Heterologously-expressed and Liposome-reconstituted Human Transient Receptor Potential Melastatin 4 Channel (TRPM4) is a Functional Tetramer

Sci Rep. 2016 Jan 20:6:19352. doi: 10.1038/srep19352.

Abstract

Mutation, irregular expression and sustained activation of the Transient Receptor Potential Channel, type Melastatin 4 (TRPM4), have been linked to various cardiovascular diseases. However, much remains unknown about the structure of this important ion channel. Here, we have purified a heterologously expressed TRPM4-eGFP fusion protein and investigated the oligomeric state of TRPM4-eGFP in detergent micelles using crosslinking, native gel electrophoresis, multi-angle laser light scattering and electron microscopy. Our data indicate that TRPM4 is tetrameric, like other TRP channels studied to date. Furthermore, the functionality of liposome reconstituted TRPM4-eGFP was examined using electrophysiology. Single-channel recordings from TRPM4-eGFP proteoliposomes showed inhibition of the channel using Flufenamic acid, a well-established inhibitor of TRPM4, suggesting that the channels are functional upon reconstitution. Our characterisation of the oligomeric structure of TRPM4 and the ability to reconstitute functional channels in liposomes should facilitate future studies into the structure, function and pharmacology of this therapeutically relevant channel.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Gene Expression*
  • Green Fluorescent Proteins
  • Humans
  • Liposomes / chemistry
  • Molecular Imaging
  • Protein Multimerization*
  • Protein Transport
  • Proteolysis
  • Recombinant Fusion Proteins
  • TRPM Cation Channels / chemistry*
  • TRPM Cation Channels / genetics*
  • TRPM Cation Channels / metabolism

Substances

  • Liposomes
  • Recombinant Fusion Proteins
  • TRPM Cation Channels
  • TRPM4 protein, human
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins