Competitive Interactions of Kv7 Channel Carboxy-Termini with PIP2 and Calmodulin

Abstract

Calmodulin (CaM) is a ubiquitous calcium sensor which binds to the carboxyl terminus of Kv7 channels, thus regulating channel function. Ca2+-dependent interactions of Kv7 with CaM mediate channel inhibition by specific G protein coupled receptors (GPCR). Yet other GPCR's inhibit Kv7 channels via the depletion of membrane phosphatidylinositol 4,5-bisphosphate (PIP2), which is required for Kv7 activity. Putative binding sites for PIP2 and CaM are in close proximity or overlap within the proximal C-termini of Kv7 channels. We investigated whether calmodulin and PIP2 binding to Kv7 channels is competitive, a phenomenon that would predict an increased affinity for Kv7-CaM interaction upon PIP2 depletion, and, conversely a decrease of Kv7 channel PIP2 affinity upon CaM binding. We performed co-immunoprecipitation between CaM and Kv7.4 overexpressed in HEKMSR cells under conditions of tonic PIP2, chronic PIP2 depletion (overexpression of PIP2 sequestering construct) and chronic PIP2 elevation (overexpression of phosphatidylinositol 5-kinase, PI5K). Sequestering PIP2 increased the co-immunoprecipitation of CaM with KCNQ4, while overproduction of PIP2 decreased CaM-binding. Next, we evaluated fluorescence recovery after photobleaching using TIRF illumination (TIRF-FRAP) between KCNQ4 and eYFP-CaM under conditions of normal, low and high membrane PIP2. When only eYFP-CaM was expressed in HEKMSR cells, TIRF-FRAP had a time constant of 43±9 s (n=24), co-expression of CaM with Kv7.4 showed an increase in the recovery time constant to 93±21 s (n=24, p≤0.05), indicating a fraction of CaM molecules are tethered to the plasma membrane by the interaction with Kv7.4. Depletion of PIP2 with wortmannin (10 μM) resulted in a further increase in recovery time to 453±165 s (n=27, p≤0.001); overexpression of PI5K resulted in the recovery time of 118±19 s (n=20, p≤0.001 vs. wortmannin). Collectively, these data strongly suggest a competitive nature for CaM and PIP2 interactions at the Kv7 C-terminus.