The role of the N terminus and transmembrane domain of TRPM8 in channel localization and tetramerization.
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.
Phelps
Christopher B
CB
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Gaudet
Rachelle
R
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D018408:Patch-Clamp Techniques; D020836:Protein Structure, Quaternary / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D017384:Sequence Deletion; D050053:TRPM Cation Channels / Q000235:genetics / Q000378:metabolism*
D050053:TRPM Cation Channels; C498947:Trpm8 protein, rat