Melissa A. Vollrath

TRPA1 Contributes to Cold, Mechanical, and Chemical Nociception but Is Not Essential for Hair-Cell Transduction

TRPA1, a member of the transient receptor potential (TRP) family of ion channels, is expressed by dorsal root ganglion neurons and by cells of the inner ear, where it has proposed roles in sensing sound, painful cold, and irritating chemicals. To test the in vivo roles of TRPA1, we generated a mouse in which the essential exons required for proper function of the Trpa1 gene were deleted. Knockout mice display behavioral deficits in response to mustard oil, to cold ( approximately 0 degrees C), and to punctate mechanical stimuli. These mice have a normal startle reflex to loud noise, a normal sense of balance, a normal auditory brainstem response, and normal transduction currents in vestibular hair cells. TRPA1 is apparently not essential for hair-cell transduction but contributes to the transduction of mechanical, cold, and chemical stimuli in nociceptor sensory neurons.

TRPA1 is a candidate for the mechanosensitive transduction channel of vertebrate hair cells.

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.

Mechanoelectrical and voltage-gated ion channels in mammalian vestibular hair cells

Mammalian vestibular afferents respond robustly to head movements at low frequencies and provide input to reflexes that control eye, head and body position. Vestibular organs have distinctive regions and hair cells: Type II cells receive bouton afferent endings and type I cells receive large calyx afferent endings. In the rodent utricle, type II cells are broadly tuned to frequencies between 10 and 30 Hz. Other recent data suggest that otolith organs function in this frequency range, which is higher than previously imagined. Some of the tuning derives from adaptation of the transducer current, which is best fitted with a double exponential decay with time constants of ∼4 and 40 ms. Further tuning is provided by basolateral conductances, principally outwardly rectifying, voltage-gated K+ conductances. The kinetics of the K+ currents tend to vary with location in the sensory epithelium and therefore may contribute to regional variation in afferent physiology. Type I hair cells have a large, negatively activating K+ conductance, gK,L, that confers a very low input resistance and therefore attenuates the receptor potential. This may reduce nonlinearity in the receptor potential, a possibly useful feature for the motor reflexes served by the vestibular system. On the other hand, the small receptor potentials together with unusually negative resting potentials are hard to reconcile with calcium-mediated quantal transmission. This problem may be overcome by factors that inhibit gK,L’s activation at resting potential. Also, the calyx may support nonquantal transmission.

Stimulus Processing by Type II Hair Cells in the Mouse Utricle

Abstract: In type II and neonatal hair cells in the mouse utricle, the receptor potentials evoked by low‐frequency sinusoidal deflections of the hair bundle are attenuated by adaptation of the mechanoelectrical transduction current and the voltage‐dependent activation of a large potassium (K)‐selective outwardly rectifying conductance, gDR. These processes may contribute to high‐pass filtering of the responses of some utricular afferents to sinusoidal linear accelerations below 2 Hz. Depolarizing receptor potentials are more attenuated by gDR than are hyperpolarizing receptor potentials. It may therefore reduce nonlinear distortion introduced by mechanoelectrical transduction, which generates larger depolarizing currents than hyperpolarizing currents.

The zebrafish pinball wizard gene encodes WRB, a tail-anchored-protein receptor essential for inner-ear hair cells and retinal photoreceptors.

The zebrafish pinball wizard (pwi) mutant is deaf and blind. The pwi phenotype includes a reduced auditory startle response and reduced visual evoked potentials, suggesting fatigue of synaptic release at ribbon synapses in hair cells and photoreceptors. The gene defective in the pwi mutant is WRB, a protein homologous to the yeast protein Get1, which is involved in the insertion of ‘tail‐anchored’ membrane proteins. Many tail‐anchored proteins are associated with synaptic vesicles, and both vesicles and synaptic ribbons are reduced in synaptic regions of hair cells in pwi. Abnormal processing of synaptic vesicle proteins important for ribbon synapses can explain the pwi phenotype.

Hair-Cell Mechanotransduction Persists in TRP Channel Knockout Mice.

Members of the TRP superfamily of ion channels mediate mechanosensation in some organisms, and have been suggested as candidates for the mechanotransduction channel in vertebrate hair cells. Some TRP channels can be ruled out based on lack of an inner ear phenotype in knockout animals or pore properties not similar to the hair-cell channel. Such studies have excluded Trpv4, Trpa1, Trpml3, Trpm1, Trpm3, Trpc1, Trpc3, Trpc5, and Trpc6. However, others remain reasonable candidates. We used data from an RNA-seq analysis of gene expression in hair cells as well as data on TRP channel conductance to narrow the candidate group. We then characterized mice lacking functional Trpm2, Pkd2, Pkd2l1, Pkd2l2 and Pkd1l3, using scanning electron microscopy, auditory brainstem response, permeant dye accumulation, and single-cell electrophysiology. In all of these TRP-deficient mice, and in double and triple knockouts, mechanotransduction persisted. Together with published studies, these results argue against the participation of any of the 33 mouse TRP channels in hair cell transduction.

The frequency dependence of receptor potentials in hair cells of the mouse utricle

The mechanoelectrical transduction currents of hair cells in the mouse utricle adapt at varying rates to step deflections of the hair bundles. We consider contributions of this adaptation process and of input resistance and membrane capacitance to the frequency dependence of the receptor potential. Whole-cell recordings of transduction current and receptor potential were made from hair cells in the excised epithelium of the mouse utricle. Hair bundles were deflected by a fluid jet with step and sinusoidal waveforms. In type II cells, the receptor potential was a bandpass function of stimulus frequency. The adaptation rate of the transduction current, measured in response to step bundle deflections, accounted for much of the roll-off in the receptor potential at low frequencies of sinusoidal deflections. Corner frequencies predicted from the adaptation time course varied from 2 to 60 Hz. Voltage-gated conductances also contributed. Roll-off of the receptor potential at the high-frequency end may largely reflect input resistance and capacitance. Corner frequencies predicted by estimated membrane time constants varied from 30 to 150 Hz. In type I cells, slower or no adaptation and shorter membrane time constants predict larger response bandwidths. Frequency tuning in vivo will reflect other factors, including the mechanical response of the otolith and otolithic membrane to head movements.