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Importance of a Conserved Sequence Motif in Transmembrane Segment S3 for the Gating of Human TRPM8 and TRPM2
For mammalian TRPM8, the amino acid residues asparagine-799 and aspartate-802 are essential for the stimulation of the channel by the synthetic agonist icilin. Both residues belong to the short sequence motif N-x-x-D within the transmembrane segment S3 highly conserved in the entire superfamily of v...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3503820/ https://www.ncbi.nlm.nih.gov/pubmed/23185472 http://dx.doi.org/10.1371/journal.pone.0049877 |
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author | Winking, Mathis Hoffmann, Daniel C. Kühn, Cornelia Hilgers, Ralf-Dieter Lückhoff, Andreas Kühn, Frank J. P. |
author_facet | Winking, Mathis Hoffmann, Daniel C. Kühn, Cornelia Hilgers, Ralf-Dieter Lückhoff, Andreas Kühn, Frank J. P. |
author_sort | Winking, Mathis |
collection | PubMed |
description | For mammalian TRPM8, the amino acid residues asparagine-799 and aspartate-802 are essential for the stimulation of the channel by the synthetic agonist icilin. Both residues belong to the short sequence motif N-x-x-D within the transmembrane segment S3 highly conserved in the entire superfamily of voltage-dependent cation channels, among them TRPM8. Moreover, they are also conserved in the closely related TRPM2 channel, which is essentially voltage-independent. To analyze the differential roles of the motif for the voltage-dependent and voltage-independent gating, we performed reciprocal replacements of the asparagine and aspartate within the S3 motif in both channels, following the proposed idea that specific electrostatic interactions with other domains take place during gating. Wild-type and mutant channels were heterologeously expressed in HEK-293 cells and channel function was analyzed by whole-cell patch-clamp analysis as well as by Ca(2+)-imaging. Additionally, the expression of the channels in the plasma membrane was tested by Western blot analysis, in part after biotinylation. For the mutations of TRPM8, responses to menthol were only compromised if also the expression of the glycosylated channel isoform was prevented. In contrast, responses to cold were consistently and significantly attenuated but not completely abolished. For TRPM2, surface expression was not significantly affected by any of the mutations but channel function was only retained in one variant. Remarkably, this was the variant of which the corresponding mutation in TRPM8 exerted the most negative effects both on channel function and expression. Furthermore, we performed an exchange of the inner pair of residues of the N-x-x-D motif between the two channels, which proved deleterious for the functional expression of TRPM8 but ineffective on TRPM2. In conclusion, the N-x-x-D motif plays specific roles in TRPM8 and TRPM2, reflecting different requirements for voltage-dependent and voltage-independent channel gating. |
format | Online Article Text |
id | pubmed-3503820 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-35038202012-11-26 Importance of a Conserved Sequence Motif in Transmembrane Segment S3 for the Gating of Human TRPM8 and TRPM2 Winking, Mathis Hoffmann, Daniel C. Kühn, Cornelia Hilgers, Ralf-Dieter Lückhoff, Andreas Kühn, Frank J. P. PLoS One Research Article For mammalian TRPM8, the amino acid residues asparagine-799 and aspartate-802 are essential for the stimulation of the channel by the synthetic agonist icilin. Both residues belong to the short sequence motif N-x-x-D within the transmembrane segment S3 highly conserved in the entire superfamily of voltage-dependent cation channels, among them TRPM8. Moreover, they are also conserved in the closely related TRPM2 channel, which is essentially voltage-independent. To analyze the differential roles of the motif for the voltage-dependent and voltage-independent gating, we performed reciprocal replacements of the asparagine and aspartate within the S3 motif in both channels, following the proposed idea that specific electrostatic interactions with other domains take place during gating. Wild-type and mutant channels were heterologeously expressed in HEK-293 cells and channel function was analyzed by whole-cell patch-clamp analysis as well as by Ca(2+)-imaging. Additionally, the expression of the channels in the plasma membrane was tested by Western blot analysis, in part after biotinylation. For the mutations of TRPM8, responses to menthol were only compromised if also the expression of the glycosylated channel isoform was prevented. In contrast, responses to cold were consistently and significantly attenuated but not completely abolished. For TRPM2, surface expression was not significantly affected by any of the mutations but channel function was only retained in one variant. Remarkably, this was the variant of which the corresponding mutation in TRPM8 exerted the most negative effects both on channel function and expression. Furthermore, we performed an exchange of the inner pair of residues of the N-x-x-D motif between the two channels, which proved deleterious for the functional expression of TRPM8 but ineffective on TRPM2. In conclusion, the N-x-x-D motif plays specific roles in TRPM8 and TRPM2, reflecting different requirements for voltage-dependent and voltage-independent channel gating. Public Library of Science 2012-11-21 /pmc/articles/PMC3503820/ /pubmed/23185472 http://dx.doi.org/10.1371/journal.pone.0049877 Text en © 2012 Winking et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Winking, Mathis Hoffmann, Daniel C. Kühn, Cornelia Hilgers, Ralf-Dieter Lückhoff, Andreas Kühn, Frank J. P. Importance of a Conserved Sequence Motif in Transmembrane Segment S3 for the Gating of Human TRPM8 and TRPM2 |
title | Importance of a Conserved Sequence Motif in Transmembrane Segment S3 for the Gating of Human TRPM8 and TRPM2 |
title_full | Importance of a Conserved Sequence Motif in Transmembrane Segment S3 for the Gating of Human TRPM8 and TRPM2 |
title_fullStr | Importance of a Conserved Sequence Motif in Transmembrane Segment S3 for the Gating of Human TRPM8 and TRPM2 |
title_full_unstemmed | Importance of a Conserved Sequence Motif in Transmembrane Segment S3 for the Gating of Human TRPM8 and TRPM2 |
title_short | Importance of a Conserved Sequence Motif in Transmembrane Segment S3 for the Gating of Human TRPM8 and TRPM2 |
title_sort | importance of a conserved sequence motif in transmembrane segment s3 for the gating of human trpm8 and trpm2 |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3503820/ https://www.ncbi.nlm.nih.gov/pubmed/23185472 http://dx.doi.org/10.1371/journal.pone.0049877 |
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