Novel Insights from Structural Analysis of Interactions of KCNQ K+ Channels with Calmodulin

Voltage-gated M-type K+ channels, made by KCNQ subunits, regulate excitability in nerve and muscle. The gating of these channels are modulated by receptors coupled to Gq/11 G proteins via several signals, such as calmodulin (CaM) and the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2), and protein kinases. Here, focusing on KCNQ4, we studied the structural and biochemical mechanisms of channel regulation by CaM, using isothermal titration calorimetry, microscale thermophoresis and nuclear magnetic resonance. As for a number of other ion channels, CaM directly interacts with the C-terminus of KCNQ channels, at their A & B helices. Using similar biochemical and structural methods, we also studied the interaction of KCNQ channels with PIP2 analogues and its cross-talk with CaM actions. Such cross-talk is made more likely by two loci of PIP2 interactions with the C-terminus near the A & B helices. Since it is still unclear how CaM and PIP2 binding cause physical rearrangements of the channels that affect gating, we probed the affinities of CaM for the A & B helices, asked whether CaM is constitutively bound to the channels, and utilized our crystal structure of CaM bound to the KCNQ4 A & B helices to gain insight into CaM-induced structural rearrangements of the C-terminus. To probe for potential cross-talk, we asked if Ca2+ binding to the CaM/C-terminal complex affects PIP2 interactions and thus, PIP2 regulation of channel gating. Supported by NIH grants R01 NS094461-01 and R01NS043394-11 to M.S.S. and NRSA training grant F31 NS090887-02/NIH to C.R.A.