The role of the N terminus and transmembrane domain of TRPM8 in channel localization and tetramerization

J Biol Chem. 2007 Dec 14;282(50):36474-80. doi: 10.1074/jbc.M707205200. Epub 2007 Oct 1.

Abstract

Transient receptor potential (TRP) channels are a family of cation channels involved in diverse cellular functions. They are composed of a transmembrane domain of six putative transmembrane segments flanked by large N- and C-terminal cytoplasmic domains. The melastatin subfamily (TRPM) channels have N-terminal domains of approximately 700 amino acids with four regions of shared homology and C-terminal domains containing the conserved TRP domain followed by a coiled-coil region. Here we investigated the effects of N- and C-terminal deletions on the cold and menthol receptor, TRPM8, expressed heterologously in Sf21 insect cells. Patch-clamp electrophysiology was used to study channel activity and revealed that only deletion of the first 39 amino acids was tolerated by the channel. Further N-terminal truncation or any C-terminal deletions prevented proper TRPM8 function. Confocal microscopy with immunofluorescence revealed that amino acids 40-86 are required for localization to the plasma membrane. Furthermore, analysis of deletion mutant oligomerization shows that the transmembrane domain is sufficient for TPRM8 assembly into tetramers. TRPM8 channels with C-terminal deletions tetramerize and localize properly but are inactive, indicating that although not essential for tetramerization and localization, the C terminus is critical for proper function of the channel sensor and/or gate.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence / genetics
  • Animals
  • Cell Line
  • Cell Membrane / genetics
  • Cell Membrane / metabolism*
  • Microscopy, Confocal
  • Patch-Clamp Techniques
  • Protein Structure, Quaternary / genetics
  • Protein Structure, Secondary / genetics
  • Protein Structure, Tertiary / genetics
  • Rats
  • Sequence Deletion
  • TRPM Cation Channels / genetics
  • TRPM Cation Channels / metabolism*

Substances

  • TRPM Cation Channels
  • Trpm8 protein, rat