Biochemical Assays of CaM and PiP2 Interactions with KCNQ Channel Domains

Abstract

Calmodulin (CaM) and phosphatidylinositol 4,5-bisphosphate (PIP2) are involved in controlling the gating of KCNQ ("M-type") K+ channels. It is well established that CaM has affinity for both the A and B helices of the KCNQ proximal C-terminus, and previous work indicates that CaM may be constitutively bound to the B helix. KCNQ channels are also suggested to contain multiple PIP2-binding sites, including a region of basic residues between the A and B helices (A-B linker) and a basic region at the C-terminal junction with the S6 transmembrane domain (S6Jx). Using a novel approach to understand the mechanisms controlling the modulation of KCNQ channels, we compared the biochemical affinities of PIP2 and CaM for these corresponding domains. Isothermal titration calorimetry (ITC) revealed a high affinity of Ca2+/CaM for a peptide corresponding to the KCNQ3 B helix (Kd ∼82 nM), whereas Ca2+/CaM bound the A helix with a much higher Kd (∼2 μM). The interactions between apo-CaM and the A or B helix peptide were not strong enough to obtain Kd values. Analytical ultracentrifugation results indicated that PIP2 directly bound to the S6Jx peptide, whereas its interaction with the A helix-B helix linker peptide is, as yet, indeterminate. These findings support the idea that CaM binds both the A and B helices of the KCNQ C-terminal domain in a Ca2+-dependent manner. Since the S6Jx domain is known to be close to the cell membrane by many structural studies of K+ channels, these data also suggest that only a very small conformational change in the C-termini of KCNQ channels is required to alter its binding to membrane PIP2. We will present more biochemical results that should help define the structural and molecular mechanism of PIP2 and CaM actions on KCNQ channels.