Biophysical interactions of PIP2 and calmodulin with KCNQ (Kv7) K+ ion channels

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 DESCRIPTION (provided by applicant): Biophysical interactions of PIP2 and calmodulin with KCNQ (Kv7) K ion channels + KCNQ potassium channels control cellular excitability, and inherited mutations in the proximal half of the C- terminus of these membrane proteins can result in cardiac arrhythmia, deafness and epilepsy. The signature "M-current" produced by KCNQ channels was first observed in sympathetic neurons, and can be inhibited by stimulation of Gq/11 muscarinic receptors. Phosphatidylinositol 4, 5-bisphosphate (PIP2) and calmodulin (CaM) are Gq/11 second messenger molecules suggested to modulate KCNQ channel function by directly binding the proximal C-terminus of KCNQ channels. As a result, many inherited mutations may interfere with KCNQ channel function by disrupting PIP2 and CaM binding to the channels. It is well established that CaM can bind both the A and B helices of the C-terminus of KCNQ channels, and previous work indicates that CaM may be constitutively bound to the channels. The precise locations of the PIP2 binding sites are less clear, but are suggested to lie on two distinct domains enriched with basic amino acids that also reside on the KCNQ proximal C-terminus. The mechanisms for how PIP2 and CaM modulate KCNQ channels, and their interactions between each other, are as yet uncertain. However, the close proximity of these binding sites to each other suggests that these molecules may engage in a rich crosstalk dynamic to modulate KCNQ channel function. The overarching hypothesis is that the complex interactions between PIP2 and CaM guide the function of KCNQ channels. This study presents a novel approach to understand the mechanisms controlling the modulation of KCNQ channels. Cutting edge biophysical methods will be used to determine the biochemical binding affinities of PIP2 and CaM for KCNQ channels, and their precise sites of action. In order to gain a comprehensive understanding of the binding affinities, the experiments in this study employ purified protein fragments corresponding to the proximal half of the KCNQ C-terminus, in addition to short peptides corresponding to the proposed binding domains. Our preliminary data show stunning differences in the binding affinities and thermodynamic parameters of calmodulin for KCNQ channels. Also compelling is that these preliminary results hint at drastic differences of PIP2 affinity for each of the proposed domains on each KCNQ channel subtype. The completion of this project is expected to provide a significant impact on many ion channel diseases, since the molecular mechanisms for controlling KCNQ channels appear common to many other ion channels. As the applicant continues to progress in her research training in the supportive environment at The University of Texas Health Science Center, we will present more results that should help define the structural and molecular mechanism of PIP2 and CaM actions on KCNQ channels.