Abstract
Phosphatidylinositol −4,5-bisphosphate (PI(4,5)P2) is an important regulator of activity of a variety of ion channels including inwardly rectifying potassium channels, KCNQ, TRP, and voltage-gated calcium channels. Several groups have provided evidence that also voltage-gated potassium (Kv) channels might be regulated by PI(4,5)P2. Taking the wide expression of Kv channels in a variety of different tissues into account, such regulation of activity by PI(4,5)P2 could potentially be of great physiological importance. To determine whether several Kv channels show a regulation of their activity by PI(4,5)P2 we have coexpressed them in tsA201-cells with either a voltage-gated lipid 5-phosphatase (VSP), a G-protein coupled receptor (M1R), or an engineered fusion protein carrying both a 4- and 5-lipid phosphatase activity (Pseudojanin, PJ). These tools deplete PI(4,5)P2 with application of depolarization, muscarinic agonists, or rapamycin, respectively. We monitored PI(4,5)P2 amounts at the plasma membrane by FRET with PH-probes from PLCδ1 simultaneously with whole-cell patch-clamp recordings. Activating VSP or PJ inhibited KCNQ2/3 channel current leaving only about 5-10 % remaining current. Activation of M1R inhibited KCNQ2/3 current a comparable amount. Thus the tools used for our assays are working. We tested with them for potential regulation of activity by PI(4,5)P2 of Kv1.1/Kvβ1.1, Kv1.3, Kv1.4 and Kv1.5/Kvβ1.3, Kv3.4, Kv4.2, Kv4.3 (both with different KChIPs and DPP6-s) and HERG/KCNE2. Interestingly, we found a substantial upregulation of current density and a removal of inactivation for Kv1.1/Kvβ1.1 and Kv3.4 upon activation of M1R, but no changes in activity upon only activating VSP or PJ. All other channels tested showed no alteration in activity in any of the assays we used. In conclusion, “physiological” depletion of PI(4,5)P2 at the plasma membrane does not seem to influence activity of most tested Kv channels. Supported by NIH grant NS08174 and the AvH Foundation.
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