Purification and membrane interactions of human KCNQ1100-370 potassium ion channel

Gunjan Dixit; Rebecca B Stowe; Alison Bates; Colleen K Jaycox; Jorge R Escobar; Benjamin D Harding; Daniel L Drew Jr; Christopher P New; Indra D Sahu; Richard E Edelmann; Carole Dabney-Smith; Charles R Sanders; Gary A Lorigan

doi:10.1016/j.bbamem.2022.184010

Purification and membrane interactions of human KCNQ1_100-370 potassium ion channel

Biochim Biophys Acta Biomembr. 2022 Nov 1;1864(11):184010. doi: 10.1016/j.bbamem.2022.184010. Epub 2022 Jul 21.

Affiliations

¹ 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.
² Department of Chemistry and Biochemistry, Miami University, 651 E. High Street, Oxford, OH 45056, USA.
³ Center for Advanced Microscopy and Imaging, Miami University, Oxford, OH 45056, USA.
⁴ Department of Biochemistry and Center for Structural Biology, Vanderbilt University, Nashville, TN 37240, USA.
⁵ 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.

PMID: 35870481
DOI: 10.1016/j.bbamem.2022.184010

Abstract

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 KCNQ1₁₀₀_-₃₇₀, and lipid bilayer dynamics upon interaction with KCNQ1₁₀₀_-₃₇₀. The recombinant human KCNQ1 was expressed in Escherichia coli and purified into n-dodecylphosphocholine (DPC) micelles. The purified KCNQ1₁₀₀_-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, ³¹P and ²H solid-state NMR spectroscopy in DPPC/POPC/POPG vesicles (MLVs) indicated a direct interaction between KCNQ₁₀₀_-₃₇₀ and the phospholipid head groups. Finally, a visual inspection of KCNQ1₁₀₀_-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.

Keywords: K(+) channel; KCNQ1; Membrane protein; NMR.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Humans
KCNQ1 Potassium Channel / metabolism
Long QT Syndrome*
Potassium / metabolism
Potassium Channels
Potassium Channels, Voltage-Gated* / metabolism
Tandem Mass Spectrometry

Substances

KCNQ1 Potassium Channel
KCNQ1 protein, human
Potassium Channels
Potassium Channels, Voltage-Gated
Potassium

Grants and funding

R35 GM126935/GM/NIGMS NIH HHS/United States