Biochemical Analysis of the Regulation of Kv7 Channels by PIP2 and Calmodulin

Abstract

Kv7 (M-type, KCNQ) channels produce an outward potassium current (M-current) and play dominant roles in control of neuronal excitability due to their threshold of activation at sub-threshold potentials. Kv7 channels have been identified as therapeutic targets to reduce neuronal excitability in certain brain disorders, such as epilepsy and chronic pain. We seek to elucidate the molecular mechanisms of Kv7-channel regulation by second-messengers used in their regulation by Gq/11-coupled receptors, and the structural determinants of the channels that are involved. Activation of Gq/11-mediated signals results in the hydrolysis of the membrane-bound lipid, PIP2 (phosphatidylinositol 4,5-bisphosphate), and an increase in calcium-bound calmodulin (Ca2+/CaM) that is associated with M-current suppression and enhanced neuronal excitability. The structure of Kv7 channels consists of a 6-transmembrane-spanning region with an extended cytoplasmic carboxy-terminus containing a proximal “regulatory domain” with two alpha helices and two regions enriched in basic amino acids that are thought to be the principal sites of PIP2- and CaM-mediated binding. Recently, we examined the affinity of PIP2 to this regulatory domain spanning from the S6-TMD to the end of the B-helix domain (KCNQ-RD). By monitoring the shift in intrinsic protein fluorescence, we observed a change in the conformation of KCNQ-RD by the presence of the water-soluble analog, diC8-PIP2. Fluorescence polarization assays showed a reduction of anisotropy of a fluorescently labeled PIP2 analog with increasing KCNQ-RD titrations. These results support that PIP2 directly binds the KCNQ-RD. Since CaM also binds in this region, we hypothesize that the interactions of CaM and PIP2 with Kv7 channels are allosterically or sterically coupled, such that the binding of one molecule affects the affinity of binding of the other, or affects the efficacy of its action in modulating channel gating.