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Ethanol inhibits Kv7.2/7.3 channel open probability by reducing the PI(4,5)P2 sensitivity of Kv7.2 subunit.

ABSTRACT: Ethanol often causes critical health problems by altering the neuronal activities of the central and peripheral nerve systems. One of the cellular targets of ethanol is the plasma membrane proteins including ion channels and receptors. Recently, we reported that ethanol elevates membrane excitability in sympathetic neurons by inhibiting Kv7.2/7.3 channels in a cell type-specific manner. Even though our studies revealed that the inhibitory effects of ethanol on the Kv7.2/7.3 channel was diminished by the increase of plasma membrane phosphatidylinositol 4,5-bisphosphate (PI (4,5)P2), the molecular mechanism of ethanol on Kv7.2/7.3 channel inhibition remains unclear. By investigating the kinetics of Kv7.2/7.3 current in high K+ solution, we found that ethanol inhibited Kv7.2/7.3 channels through a mechanism distinct from that of tetraethylammonium (TEA) which enters into the pore and blocks the gate of the channels. Using a non-stationary noise analysis (NSNA), we demonstrated that the inhibitory effect of ethanol is the result of reduction of open probability (PO) of the Kv7.2/7.3 channel, but not of a single channel current (i) or channel number (N). Finally, ethanol selectively facilitated the kinetics of Kv7.2 current suppression by voltage-sensing phosphatase (VSP)-induced PI(4,5)P2 depletion, while it slowed down Kv7.2 current recovery from the VSP-induced inhibition. Together our results suggest that ethanol regulates neuronal activity through the reduction of open probability and PI(4,5)P2 sensitivity of Kv7.2/7.3 channels. [BMB Reports 2021; 54(6): 311-316].

SUBMITTER: Kim KW

PROVIDER: S-EPMC8249878 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Low-threshold voltage-gated M-type potassium channels (M channels) are tetraheteromers, commonly of two Kv7.2 and two Kv7.3 subunits. Though gated by voltage, the channels have an absolute requirement for binding of the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)) to open. We have investigated the quantitative relation between the concentration of a water-soluble PI(4,5)P(2) analog, dioctanoyl-PI(4,5)P(2) (DiC(8)-PI(4,5)P(2)), and channel open probability (P(open)) by fast application of increasing concentrations of DiC(8)-PI(4,5)P(2) to the inside face of membrane patches excised from Chinese hamster ovary cells expressing M channels as heteromeric Kv7.2/7.3 subunits. The rationale for the experiments is that this will mimic the effect of changes in membrane PI(4,5)P(2) concentration. Single-channel conductances from channel current-voltage relations in cell-attached mode were 9.2 ± 0.1 pS with a 2.5-mM pipette [K(+)]. Plots of P(open) against DiC(8)-PI(4,5)P(2) concentration were best fitted using a two-component concentration-P(open) relationship with high and low affinity, half-maximal effective concentration (EC(50)) values of 1.3 ± 0.14 and 75.5 ± 2.5 µM, respectively, and Hill slopes of 1.4 ± 0.06. In contrast, homomeric channels from cells expressing only Kv7.2 or Kv7.3 constructs yielded single-component curves with EC(50) values of 76.2 ± 19.9 or 3.6 ± 1.0 µM, respectively. When wild-type (WT) Kv7.2 was coexpressed with a mutated Kv7.3 subunit with >100-fold reduced sensitivity to PI(4,5)P(2), the high-affinity component of the activation curve was lost. Fitting the data for WT and mutant channels to an activation mechanism with independent PI(4,5)P(2) binding to two Kv7.2 and two Kv7.3 subunits suggests that the two components of the M-channel activation curve correspond to the interaction of PI(4,5)P(2) with the Kv7.3 and Kv7.2 subunits, respectively, that channels can open when only the two Kv7.3 subunits have bound DiC(8)-PI(4,5)P(2), and that maximum channel opening requires binding to all four subunits.

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Ethanol inhibits Kv7.2/7.3 channel open probability by reducing the PI(4,5)P2 sensitivity of Kv7.2 subunit.

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