Synergistic activation of G protein-gated inwardly rectifying potassium channels by the betagamma subunits of G proteins and Na(+) and Mg(2+) ions

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.

Figure 3

Mg²⁺ ions gate the ATP-modified GIRK channels. NP _o, NF _o, and MT _o 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. NP _o, NF _o, and MT _o 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 K_ACh by MgATP and Na⁺. (A) Single channel activity (top; NP _o, bin = 5 s) plotted as a function of time. The data were obtained from an inside-out patch excised from an atrial cell. The K_ACh 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) NP _o plot of K_ACh 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 (NP _o, 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 NP _o for seven patches is plotted for different conditions. Steady state channel activity after activation by GTPγS was taken as reference (GTPγS) and NP _o 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 NP _o 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) NP _o 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. V_m = −80 mV. 5 μM acetylcholine was present in the pipette. (D) The mean NP _o for nine patches are plotted for different conditions. Steady state channel activity after β₁γ₇ activation (after β₁γ₇ washout) was taken as reference and NP _o 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 NP _o 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 G_βγ and/or Na⁺ and Mg²⁺ ions. (C_o) 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 (NP _o) 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 (NP _o, 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 NP _o of four experiments such as that shown in A. The mean NP _o 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) K_ACh 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 G_βγ, Na⁺, and Mg²⁺ ions in activating GIRK channels. (A) The mean NP _o 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 NP _o 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 NP _o was 0.43 ± 0.14. 10 mM Na⁺ ions gave a mean NP _o of 0.40 ± 0.12. When applied together, in the presence of PIP₂, Mg²⁺ and Na⁺ ions (10 mM each) yielded a mean NP _o of 2.08 ± 0.52. (B) Mean NP _o plots for six inside-out patches from oocytes expressing GIRK1/GIRK4. V_m 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 NP _o 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 NP _o of 0.12 ± 0.045. After GTPγS washout, Mg²⁺ ions gave a mean NP _o of 0.12 ± 0.03. 10 mM Na⁺ ions gave a mean NP _o of 0.23 ± 0.06. Coapplication of Mg²⁺ and Na⁺ ions resulted in a mean NP _o of 0.26 ± 0.08. (C) Mean NP _o plots for six patches. The inside-out patches were excised from oocytes expressing GIRK1/GIRK4. V_m 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 NP _o 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 NP _o 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 NP _o of 0.2 ± 0.09. GTPγS gave a mean NP _o 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 NP _o of 0.42 ± 0.19 was obtained. Mg²⁺ and Na⁺ ions applied together resulted in NP _o of 1.6 ± 0.49.