KCNQ (Kv7, M-type) K+ channels are key regulators of neuronal excitability. KCNQ channels are modulated by specific G protein coupled receptors which inhibit the channel via (1) a depletion in membrane phosphatidylinositol 4,5-bisphosphate (P1P2) or (2) via Ca2+-calrnodulin (CaM). Despite the suggested close proximity (or even overfap) of PIP2 and CaM binding sites within KCNQ carboxy termini, the relationships between these two mechanisms of channel regulation are not known. We investigated the competitive interaction of CaM and PIP2 with the high-PIP2-affinity KCNQ3 and low-PIP2- affinity KCNQ4 channels using a combination of electrophysiological, imaging and biochemical approaches. Using fluorescence recovery after photobleaching under total internal reflection fluorescent illumination (TIRF-FRAP) we show that chronic PIP2 depletion (by overexpression of 5'phosphatase or application of wortmannin) significantly enhances KCNQ4 interaction with CaM while KCNQ3/CaM interaction was not affected. Conversely an increase in PIP2 did not change the KCNQ4/CaM interaction, relative to those cells overexpressing KCNQ4+CaM only. These findings were confirmed in our biochemical experiments, suggesting that co-immunoprecipitation of KCNQ4, but not KCNQ3, by CaM is enhanced by PIP, depletion and reduced upon an increase in membrane PIP2 (achieved by overexpression of PI5-kinase).