Regulation of Kv7 (KCNQ) K+ channel open probability by phosphatidylinositol 4,5-bisphosphate

Abstract

Voltage-gated Kv7 (KCNQ) channels underlie important K+ currents, including the neuronal M current, and are thought to be sensitive to membrane phosphatidylinositol 4,5-bisphosphate (PIP2) and PIP2 depletion to underlie muscarinic receptor inhibition. We studied regulation of Kv7.2-7.4 channels by PIP2 in Chinese hamster ovary (CHO) cells using single-channel and whole-cell patch clamp and biochemical analysis. Maximal open probabilities (Po) of Kv7.2-Kv7.4 homomultimers and of Kv7.2/7.3 heteromultimers were found to be strongly dependent on the [diC8-PIP2] applied to inside-out patches, with differential apparent affinities that correlate with their maximal Po in on-cell mode. Unitary conductance was not affected by PIP2. Raising tonic [PIP2] by coexpression of phosphatidylinositol (4)5-kinase increased the maximal Po of both Kv7.2 and Kv7.2/7.3 channels studied in on-cell patches and increased whole-cell Kv7.2, but not Kv7.3, current amplitudes. In cells coexpressed with muscarinic M1 receptors, bath application of muscarinic agonist reduced the maximal Po of Kv7.2/7.3 channels isolated in on-cell patches. Coexpression of a PIP2 sequestering construct moderately reduced whole-cell Kv7.2/7.3 currents, and coexpression of a construct containing a PIP2 phosphatase nearly abolished them. Finally, biochemical analysis of anionic phospholipids in CHO cells stably expressing M1 receptors shows that PIP2 and PIP are nearly depleted 1 min after muscarinic stimulation, with an unexpected rebound after 10 min. These results strongly support the direct regulation of Kv7 channels by PIP2 and its depletion as the mechanism of muscarinic suppression of M channels. Divergent apparent affinities of Kv7.2-7.4 channels for PIP2 may underlie their highly differential maximal Po observed in cell-attached patches.

Figure 1.

DiC8-PIP₂ stabilizes the opening of Kv7.2/7.3 channels. A, Current records from a patch containing a single Kv7.2/7.3 channel in cell-attached mode (On-cell) and in inside-out mode with various concentrations of diC8-PIP₂ in the cytoplasmically facing bath solution. B, Each 3 s sweep during the experiment was analyzed for channel P_o, and the time course of the P_o during the experiment is plotted. The transition from on-cell to inside-out mode is indicated by the arrow, and the applications of different diC8-PIP₂ concentrations are indicated by the bars.

Figure 2.

A-C, Individual sweeps from patches containing a single Kv7.2 (A), Kv7.3 (B), or Kv7.4 (C) channel in the presence of the indicated concentration of diC8-PIP₂. The records were filtered at 500 Hz (A, B) and 200 Hz (C).

Figure 3.

A, Dose-response relationship of the P_o for Kv7.2 (squares), Kv7.3 (triangles), Kv7.4 (inverted triangles), and Kv7.2/7.3 (circles) channels as a function of diC8-PIP₂ concentration in inside-out patches. The data were fit by a Hill equation (see Materials and Methods), with data errors taken into account by the fitting routine for all channels, except Kv7.2/7.3. The parameters of the fits are given in the text. B, The unitary current amplitudes for Kv7.2/7.3 channels over a range of voltages, either in the absence or presence of diC8-PIP₂ (30 μm) in the bath, are plotted. The data were fit linearly, yielding single-channel conductances given in the text.

Figure 4.

Overexpression of PI(4)5-kinase increases the P_o of Kv7 channels in patches and increases whole-cell Kv7.2 but not Kv7.3, currents. A, Individual sweeps from a patch containing either a single Kv7.2/7.3 or a Kv7.2 channel from control CHO cells or those cotransfected with PI(4)5-kinase are shown. Recordings were first made in on-cell modes, followed by excision to inside-out modes. O, Open channel levels; C, closed channel levels. B, Bars show the mean P_o at 0 mV for all of the on-cell patches. C, Bars show whole-cell current amplitudes from CHO cells transfected with Kv7.2 or Kv7.3 alone or together with PI(4)5-kinase. Traces from representative experiments are displayed above. The voltage protocol used is as shown.

Figure 5.

Stimulation of M₁ receptors by bath application of agonist reduces the P_o of channels in cell-attached patches. CHO cells were cotransfected with Kv7.2+7.3 channels and M₁ receptors. A, Sweeps show channel activities from a patch containing two Kv7.2/3 channels before, during, and after bath application of oxo-M (10 μm). B, Bars show the mean unitary P_o from four experiments such as in A. O, Open channel levels; C, closed channel levels.

Figure 6.

Expression of PIP₂ sequestering or degrading constructs reduces whole-cell Kv7.2/7.3 current amplitudes. CHO cells were cotransfected with Kv7.2+Kv7.3 together with either GFP only (control) or the PLCδ PH, Lyn-PH-PP, or Akt-PH constructs. Currents were elicited using the indicated voltage protocol in whole-cell configuration. A, Plotted are superimposed current traces from four different cells transfected with each different construct. B, Bars show the mean whole-cell current amplitudes for these four groups of cells. ^**p < 0.01; ^***p < 0.001.

Figure 7.

Effects of muscarinic stimulation on anionic phospholipids in CHO cells expressing M₁ receptors. Anionic phospholipids were determined from their deacylation products by anion exchange HPLC with suppressed conductivity detection. Data are compiled for application of the cholinergic agonist carbachol (100 μm) for 1 or 10 min. Bars show the mean PIP₂, PIP, PI, or PA, expressed as a percentage of total membrane phosphoinositides. The left axis refers to %PIP₂ and %PIP, and the right axis refers to %PI and %PA.