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PCR Cloning and Subcloning> | TMC1 orthologs (Genscript) were subcloned into a pcDNA3.1(+1) vector containing a mammalian cytomegalovirus promoter (CMV) and a C-terminal HRV3C cleavage site followed by the human influenza hemagglutinin (HA) tag, and expressed in HEK293FT cells. | Get A Quote |
The proteins that form the permeation pathway of mechanosensory transduction channels in inner-ear hair cells have not been definitively identified. Genetic, anatomical, and physiological evidence support a role for transmembrane channel-like protein (TMC) 1 in hair cell sensory transduction, yet the molecular function of TMC proteins remains unclear. Here, we provide biochemical evidence suggesting TMC1 assembles as a dimer, along with structural and sequence analyses suggesting similarity to dimeric TMEM16 channels. To identify the pore region of TMC1, we used cysteine mutagenesis and expressed mutant TMC1 in hair cells of Tmc1/2-null mice. Cysteine-modification reagents rapidly and irreversibly ... More