title: Expression, purification, and projection structure by single particle electron microscopy of functional human TRPM4 heterologously expressed in Xenopus laevis oocytes.
abstract: 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.
authors: Institute of Biochemistry and Molecular Medicine (IBMM), NCCR TransCure, University of Bern, Bern, Switzerland. Electronic address: benjamin.clemencon@ibmm.unibe.ch.
authors: Benjamin
authors: B
authors: Clémençon
authors: Institute of Biochemistry and Molecular Medicine (IBMM), NCCR TransCure, University of Bern, Bern, Switzerland.
authors: Michael
authors: M
authors: Fine
authors: Department of Obstetrics and Gynecology, University Hospital of Bern, Bern, Switzerland; Department of Clinical Research, University of Bern, Bern, Switzerland.
authors: Benjamin
authors: B
authors: Lüscher
authors: Department of Obstetrics and Gynecology, University Hospital of Bern, Bern, Switzerland; Department of Clinical Research, University of Bern, Bern, Switzerland.
authors: Marc U
authors: MU
authors: Baumann
authors: Department of Obstetrics and Gynecology, University Hospital of Bern, Bern, Switzerland; Department of Clinical Research, University of Bern, Bern, Switzerland.
authors: Daniel V
authors: DV
authors: Surbek
authors: Department of Clinical Research, NCCR TransCure, University of Bern, Bern, Switzerland.
authors: Hugues
authors: H
authors: Abriel
authors: Institute of Biochemistry and Molecular Medicine (IBMM), NCCR TransCure, University of Bern, Bern, Switzerland.
authors: Matthias A
authors: MA
authors: Hediger
mesh_terms: D000818:Animals; D046529:Microscopy, Electron, Transmission; D009865:Oocytes / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D050053:TRPM Cation Channels / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D011994:Recombinant Proteins; D050053:TRPM Cation Channels; C437888:TRPM4 protein, human
keywords: Hi clamp two-electrode voltage clamp (TEV) system; Human TRPM4; Membrane protein purification; Roboinject system; Single particle analysis (SPA); Transmission electron microscopy (TEM); Xenopus laevis oocytes