The conversion of physical forces into electrical and biochemical signals is known as mechanotransduction. Mechanotransduction is essential for various physiological processes such as the conscious sensation of touch, proprioception and hearing, and the unconscious sensation of blood flow. TACAN (Tension-Activated-ChANnel) has recently been identified as one of the principle pain-sensing channels and is a candidate mechanosensitive (MS) ion channel found in nociceptors. TACAN does not share significant sequence similarity to any known class of ion channel and thus represents a novel class of mechanosensitive channels. Presently, the three-dimensional structure of TACAN and its gating mechanism are unknown.
TACAN DNA containing an N-terminal 6XHis-tag has been cloned into a pET-28a vector. Based on secondary structure predictions using different online servers, TACAN is expected to comprise 5 or 6 transmembrane helices, a 138 residue N-terminal domain and a 14 residue C-terminal domain that includes a cluster of 9 basic residues. Expression of TACAN was tested in BL21 (DE3), Rosetta 2 (DE3) and Rosetta (DE3) PlysS. The latter showed the best yield. TACAN DNA containing an N-terminal His tag followed by a thrombin cleavage site was cloned in pET-28a vector using EcoR1 and Xba1. It was transformed and expressed in Rosetta (DE3) PlysS strain and purified in 0.02% DDM using TALON metal affinity resin followed by Size Exclusion Chromatography. Purified fractions were then used for negative staining and cryo-EM.
This study will help obtain knowledge about the structure and architecture of the transmembrane domain, the importance of the N-terminal and C-terminal domains in mechanosensitivity and the essential residues lining the pore-forming helices. The structure of TACAN is essential to understanding the underlying mechanisms of function of this channel which will increase our knowledge of channel structure and open the path to developing potential inhibitors and drugs for chronic pain.