The phosphoinositide sensitivity of the K(v) channel family

Abstract

Recently, we screened several KV channels for possible dependence on plasma membrane phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2). The channels were expressed in tsA-201 cells and the PI(4,5)P 2 was depleted by several manipulations in whole-cell experiments with parallel measurements of channel activity. In contrast to reports on excised-patches using Xenopus laevis oocytes, we found only KV 7, but none of the other tested KV channels, to be strongly dependent on PI(4,5)P 2. We now have extended our study to KV 1.2 channels, a KV channel we had not previously tested, because a new published study on excised patches showed regulation of the voltage-dependence of activation by PI(4,5)P 2. In full agreement with those published results, we found a reduction of current amplitude by ~20% after depletion of PI(4,5)P 2 and a small left shift in the activation curve of KV 1.2 channels. We also found a small reduction of KV 11.1 (hERG) currents that was not accompanied by a gating shift. In conclusion, our whole-cell methods yield a PI(4,5)P 2-dependence of KV 1.2 currents in tsA-201 cells that is comparable to findings from excised patches of Xenopus laevis oocytes. We discuss possible physiological rationales for PI(4,5)P 2 sensitivity of some ion channels and insensitivity of others.

Keywords: KV1.2; Voltage-gated potassium channel; phosphatidylinositol 4,5-bisphosphate.

Figure 1. PI(4,5)P₂ dependence of K_V1.2 channels. (A) K_V1.2 channels and M₁R were transiently expressed in tsA-201 cells. Currents were recorded in the whole-cell configuration (pulse protocol shown above current traces). Figure shows current traces before (solid black) and after (dashed gray) application of 10 µM Oxo-M. (B) Time course of K_V1.2 mediated currents at +20 mV for the experiment shown in (A). (C) Current traces of K_V1.2 channels coexpressed with pseudojanin-YFP and LDR-CFP before (solid black) and after (dashed gray) addition of rapamycin. (D) Percentage inhibition of K_V1.2 channels by activation of M₁R or by recruitment of pseudojanin to the plasma membrane. (E) Representative G-V curve generated from test pulses to membrane potentials of -80 to +60 mV from a holding potential of -80 mV before (solid black) and after (dashed gray) activation of M₁R by Oxo-M application. (F) Voltage of half-maximal activation (V_0.5) for K_V1.2 channels before and after M₁R-activation. *p < 0.05.

Figure 2. Voltage-dependence of activation of hERG channels before and after PI(4,5)P₂ depletion. (A) Figure shows current traces for hERG channels expressed in tsA-201 cells together with M₁R before (solid black) and after (dashed gray) application of 10 µM Oxo-M. Pulse protocols as shown above current traces. (B) Time course of hERG channel mediated current at +40 mV from the experiment shown in (A). (C) Voltages of half-maximal activation (V_0.5) of hERG channels before and after M₁R-activation. (D) Current traces for hERG channels expressed together with pseudojanin-YFP (PJ) and LDR-CFP before (solid black) and after (dashed gray) recruitment of PJ to the plasma membrane by rapamycin-application. (E) Representative G-V curve of hERG channels generated from test pulses to membrane potentials of -80 to +60 mV from a holding potential of -80 mV before (solid black) and after (dashed gray) recruitment of PJ to the plasma membrane. (F) Voltages of half-maximal activation (V_0.5) for hERG channels before and after PJ-recruitment.