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

Protein Expr Purif. 2014 Mar;95:169-76. doi: 10.1016/j.pep.2013.11.017. Epub 2013 Dec 10.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Microscopy, Electron, Transmission
  • Oocytes / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification*
  • Recombinant Proteins / metabolism*
  • TRPM Cation Channels / chemistry
  • TRPM Cation Channels / genetics
  • TRPM Cation Channels / isolation & purification*
  • TRPM Cation Channels / metabolism*
  • Xenopus laevis

Substances

  • Recombinant Proteins
  • TRPM Cation Channels
  • TRPM4 protein, human