title: Bidirectional modulation of thermal and chemical sensitivity of TRPM8 channels by the initial region of the N-terminal domain.
abstract: TRPM8, a nonselective cation channel activated by cold, voltage, and cooling compounds such as menthol, is the principal molecular detector of cold temperatures in primary sensory neurons of the somatosensory system. The N-terminal domain of TRPM8 consists of 693 amino acids, but little is known about its contribution to channel function. Here, we identified two distinct regions within the initial N terminus of TRPM8 that contribute differentially to channel activity and proper folding and assembly. Deletion or substitution of the first 40 residues yielded channels with augmented responses to cold and menthol. The thermal threshold of activation of these mutants was shifted 2 °C to higher temperatures, and the menthol dose-response curve was displaced to lower concentrations. Site-directed mutagenesis screening revealed that single point mutations at positions Ser-26 or Ser-27 by proline caused a comparable increase in the responses to cold and menthol. Electrophysiological analysis of the S27P mutant revealed that the enhanced sensitivity to agonists is related to a leftward shift in the voltage dependence of activation, increasing the probability of channel openings at physiological membrane potentials. In addition, we found that the region encompassing positions 40-60 is a key element in the proper folding and assembly of TRPM8. Different deletions and mutations within this region rendered channels with an impaired function that are retained within the endoplasmic reticulum. Our results suggest a critical contribution of the initial region of the N-terminal domain of TRPM8 to thermal and chemical sensitivity and the proper biogenesis of this polymodal ion channel.
authors.affiliation: From the Departamento de Biología, Facultad de Química y Biología, Universidad de Santiago de Chile, 9160000 Santiago, Chile and the Instituto de Neurociencias de Alicante, Universidad Miguel Hernández-Consejo Superior de Investigaciones Científicas, 03550 Alicante, Spain maria.pertusa@usach.cl.
authors.forename: María
authors.initials: M
authors.lastname: Pertusa
authors.affiliation: From the Departamento de Biología, Facultad de Química y Biología, Universidad de Santiago de Chile, 9160000 Santiago, Chile and.
authors.forename: Alejandro
authors.initials: A
authors.lastname: González
authors.affiliation: From the Departamento de Biología, Facultad de Química y Biología, Universidad de Santiago de Chile, 9160000 Santiago, Chile and.
authors.forename: Paulina
authors.initials: P
authors.lastname: Hardy
authors.affiliation: From the Departamento de Biología, Facultad de Química y Biología, Universidad de Santiago de Chile, 9160000 Santiago, Chile and.
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Madrid
authors.affiliation: the Instituto de Neurociencias de Alicante, Universidad Miguel Hernández-Consejo Superior de Investigaciones Científicas, 03550 Alicante, Spain.
authors.forename: Félix
authors.initials: F
authors.lastname: Viana
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D030161:Avian Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002645:Chickens; D003080:Cold Temperature; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D008610:Menthol / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D050053:TRPM Cation Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D030161:Avian Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D050053:TRPM Cation Channels; C544443:TRPM2 protein, mouse; C425966:TRPM8 protein, human; C492297:TRPM8 protein, mouse; D008610:Menthol
keywords: Calcium Imaging; Cold Sensitivity; Ion Channel; Membrane Protein; Menthol; N Terminus; Neuroscience; TRPM8; Transient Receptor Potential Channels (TRP Channels)