Expression, purification, and projection structure by single particle electron microscopy of functional human TRPM4 heterologously expressed in Xenopus laevis oocytes

Clemençon, Benjamin; Fine, Michael; Lüscher, Benjamin; Baumann, Marc; Surbek, Daniel; Abriel, Hugues; Hediger, Matthias (2014). Expression, purification, and projection structure by single particle electron microscopy of functional human TRPM4 heterologously expressed in Xenopus laevis oocytes. Protein expression and purification, 95, pp. 169-176. Elsevier 10.1016/j.pep.2013.11.017

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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.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Biochemistry and Molecular Medicine
04 Faculty of Medicine > Department of Gynaecology, Paediatrics and Endocrinology (DFKE) > Clinic of Gynaecology
04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR) > DBMR Forschung Mu35 > Forschungsgruppe Ionenkanalkrankheiten
04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR) > DBMR Forschung Mu35 > Forschungsgruppe Ionenkanalkrankheiten

UniBE Contributor:

Clemençon, Benjamin; Fine, Michael; Lüscher, Benjamin; Baumann, Marc; Surbek, Daniel; Abriel, Hugues and Hediger, Matthias

Subjects:

600 Technology > 610 Medicine & health
500 Science > 570 Life sciences; biology

ISSN:

1096-0279

Publisher:

Elsevier

Language:

English

Submitter:

Patrizia Catucci

Date Deposited:

23 Jul 2014 15:42

Last Modified:

11 Nov 2015 13:43

Publisher DOI:

10.1016/j.pep.2013.11.017

PubMed ID:

24333049

Uncontrolled 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

BORIS DOI:

10.7892/boris.43301

URI:

https://boris.unibe.ch/id/eprint/43301

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