Petit-Jacques J et al. (1999), Synergistic activation of G protein-gated inwar...

XB-ART-12092

J Gen Physiol 1999 Nov 01;1145:673-84.

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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.

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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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Species referenced: Xenopus
Genes referenced: bag3 kcnj3 kcnj5

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	Figure 3. Mg2+ ions gate the ATP-modified GIRK channels. NPo, NFo, and MTo plots (bin = 5 s) of GIRK channel activity in an inside-out patch excised from an oocyte expressing GIRK1/GIRK4. 10 mM Mg2+, 2.5 mM MgATP, and 20 mM Na+ were applied via the bath as indicated by the bars. The membrane was clamped at −80 mV and 5 μM acetylcholine was present in the pipette. NPo, NFo, and MTo in 10 mM Mg2+ were calculated such that the amplitude levels were set to match the reduced amplitude of the channel openings in 10 mM Mg2+.
	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).
	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.
	Figure 8. A model depicting gating of GIRK channels by the combination of PIP2 with gating molecules Gβγ and/or Na+ and Mg2+ ions. (Co) Channel closed state, in the absence of PIP2 in the plasma membrane and gating molecules. (C1) Channel closed state, in the presence of PIP2 in the membrane GIRK channels experience weak interactions that in the absence of gating molecules are not of sufficient strength to gate the channel. (C2) Channel closed state, gating molecules can interact with the channel at distinct sites but in the absence of PIP2 they fail to gate the channel. (O) Channel open state, gating molecules in the presence of membrane PIP2 can activate the channel and show synergism.
	Figure 4. Concentration dependence of GIRK channel activation on internal Mg2+ ions. Normalized activity (NPo) of GIRK1/GIRK4 channels is plotted for different Mg2+ concentrations. Inside-out patches were exposed to 2.5 μM PIP2 before and during application of Mg2+. Responses were expressed in fold increase above the activity in the presence of PIP2 and were normalized to those recorded in the presence of 1 mM Mg2+. Vertical bars represent SEM. The responses obtained at the low concentrations of Mg2+ tested (<1 mM) were significantly higher than those with PIP2 alone (P < 0.05, paired t test). The holding potential was at −80 mV. 5 μM ACh was present in the pipette. *Significant differences from 1 mM Mg2+ (P < 0.01; paired t test).
	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.
	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.
	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.

References [+] :

Baukrowitz, PIP2 and PIP as determinants for ATP inhibition of KATP channels. 1998, Pubmed, Xenbase

Baukrowitz, PIP2 and PIP as determinants for ATP inhibition of KATP channels. 1998, Pubmed , Xenbase
Chan, A recombinant inwardly rectifying potassium channel coupled to GTP-binding proteins. 1996, Pubmed , Xenbase
Chen, A region of adenylyl cyclase 2 critical for regulation by G protein beta gamma subunits. 1995, Pubmed
Chuang, Regulation of IRK3 inward rectifier K+ channel by m1 acetylcholine receptor and intracellular magnesium. 1997, Pubmed , Xenbase
Corey, Number and stoichiometry of subunits in the native atrial G-protein-gated K+ channel, IKACh. 1998, Pubmed
Fan, Anionic phospholipids activate ATP-sensitive potassium channels. 1997, Pubmed
Flanagan, The structure of divalent cation-induced aggregates of PIP2 and their alteration by gelsolin and tau. 1997, Pubmed
Hamill, Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. 1981, Pubmed
He, Identification of a potassium channel site that interacts with G protein betagamma subunits to mediate agonist-induced signaling. 1999, Pubmed
Hilgemann, Regulation of cardiac Na+,Ca2+ exchange and KATP potassium channels by PIP2. 1996, Pubmed
Ho, Molecular determinants for sodium-dependent activation of G protein-gated K+ channels. 1999, Pubmed , Xenbase
Huang, Evidence that direct binding of G beta gamma to the GIRK1 G protein-gated inwardly rectifying K+ channel is important for channel activation. 1995, Pubmed , Xenbase
Huang, Binding of the G protein betagamma subunit to multiple regions of G protein-gated inward-rectifying K+ channels. 1997, Pubmed
Huang, Direct activation of inward rectifier potassium channels by PIP2 and its stabilization by Gbetagamma. 1998, Pubmed , Xenbase
Isenberg, Calcium tolerant ventricular myocytes prepared by preincubation in a "KB medium". 1982, Pubmed
Koch, The binding site for the beta gamma subunits of heterotrimeric G proteins on the beta-adrenergic receptor kinase. 1993, Pubmed
Krapivinsky, The G-protein-gated atrial K+ channel IKACh is a heteromultimer of two inwardly rectifying K(+)-channel proteins. 1995, Pubmed , Xenbase
Krapivinsky, G beta gamma binds directly to the G protein-gated K+ channel, IKACh. 1995, Pubmed
Kunkel, Identification of domains conferring G protein regulation on inward rectifier potassium channels. 1995, Pubmed , Xenbase
Kurachi, Role of intracellular Mg2+ in the activation of muscarinic K+ channel in cardiac atrial cell membrane. 1986, Pubmed
Kurachi, G protein activation of cardiac muscarinic K+ channels. 1992, Pubmed
Logothetis, The beta gamma subunits of GTP-binding proteins activate the muscarinic K+ channel in heart. , Pubmed
Logothetis, Incremental reductions of positive charge within the S4 region of a voltage-gated K+ channel result in corresponding decreases in gating charge. 1992, Pubmed , Xenbase
Matsuda, Magnesium gating of the inwardly rectifying K+ channel. 1991, Pubmed
Nichols, Inward rectifier potassium channels. 1997, Pubmed
Reuveny, Activation of the cloned muscarinic potassium channel by G protein beta gamma subunits. 1994, Pubmed , Xenbase
Shyng, Membrane phospholipid control of nucleotide sensitivity of KATP channels. 1998, Pubmed
Silverman, Subunit stoichiometry of a heteromultimeric G protein-coupled inward-rectifier K+ channel. 1996, Pubmed , Xenbase
Stühmer, Electrophysiological recording from Xenopus oocytes. 1992, Pubmed , Xenbase
Sui, Na+ activation of the muscarinic K+ channel by a G-protein-independent mechanism. 1996, Pubmed , Xenbase
Sui, Activation of the atrial KACh channel by the betagamma subunits of G proteins or intracellular Na+ ions depends on the presence of phosphatidylinositol phosphates. 1998, Pubmed , Xenbase
Vivaudou, Skeletal muscle ATP-sensitive K+ channels recorded from sarcolemmal blebs of split fibers: ATP inhibition is reduced by magnesium and ADP. 1991, Pubmed
Zhainazarov, Effects of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate on a Na+-gated nonselective cation channel. 1999, Pubmed
Zhang, Activation of inwardly rectifying K+ channels by distinct PtdIns(4,5)P2 interactions. 1999, Pubmed , Xenbase