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Jan 08, 2019
Biochemistry
KCNQ1 (Kv7.1 or KvLQT1) is a potassium ion channel protein found in the heart, ear and other tissues. In complex with the KCNE1 accessory protein it plays a role during the repolarization phase of the cardiac action potential. Mutations in the channel have been associated with several diseases including congenital deafness, sudden infant death syndrome, and long QT syndrome. NMR structural studies in detergent micelles and a cryo-EM structure of KCNQ1 from Xenopus laevis have shown that the voltage sensor domain (Q1-VSD) of the channel has four transmembrane helices, S1-S4, showing an overall structural similarity with other VSDs characterized to date. In this study, we describe a reliable method for the reconstitution of Q1-VSD into POPC/POPG lipid bilayered vesicles. Here, site-directed spin labeling EPR spectroscopy was used to probe the structural dynamics and topology of the numerous residues of Q1-VSD in POPC/POPG lipid bilayered vesicles. Spin-labeled cysteine mutants F130C and F232C were found to be located inside the vesicles, while Q147C and F222C were found to be solvent accessible. The dynamics of the bilayer vesicles upon incorporation of Q1-VSD were studied using 31P solid-state NMR spectroscopy by varying the protein to lipid molar ratios confirming the incorporation of the protein within the bilayer vesicles. Circular dichroism spectroscopic data showed that the α-helical content of Q1-VSD is higher for the protein reconstituted in vesicles relative to the previous studies using DPC detergent micelles. This study provides an insight into the structural topology and dynamics of the voltage sensor domain of the potassium ion channel reconstituted in a lipid bilayer environment, forming the basis for more advanced structural and functional studies.

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