A Molecular Determinant For The Rectification And Study On The Mechanism Of TRPA1Channel

Objective：to study the molecular determinant for the rectification ofTRPA1channel. And preliminarily discussed the mechanism. Made thefoundation for further study of the nonselectivity cat ion channelTRPA1.Methods: The Leu906is mutated to cystine. The TRPA1and mutantschannels were9:1co-transfection with the EGFP vector, coding forgreen fluorescent protein. electrophysiological recordings wereperformed between24and36h after transfection. Single amino acidsin the pore region of TRPA1were mutated using the standard PCRoverlap extension technique, and the nucleotide sequences of allmutants were verified by sequencing of the corresponding cDNAs.Substitution of extracellular and intracellular divalent cations(Ca²⁺,Mg²⁺) to investigate rectification of TRPA1channel and mutantchannels.Result：（1）a conserved hydrophobic amino acid, Leu906in the S5-P-S6 region, determines rectification. Mutation of the Leu to Cys （L906C）converts the channel to inward rectification.（2）extracellular and intracellular divalent cations (Ca²⁺,Mg²⁺)increase the rectification of TRPA1channel and L906C mutantchannels. However,there is no change of rectification property.（3）L906C is inactivated at positive potentials in a time-andvoltage-dependent manner.（4）L906C mutation decreases the inhibitory effect of TRPA1poreblocker HC030031and RR.Conclusion： Different from wild-type, the inward rectification ofL906C is not modulated by calcium any more. In addition, the mutantdisplays a voltage-dependent inactivation which is absent in thewild-type channel. The deceased sensitivity to the blockers is consistentwith the idea that L906locates in the pore region and thus playessential role in the channel gating.L906, the residue plays essentialrole in TRPA1gating and determines the intrinsic rectificationfundamentally. Our findings provide new insight into the rectificationmechanism and the structure of pore region of the TRPA1channel.