期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 104, 期 39, 页码 15555-15559出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0704039104
关键词
aromatic residue anchoring; gating machinery; mechanosensitivity; yeast calcium signal; vacuolar membrane
资金
- NIGMS NIH HHS [GM054867, R01 GM047856, R01 GM054867, GM047856] Funding Source: Medline
Transient receptor potential (TRP) channels are first elements in sensing chemicals, heat, and force and are widespread among protists and fungi as well as animals. Despite their importance,the arrangement and roles of the amino acids that constitute the TRIP channel gate are unknown. The yeast TRPY1 is activated in vivo by osmotically induced vacuolar membrane deformation and by cytoplasmic Ca2+. After a random mutagenesis, we isolated TRPY1 mutants that responded more strongly to mild osmotic upshocks. One such gain-of-function mutant has a Y458H substitution at the C terminus of the predicted sixth transmembrane helix. Direct patch-clamp examination of vacuolar membranes showed that Y458H channels were already active with little stimulus and showed marked flickers between the open and intraburst closed states. They remained responsive to membrane stretch force and to Ca2+, indicating primary defects in the gate region but not in the sensing of gating principles. None of the other 18 amino acid replacements engineered here showed normal channel kinetics except the two aromatic substitutions, Y458F and Y458W. The Y458 of TRPY1 has its aromatic counterpart in mammalian TRPM. Furthermore, conserved aromatics one a-helical turn downstream from this point are also found in animal TRPC, TRPN, TRPP, and TRPML, suggesting that gate anchoring with aromatics may be common among many TRIP channels. The possible roles of aromatics at the end of the sixth transmembrane helix are discussed.
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