Regulation of M-type (Kv7) potassium channels by transmitters and messengers: is PIP2 the final common pathway?. (360G-Wellcome-085419_Z_08_Z)

Our question is: do different intracellular and extracellular Kv7 channel regulators act by changing the ability of membrane PIP2 to activate the channels? We will use two complementary approaches to answer this electrophysiological recording of M/Kv7 channel behaviour, and biochemical measurements of PIP2 binding to Kv7 fusion proteins. The rationale behind the electrophysiological experiments is that, if different intracellular and extracellular signals ultimately converge on changes in PIP2 channel interaction, they should show similar and predictable effects on channel kinetic behaviour. We have developed a provisional model of PIP2 regulated channel kinetics and will assess how far this predicts the changes produced by altering the amounts or availability of PIP2, and compare this with the effects of other regulators and of different transmitters. This will be done using sympathetic neurons in which the various regulators, receptors and transducers common to nerve cells are al ready extant. Additionally, we will test responses of Kv7.2/7.3 channels, channel subunits and mutated subunits expressed in CHO cells to PIP2 and other regulators. Quantitative binding studies of Kv7.2 and Kv7.3 C-terminus fusion proteins (already prepared) to PIP2 and other anionic phospholipids will be undertaken using phospholipid arrays and surface plasmon resonance.