Structural modeling and patch-clamp analysis of pain-related mutation TRPA1-N855S reveal inter-subunit salt bridges stabilizing the channel open state.
The ankyrin transient receptor potential channel TRPA1 is a polymodal sensor for noxious stimuli, and hence a promising target for treating chronic pain. This tetrameric six-transmembrane segment (S1-S6) channel can be activated by various pungent chemicals, such as allyl isothiocyanate or cinnamaldehyde, but also by intracellular Ca(2+) or depolarizing voltages. Within the S4-S5 linker of human TRPA1, a gain-of-function mutation, N855S, was recently found to underlie familial episodic pain syndrome, manifested by bouts of severe upper body pain, triggered by physical stress, fasting, or cold. To clarify the structural basis for this channelopathy, we derive a structural model of TRPA1 by combining homology modeling, molecular dynamics simulations, point mutagenesis and electrophysiology. In the vicinity of N855, the model reveals inter-subunit salt bridges between E854 and K868. Using the heterologous expression of recombinant wild-type and mutant TRPA1 channels in HEK293T cells, we indeed found that the charge-reversal mutants E854R and K868E exhibited dramatically reduced responses to chemical and voltage stimuli, whereas the charge-swapping mutation E854R/K868E substantially rescued their functionalities. Moreover, mutation analysis of highly conserved charged residues within the S4-S5 region revealed a gain-of-function phenotype for R852E with an increased basal channel activity, a loss of Ca(2+)-induced potentiation and an accelerated Ca(2+)-dependent inactivation. Based on the model and on a comparison with the recently revealed atomic-level structure of the related channel TRPV1, we propose that inter-subunit salt bridges between adjacent S4-S5 regions are crucial for stabilizing the conformations associated with chemically and voltage-induced gating of the TRPA1 ion channel.
Division of Biomolecular Physics, Institute of Physics, Faculty of Mathematics and Physics, Charles University in Prague, Ke Karlovu 5, 121 16 Prague 2, Czech Republic.
Vlastimil
V
Zíma
Department of Cellular Neurophysiology, Institute of Physiology of the Czech Academy of Sciences, Videnska 1083, 14220 Prague 4, Czech Republic.
Katja
K
Witschas
Department of Cellular Neurophysiology, Institute of Physiology of the Czech Academy of Sciences, Videnska 1083, 14220 Prague 4, Czech Republic; Department of Biochemistry, Faculty of Science, Charles University in Prague, Albertov 6, 128 43 Prague 2, Czech Republic.
Anna
A
Hynkova
Department of Cellular Neurophysiology, Institute of Physiology of the Czech Academy of Sciences, Videnska 1083, 14220 Prague 4, Czech Republic.
Lucie
L
Zímová
Division of Biomolecular Physics, Institute of Physics, Faculty of Mathematics and Physics, Charles University in Prague, Ke Karlovu 5, 121 16 Prague 2, Czech Republic.
Ivan
I
Barvík
Department of Cellular Neurophysiology, Institute of Physiology of the Czech Academy of Sciences, Videnska 1083, 14220 Prague 4, Czech Republic. Electronic address: vlachova@biomed.cas.cz.
Viktorie
V
Vlachova
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004558:Electric Stimulation; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D017879:Isothiocyanates / Q000494:pharmacology; D008564:Membrane Potentials / Q000235:genetics*; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics*; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary; D012694:Serine / Q000235:genetics; D000074025:TRPA1 Cation Channel; D014162:Transfection; D050051:Transient Receptor Potential Channels / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D015220:Calcium Channels; D017879:Isothiocyanates; D009419:Nerve Tissue Proteins; D000074025:TRPA1 Cation Channel; C473350:TRPA1 protein, human; D050051:Transient Receptor Potential Channels; D012694:Serine; C041942:2,3,4-tri-O-acetylarabinopyranosyl isothiocyanate; D001216:Asparagine; D002118:Calcium
Ankyrin receptor subtype 1; Homology modeling; Molecular dynamics; Mutagenesis; S4–S5-linker; Transient receptor potential