TY - JOUR
T1 - Expression, purification, and projection structure by single particle electron microscopy of functional human TRPM4 heterologously expressed in Xenopus laevis oocytes
AU - Clémençon, Benjamin
AU - Fine, Michael
AU - Lüscher, Benjamin
AU - Baumann, Marc U.
AU - Surbek, Daniel V.
AU - Abriel, Hugues
AU - Hediger, Matthias A.
N1 - Funding Information:
This study was supported by the Swiss National Science Foundation (SNSF) through the National Centre of Competence in Research (NCCR) TransCure and The TransCure International Fellowship Program (IFP TransCure), funded in part by an FP7 European Marie Curie Actions Grant.
Copyright:
Copyright 2014 Elsevier B.V., All rights reserved.
PY - 2014/3
Y1 - 2014/3
N2 - Despite efforts implicating the cationic channel transient receptor potential melastatin member 4 (TRPM4) to cardiac, nervous, and immunological pathologies, little is known about its structure and function. In this study, we optimized the requirements for purification and extraction of functional human TRPM4 protein and investigated its supra-molecular assembly. We selected the Xenopus laevis oocyte expression system because it lacks endogenous TRPM4 expression, it is known to overexpress functional human membrane channels, can be used for structure-function analysis within the same system, and is easily scaled to improve yield and develop moderate throughput capabilities through the use of robotics. Negative-stain electron microscopy (EM) revealed various sized low-resolution particles. Single particle analysis identified the majority of the projections represented the monomeric form with additional oligomeric structures potentially characterized as tetramers. Two-electrode voltage clamp electrophysiology demonstrated that human TRPM4 is functionally expressed at the oocyte plasma membrane. This study opens the door for medium-throughput screening and structure-function determination of this important therapeutically relevant target.
AB - Despite efforts implicating the cationic channel transient receptor potential melastatin member 4 (TRPM4) to cardiac, nervous, and immunological pathologies, little is known about its structure and function. In this study, we optimized the requirements for purification and extraction of functional human TRPM4 protein and investigated its supra-molecular assembly. We selected the Xenopus laevis oocyte expression system because it lacks endogenous TRPM4 expression, it is known to overexpress functional human membrane channels, can be used for structure-function analysis within the same system, and is easily scaled to improve yield and develop moderate throughput capabilities through the use of robotics. Negative-stain electron microscopy (EM) revealed various sized low-resolution particles. Single particle analysis identified the majority of the projections represented the monomeric form with additional oligomeric structures potentially characterized as tetramers. Two-electrode voltage clamp electrophysiology demonstrated that human TRPM4 is functionally expressed at the oocyte plasma membrane. This study opens the door for medium-throughput screening and structure-function determination of this important therapeutically relevant target.
KW - Hi clamp two-electrode voltage clamp (TEV) system
KW - Human TRPM4
KW - Membrane protein purification
KW - Roboinject system
KW - Single particle analysis (SPA)
KW - Transmission electron microscopy (TEM)
KW - Xenopus laevis oocytes
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U2 - 10.1016/j.pep.2013.11.017
DO - 10.1016/j.pep.2013.11.017
M3 - Article
C2 - 24333049
AN - SCOPUS:84892735135
SN - 1046-5928
VL - 95
SP - 169
EP - 176
JO - Protein Expression and Purification
JF - Protein Expression and Purification
ER -