Purification and membrane interactions of human KCNQ1 100-370 potassium ion channel.

Autor: Dixit G; Department of Chemistry and Biochemistry, Miami University, 651 E. High Street, Oxford, OH 45056, USA; Cell, Molecular and Structural Biology Program, Department of Chemistry & Biochemistry, Miami University, Oxford, OH 45056, USA., Stowe RB; Department of Chemistry and Biochemistry, Miami University, 651 E. High Street, Oxford, OH 45056, USA., Bates A; Department of Chemistry and Biochemistry, Miami University, 651 E. High Street, Oxford, OH 45056, USA., Jaycox CK; Department of Chemistry and Biochemistry, Miami University, 651 E. High Street, Oxford, OH 45056, USA., Escobar JR; Department of Chemistry and Biochemistry, Miami University, 651 E. High Street, Oxford, OH 45056, USA; Cell, Molecular and Structural Biology Program, Department of Chemistry & Biochemistry, Miami University, Oxford, OH 45056, USA., Harding BD; Department of Chemistry and Biochemistry, Miami University, 651 E. High Street, Oxford, OH 45056, USA., Drew DL Jr; Department of Chemistry and Biochemistry, Miami University, 651 E. High Street, Oxford, OH 45056, USA., New CP; Department of Chemistry and Biochemistry, Miami University, 651 E. High Street, Oxford, OH 45056, USA; Cell, Molecular and Structural Biology Program, Department of Chemistry & Biochemistry, Miami University, Oxford, OH 45056, USA., Sahu ID; Department of Chemistry and Biochemistry, Miami University, 651 E. High Street, Oxford, OH 45056, USA., Edelmann RE; Center for Advanced Microscopy and Imaging, Miami University, Oxford, OH 45056, USA., Dabney-Smith C; Department of Chemistry and Biochemistry, Miami University, 651 E. High Street, Oxford, OH 45056, USA; Cell, Molecular and Structural Biology Program, Department of Chemistry & Biochemistry, Miami University, Oxford, OH 45056, USA., Sanders CR; Department of Biochemistry and Center for Structural Biology, Vanderbilt University, Nashville, TN 37240, USA., Lorigan GA; Department of Chemistry and Biochemistry, Miami University, 651 E. High Street, Oxford, OH 45056, USA; Cell, Molecular and Structural Biology Program, Department of Chemistry & Biochemistry, Miami University, Oxford, OH 45056, USA. Electronic address: gary.lorigan@miamioh.edu.
Jazyk: angličtina
Zdroj: Biochimica et biophysica acta. Biomembranes [Biochim Biophys Acta Biomembr] 2022 Nov 01; Vol. 1864 (11), pp. 184010. Date of Electronic Publication: 2022 Jul 21.
DOI: 10.1016/j.bbamem.2022.184010
Abstrakt: KCNQ1 (Kv7.1 or KvLQT1) is a voltage-gated potassium ion channel that is involved in the ventricular repolarization following an action potential in the heart. It forms a complex with KCNE1 in the heart and is the pore forming subunit of slow delayed rectifier potassium current (I ks ). Mutations in KCNQ1, leading to a dysfunctional channel or loss of activity have been implicated in a cardiac disorder, long QT syndrome. In this study, we report the overexpression, purification, biochemical characterization of human KCNQ1 100 - 370 , and lipid bilayer dynamics upon interaction with KCNQ1 100 - 370 . The recombinant human KCNQ1 was expressed in Escherichia coli and purified into n-dodecylphosphocholine (DPC) micelles. The purified KCNQ1 100 - 370 was biochemically characterized by SDS-PAGE electrophoresis, western blot and nano-LC-MS/MS to confirm the identity of the protein. Circular dichroism (CD) spectroscopy was utilized to confirm the secondary structure of purified protein in vesicles. Furthermore, 31 P and 2 H solid-state NMR spectroscopy in DPPC/POPC/POPG vesicles (MLVs) indicated a direct interaction between KCNQ 100 - 370 and the phospholipid head groups. Finally, a visual inspection of KCNQ1 100 - 370 incorporated into MLVs was confirmed by transmission electron microscopy (TEM). The findings of this study provide avenues for future structural studies of the human KCNQ1 ion channel to have an in depth understanding of its structure-function relationship.
Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Gary Lorigan reports financial support was provided by NIGMS. Gary Lorigan reports financial support was provided by NIH.
(Copyright © 2022. Published by Elsevier B.V.)
Databáze: MEDLINE
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