Distinct subunit contributions to the activation of M-type potassium channels by PI(4,5)P2

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 (EC50) values of 1.3 +/- 0.14 and 75.5 +/- 2.5 mu M, 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 EC50 values of 76.2 +/- 19.9 or 3.6 +/- 1.0 mu M, 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.