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

Benjamin Clémençon, Michael Fine, Benjamin Lüscher, Marc U. Baumann, Daniel V. Surbek, Hugues Abriel, Matthias A. Hediger

Research output: Contribution to journalArticle

7 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)169-176
Number of pages8
JournalProtein Expression and Purification
Volume95
DOIs
StatePublished - Mar 1 2014
Externally publishedYes

Fingerprint

Xenopus laevis
Oocytes
Electron Microscopy
Transient Receptor Potential Channels
Electrophysiology
Robotics
Ion Channels
Electrodes
Coloring Agents
Cell Membrane
Pathology
human TRPM4 protein

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

ASJC Scopus subject areas

  • Biotechnology

Cite this

Expression, purification, and projection structure by single particle electron microscopy of functional human TRPM4 heterologously expressed in Xenopus laevis oocytes. / Clémençon, Benjamin; Fine, Michael; Lüscher, Benjamin; Baumann, Marc U.; Surbek, Daniel V.; Abriel, Hugues; Hediger, Matthias A.

In: Protein Expression and Purification, Vol. 95, 01.03.2014, p. 169-176.

Research output: Contribution to journalArticle

Clémençon, Benjamin ; Fine, Michael ; Lüscher, Benjamin ; Baumann, Marc U. ; Surbek, Daniel V. ; Abriel, Hugues ; Hediger, Matthias A. / Expression, purification, and projection structure by single particle electron microscopy of functional human TRPM4 heterologously expressed in Xenopus laevis oocytes. In: Protein Expression and Purification. 2014 ; Vol. 95. pp. 169-176.
@article{360559823ab9481d8695f510109817f8,
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.",
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",
author = "Benjamin Cl{\'e}men{\cc}on and Michael Fine and Benjamin L{\"u}scher and Baumann, {Marc U.} and Surbek, {Daniel V.} and Hugues Abriel and Hediger, {Matthias A.}",
year = "2014",
month = "3",
day = "1",
doi = "10.1016/j.pep.2013.11.017",
language = "English (US)",
volume = "95",
pages = "169--176",
journal = "Protein Expression and Purification",
issn = "1046-5928",
publisher = "Academic Press Inc.",

}

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.

PY - 2014/3/1

Y1 - 2014/3/1

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

UR - http://www.scopus.com/inward/record.url?scp=84892735135&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84892735135&partnerID=8YFLogxK

U2 - 10.1016/j.pep.2013.11.017

DO - 10.1016/j.pep.2013.11.017

M3 - Article

C2 - 24333049

AN - SCOPUS:84892735135

VL - 95

SP - 169

EP - 176

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

ER -