Activation of the atrial KACh channel by the betagamma subunits of G proteins or intracellular Na+ ions depends on the presence of phosphatidylinositol phosphates

Abstract

The betagamma subunits of GTP-binding proteins (Gbetagamma) activate the muscarinic K+ channel (KACh) in heart by direct binding to both of its component subunits. KACh channels can also be gated by internal Na+ ions. Both activation mechanisms show dependence on hydrolysis of intracellular ATP. We report that phosphatidylinositol 4,5-bisphosphate (PIP2) mimics the ATP effects and that depletion or block of PIP2 retards the stimulatory effects of Gbetagamma subunits or Na+ ions on channel activity, effects that can be reversed by restoring PIP2. Thus, regulation of KACh channel activity may be crucially dependent on PIP2 and phosphatidylinositol signaling. These striking functional results are in agreement with in vitro biochemical studies on the PIP2 requirement for Gbetagamma stimulation of G protein receptor kinase activity, thus implicating phosphatidylinositol phospholipids as a potential control point for Gbetagamma-mediated signal transduction.

Figure 1

PIP₂ mimics the MgATP dependence of the rundown of G protein-mediated activation as well as channel sensitization to gating by internal Na⁺ ions. (A) NP_o plot of K_ACh channel activity in an inside-out patch from a chicken embryonic atrial myocyte, showing that in the presence of ATP (2 mM, sodium salt, in the presence of 0.6 mM Mg²⁺ in the bath solution), GTP (100 μM) produced sustained activity (10 μM acetylcholine was present in the pipette). Withdrawal of ATP caused a gradual rundown of the GTP-induced activity. As can be seen at the beginning of the record ATP perfusion alone did not activate the channel. Application of each nucleotide is indicated by the bars. Representative single-channel currents are shown before and after the rundown of the activity, as indicated at time points a and b during the course of the experiment. (B) Plot similar to that in A, showing that PIP₂ restored the ability of GTP to stimulate channel activity and mimicked ATP in its ability to prevent rundown of the GTP-induced channel activity. Acetylcholine at 5 μM was present in the pipette solution. Perfusion of the inside-out patch with GTP (100 μM) and PIP₂ (5 μM) in the bath solution is indicated by bars. The arrows labeled a and b show time points during which rundown and restored single channel activity is shown, respectively. PIP₂, like ATP, did not stimulate channel activity when perfused alone. (C) NP_o and MT_o plots of K_ACh channel activity in inside-out patches from Xenopus oocyte coinjected with GIRK1/GIRK4 cRNAs. In the absence of MgATP, the channels did not respond to Na⁺ stimulation (20 mM). MgATP (5 mM) modified channels to a longer-lived open state but produced little activation. After this effect, channels responded to Na⁺ by increasing NP_o with no further significant change in MT_o. (D) Experiment similar to that in A from another patch showing that PIP₂ (1 μM) produced similar effects on MT_o and sensitization to gating by intracellular Na⁺ (20 mM). PIP₂ application, unlike ATP, had long lasting effects and, therefore, it was not necessary to apply it with Na⁺ ions for maximal effects.

Figure 2

PIP₂-Ab blocks the MgATP/Na or GTP[γS] activation of K_ACh channels. (A) Single-channel records and analyzed activity (NP_o) plots at the same compressed time scale, from an inside-out patch obtained from a Xenopus oocyte expressing K_ACh (GIRK1/GIRK4). K_ACh activation with MgATP/Na (5/20 mM) was blocked by PIP₂-antibody (titer, 1:1 or 1:500 dilution from manufacturer’s stock) rendering further MgATP/Na perfusion ineffective. (B) Plot similar to that in A showing GTP[γS]-mediated persistent activation (10 μM) and PIP₂-Ab (titer, 1:2 or 1:250 dilution from manufacturer’s stock) block from an inside-out patch obtained from an atrial cell (5 μM acetylcholine was present in the pipette solution).

Figure 3

Depletion of PIP₂ by PLCs blocks MgATP and G_βγ stimulation of K_ACh channel activity and restoration of PIP₂ reverses these effects. (A) Single-channel NP_o and MT_o plots as a function of time in the experiment were obtained from an inside-out patch of a Xenopus oocyte expressing GIRK1/GIRK4. The K_ACh channels were first activated by application of MgATP/Na (5/20 mM). Both NP_o and MT_o were increased by MgATP/Na. Subsequent treatment of the inside-out patch with phosphatidylinositol-specific PLC (1 unit/ml) for approximately 2.5 min, significantly reduced activation by MgATP/Na, causing a concomitant decrease in MT_o. (B) Single-channel NP_o and MT_o plots, obtained from an inside-out patch of a Xenopus oocyte expressing GIRK1/GIRK4. Soon after excision, G_βγ application (20 nM) caused persistent channel activation. Subsequent treatment of the patch with PLC-β2 (5 μg/ml) inhibited activity with a concomitant decrease in MT_o. (C) NP_o plot as a function of time in the experiment from an inside-out patch of Xenopus oocyte expressing GIRK1/GIRK4. The patch was exposed to PLC-β2 (5 μg/ml) for a period greater than 5 min, before G_βγ (20 nM) application. PLC-β2 treatment greatly retarded G_βγ effectiveness. Subsequent perfusion with PIP₂ (5 μM) revealed high channel activity, thus rescuing the G_βγ action. The number of active K_Ach channels in the membrane was greater than 5, thus precluding analysis of MT_o [see Sui et al. (13)]. (D) Single-channel NP_o and MT_o plots, obtained from an inside-out patch of Xenopus oocyte expressing GIRK1/GIRK4. Perfusion of the patch with ATP-free solutions for several minutes rendered G_βγ (20 nM) ineffective. Subsequent addition of MgATP (5 mM) to the membrane revealed high channel activity, thus rescuing the G_βγ action, which was presumably still bound but ineffective.

Figure 4

Two-gate model of K_ACh activation. State 1. In the absence of MgATP and Na⁺ ions or G_βγ subunits both channel gates are closed. State 2. In the presence of MgATP, inositol phospholipids are phosphorylated by lipid kinases to produce phosphoinositides. Direct interaction of phosphoinositides with the channel protein results in opening of the bottom gate. However, K⁺ ion permeation is limited or absent, because the top gate is closed. State 3. In the presence of the channel gating molecules (Na⁺ ions or G_βγ complex) but in the absence of MgATP, the top gate is open but K⁺ ion permeation is limited or absent, because the bottom gate is closed. State 4. In the presence of intact phosphoinositides and gating molecules (Na⁺ ions or G_βγ subunits), both gates are open resulting in K⁺ ion permeation.