The function of BK channels extracted and purified within SMALPs

Abstract

Human BK channels are large voltage and Ca2+-activated K+ channels, involved in several important functions within the body. The core channel is a tetramer of α subunits, and its function is modulated by the presence of β and γ accessory subunits. BK channels composed of α subunits, as well as BK channels composed of α and β1 subunits, were successfully solubilised from HEK cells with styrene maleic acid (SMA) polymer and purified by nickel affinity chromatography. Native SMA-PAGE analysis of the purified proteins showed the α subunits were extracted as a tetramer. In the presence of β1 subunits, they were co-extracted with the α subunits as a heteromeric complex. Purified SMA lipid particles (SMALPs) containing BK channel could be inserted into planar lipid bilayers (PLB) and single channel currents recorded, showing a high conductance (≈ 260 pS), as expected. The open probability was increased in the presence of co-purified β1 subunits. However, voltage dependent gating of the channel was restricted. In conclusion, we have demonstrated that SMA can be used to effectively extract and purify large, complex, human ion channels, from low expressing sources. That these large channels can be incorporated into PLB from SMALPs and display voltage-dependent channel activity. However, the SMA appears to reduce the voltage dependent gating of the channels.

Publication DOI: https://doi.org/10.1042/BCJ20210628
Divisions: College of Health & Life Sciences > School of Biosciences
College of Health & Life Sciences > School of Biosciences > Cellular and Molecular Biomedicine
College of Health & Life Sciences
Additional Information: © 2022 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Funding: Aston University (Aston) - Life & Health Sciences studentship; UKRI | Biotechnology and Biological Sciences Research Council (BBSRC) - BB/S008160/1
Uncontrolled Keywords: BK channel,SMALP,SMA–PAGE,detergent-free,planar lipid bilayer,Biochemistry,Molecular Biology,Cell Biology
Publication ISSN: 1470-8728
Last Modified: 29 May 2024 16:02
Date Deposited: 25 Jul 2022 10:40
Full Text Link:
Related URLs: https://portlan ... ed-and-purified (Publisher URL)
http://www.scop ... tnerID=8YFLogxK (Scopus URL)
PURE Output Type: Article
Published Date: 2022-08-12
Published Online Date: 2022-07-25
Accepted Date: 2022-07-19
Authors: Rothnie, Alice (ORCID Profile 0000-0002-4259-7015)
Patel, Jaimin
Pollock, Naomi L.
Maher, Jacqueline
Allen, Marcus

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