ABSTRACT
Mechanical deflection of the sensory hair bundles of receptor cells in the inner ear causes ion channels located at the tips of the bundle to open, thereby initiating the perception of sound. Although some protein constituents of the transduction apparatus are known, the mechanically gated transduction channels have not been identified in higher vertebrates. Here, we investigate TRP (transient receptor potential) ion channels as candidates and find one, TRPA1 (also known as ANKTM1), that meets criteria for the transduction channel. The appearance of TRPA1 messenger RNA expression in hair cell epithelia coincides developmentally with the onset of mechanosensitivity. Antibodies to TRPA1 label hair bundles, especially at their tips, and tip labelling disappears when the transduction apparatus is chemically disrupted. Inhibition of TRPA1 protein expression in zebrafish and mouse inner ears inhibits receptor cell function, as assessed with electrical recording and with accumulation of a channel-permeant fluorescent dye. TRPA1 is probably a component of the transduction channel itself.
Subject(s)
Hair Cells, Auditory/metabolism , Hearing/physiology , Ion Channels/metabolism , Mechanotransduction, Cellular/physiology , Vertebrates/metabolism , Zebrafish Proteins/metabolism , Adenoviridae/genetics , Animals , Animals, Newborn , Antibodies/immunology , Ear, Inner/metabolism , Gene Expression Regulation, Developmental , In Situ Hybridization , Ion Channels/biosynthesis , Ion Channels/genetics , Ion Channels/immunology , Mice , Oligonucleotides/genetics , Oligonucleotides/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rana catesbeiana , TRPA1 Cation Channel , Transient Receptor Potential Channels , Zebrafish/metabolism , Zebrafish Proteins/biosynthesis , Zebrafish Proteins/genetics , Zebrafish Proteins/immunologyABSTRACT
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 altered permeation properties of mechanosensory transduction. We propose that TMC1 is structurally similar to TMEM16 channels and includes ten transmembrane domains with four domains, S4-S7, that line the channel pore. The data provide compelling evidence that TMC1 is a pore-forming component of sensory transduction channels in auditory and vestibular hair cells.