubated with 5 μM PIP2 and the current traces shown were recorded after the washout of PIP2. The arrow on top of the second current trace visualizes the switch between bath solutions containing 20 mM Mg2+ and 20 mM Na+ + 1 mM Mg2+. Associated all-point histogram plots are shown for the first (20 mM Mg2+) and third (20 mM Na+ + 1 mM Mg2+) current traces. Data points are shown on a logarithmic scale ranging from 20 to 50,000.
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Related In: Results  -  Collection

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Figure 6: High concentrations of Mg2+ ions reduce the GIRK single-channel currents. (A) Single-channel records of GIRK channels in an inside-out patch excised from an oocyte expressing GIRK1/GIRK4. The membrane was clamped at −120 mV and 5 μM acetylcholine was present in the pipette. The channel was preactivated by 10 μM GTPγS and the current traces shown were recorded after washout of the GTP analogue. The switch between bath solutions containing 1 and 20 mM Mg2+ is visualized by the arrow (and by the corresponding electrical artifact on the record) on top of the second current trace. Associated all-point histogram plots indicate the amplitudes resulting from the various activity levels ranging from closed to multiple open levels and are shown for the first (1 mM Mg2+) and third (20 mM Mg2+) current traces. Data points are shown on a logarithmic scale ranging from 5 to 50,000. (B) KACh channel activity in an inside-out patch from a cardiac cell. The membrane was held at −90 mV and the pipette contained 5 μM acetylcholine. The patch was preincubated with 5 μM PIP2 and the current traces shown were recorded after the washout of PIP2. The arrow on top of the second current trace visualizes the switch between bath solutions containing 20 mM Mg2+ and 20 mM Na+ + 1 mM Mg2+. Associated all-point histogram plots are shown for the first (20 mM Mg2+) and third (20 mM Na+ + 1 mM Mg2+) current traces. Data points are shown on a logarithmic scale ranging from 20 to 50,000.
Mentions: We observed, particularly at high concentrations (>5 mM), that internal Mg2+ ions reduced the amplitude of single GIRK channel currents. In Fig. 6 A, the activity of the coexpressed channel subunits GIRK1/GIRK4 from an inside-out patch was recorded at −120 mV. After activation by 10 μM GTPγS, channel activity was recorded in a solution containing 1 mM Mg2+ ions, showing an approximate amplitude of −3.2 pA. When the solution applied to the patch was switched to one containing 20 mM Mg2+ ions, the amplitude of the single openings was rapidly reduced to a lower value, approximately −2.5 pA (n = 5). In Fig. 6 B, the activity of native KACh channels in an inside-out patch from an atrial cell was recorded at −90 mV. After exposure to 5 μM PIP2, the patch was perfused with a solution containing 20 mM Mg2+ ions, giving an amplitude of approximately −2.2 pA. When the solution applied to the patch was switched to one containing 20 mM Na+ and 1 mM Mg2+ ions, the channel amplitude immediately increased to a value of approximately −3.5 pA. This amplitude was also obtained in control conditions, where 1 mM Mg2+ ions were present (n = 5). The reduction in the single-channel amplitude was observed at various voltages. Since it was present at negative potentials (i.e., −80, −90, and −120 mV) where no rectification occurs, it is likely to proceed by a mechanism distinct from that of the rectification phenomenon. Mg2+ ions at high concentrations also decreased the amplitude of GIRK single channels when applied together with Na+ ions (data not shown). Thus, regardless of their ability to gate GIRK channels (see Fig. 3 and Fig. 7), Mg2+ ions at high concentrations (>5 mM) show a clear inhibition on single-channel current amplitudes. These data suggest that the inhibitory effect of Mg2+ ions on the single-channel amplitude was not dependent on their ability to gate the channel.

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