Mechanosensory channels of sensory cells mediate the sensations of hearing, touch, and some forms of pain. The TRPA1 (a member of the TRP family of ion channel proteins) channel is activated by pain-producing chemicals, and its inhibition impairs hair cell mechanotransduction. As shown here and previously, TRPA1 is expressed by hair cells as well as by most nociceptors (small neurons of dorsal root, trigeminal, and nodose ganglia) and localizes to their sensory terminals (mechanosensory stereocilia and peripheral free nerves, respectively). Thus, TRPA1 channels are proposed to mediate transduction in both hair cells and nociceptors. Accordingly, we find that heterologously expressed TRPA1 display channel behaviors expected for both auditory and nociceptive transducers. First, TRPA1 and the hair cell transducer share a unique set of pore properties not described for any other channel (block by gadolinium, amiloride, gentamicin, and ruthenium red, a ranging conductance of ϳ100 pS that is reduced to 54% by calcium, permeating calcium-induced potentiation followed by closure, and reopening by depolarization), supporting a direct role of TRPA1 as a pore-forming subunit of the hair cell transducer. Second, TRPA1 channels inactivate in hyperpolarized cells but remain open in depolarized cells. This property provides a mechanism for the lack of desensitization, coincidence detection, and allodynia that characterize pain by allowing a sensory neuron to respond constantly to sustained stimulation that is suprathreshold (i.e., noxious) and yet permitting the same cell to ignore sustained stimulation that is subthreshold (i.e., innocuous). Our results support a TRPA1 role in both nociceptor and hair cell transduction.

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.

In mammals, hair cell loss causes irreversible hearing and balance impairment because hair cells are terminally differentiated and do not regenerate spontaneously. By profiling gene expression in developing mouse vestibular organs, we identified the retinoblastoma protein (pRb) as a candidate regulator of cell cycle exit in hair cells. Differentiated and functional mouse hair cells with a targeted deletion of Rb1 undergo mitosis, divide, and cycle, yet continue to become highly differentiated and functional. Moreover, acute loss of Rb1 in postnatal hair cells caused cell cycle reentry. Manipulation of the pRb pathway may ultimately lead to mammalian hair cell regeneration.

The recently defined DEG͞ENaC superfamily of sodium channels includes subunits of the amiloridesensitive epithelial sodium channel (ENaC) of vertebrate colon, lung, kidney, and tongue, a molluscan FMRFamidegated channel (FaNaC), and the nematode degenerins, which are suspected mechanosensory channels. We have identified two new members of this superfamily (BNaC1 and BNaC2) in a human brain cDNA library. Phylogenetic analysis indicates they are equally divergent from all other members of the DEG͞ENaC superfamily and form a new branch or family. Human BNaC1 maps to 17q11.2-12 and hBNaC2 maps to 12q12. Northern blot and mouse brain in situ hybridizations indicate that both genes are coexpressed in most if not all brain neurons, although their patterns of expression vary slightly, and are expressed early in embryogenesis and throughout life. By analogy to the ENaCs and the degenerins, which form heteromultimeric channels, BNaC1 and BNaC2 may be subunits of the same channel.The recently defined DEG͞ENaC superfamily of sodium channels contains to date 17 proteins (not counting the many orthologs found in different vertebrate species) that have similar sequences and the same predicted structure: intracellular N and C termini, two hydrophobic membrane-spanning regions, and a large extracellular loop, which contains many cysteine residues with conserved spacing. This topology has been experimentally demonstrated for members of two different branches: ␣ENaC

In Caenorhabditis elegans necrosis-like neuronal death is induced by gain-of-function (gf) mutations in two genes, mec-4 and deg-1, that encode proteins similar to subunits of the vertebrate amiloride-sensitive epithelial Na+ channel. We have determined the progress of cellular pathology in dying neurons via light and electron microscopy. The first detectable abnormality is an infolding of the plasma membrane and the production of small electron-dense whorls. Later, cytoplasmic vacuoles and larger membranous whorls form, and the cell swells. More slowly, chromatin aggregates and the nucleus invaginates. Mitochondria and Golgi are not dramatically affected until the final stages of cell death when organelles, and sometimes the cells themselves, lyse. Certain cells, including some muscle cells in deg-1 animals, express the abnormal gene products and display a few membrane abnormalities but do not die. These cells either express the mutant genes at lower levels, lack other proteins needed to form inappropriately functioning channels, or are better able to compensate for the toxic effects of the channels. Overall, the ultrastructural changes in these deaths suggest that enhanced membrane cycling precedes vacuolation and cell swelling. The pathology of mec-4(gf) and deg-1(gf) cells shares features with that of genetic disorders with alterations in channel subunits, such as hypokalemic periodic paralysis in humans and the weaver mouse, and with degenerative conditions, e.g., acute excitotoxic death. The initial pathology in all of these conditions may reflect attempts by affected cells to compensate for abnormal membrane proteins or functions.

Mammalian brain sodium channel (BNaC, also known as BNC/ ASIC) proteins form acid-sensitive and amiloride-blockable sodium channels that are related to putative mechanosensory channels. Certain BNaC isoforms are expressed exclusively in dorsal root ganglia (DRG) and have been proposed to form the ion channels mediating tissue acidosis-induced pain. With antibody labeling, we find that the BNaC1␣ isoform is expressed by most large DRG neurons (low-threshold mechanosensors not involved in acid-induced nociception) and few small nociceptor neurons (which include high-threshold mechanoreceptors). BNaC1␣ is transported from DRG cell bodies to sensory terminals in the periphery, but not to the spinal cord, and is located specifically at specialized cutaneous mechanosensory terminals, including Meissner, Merkel, penicillate, reticular, lanceolate, and hair follicle palisades as well as some intraepidermal and free myelinated nerve endings. Accordingly, BNaC1␣ channels might participate in the transduction of touch and painful mechanical stimuli.

Varitint-waddler (Va andVaA very common cause of deafness is the loss of hair cells, which degenerate because of environmental factors or genetic mutations. Varitint-waddler (Va) mice have a mutation that causes an alanine-to-proline substitution (A419P) in the fifth transmembrane domain of TRPML3, a presumed ion channel. A second allele, Va J , contains the A419P mutation in cis to an I362T mutation (1). The earliest sign of inner ear damage in Va mice is hair cell degeneration, which begins in embryogenesis as hair cells differentiate and continues postnatally. During degeneration, the hair cells bulge out of the apical side of the epithelium and become extruded. Eventually these mice also develop abnormalities in supporting cells, in the tectorial membrane, and in the stria vascularis and in the endocochlear potential that they help generate. The defects of Va J mice are similar although less severe, suggesting that the mutation I362T attenuates the effects of A419P (1-3). A comprehensive examination of TRPML3 expression in inner ear has not been published. Expression of TRPML3 protein in hair cells was suggested by immunocytochemical methods, although results for other inner ear cell types were not reported (1).Mutations in two other TRP channels, Drosophila TRP and human TRPML1 (mutations in which cause mucolipidosis type IV), are associated with degeneration of the retina. In both cases, the channels seem necessary for cellular viability, because recessive, loss-of-function mutations cause degeneration (4, 5).By contrast, the Va and Va J mutations in TRPML3 are semidominant, although it remains unclear whether this is due to gain-of-function, haploinsufficiency, or dominant-negative effects (1, 6). ResultsWe first established which cells expressed TRPML3 in the inner ear of wild-type mice. In situ hybridization on mouse inner ear using probes from two nonoverlapping regions of TRPML3 mRNA gave identical results [ Fig. 1 and supporting information (SI) Fig. 6]. TRPML3 mRNA was most abundant in the marginal cells of the cochlear stria vascularis and the dark cells of the vestibule (Fig. 1 i, l, and m and SI Fig. 6 i, l, and m). Both cell types are involved in producing the endolymph and therefore the endocochlear potential (7-9). This expression pattern is in keeping with the reduced stria vascularis and the rounding up and loss of the cytoplasmic processes of its marginal cells in Va/Va and Va/ϩ mice (2) and with the reduced endocochlear potentials of the Va J mutants (1, 3).In addition, other cells that line the cochlear scala media and the connected vestibular endolymphatic spaces expressed TRPML3 mRNA, including (i) supporting cells at the marginal part of the lesser epithelial ridge (Hensen and Claudius cells), which in Va and Va J mice (homozygotes and heterozygotes) appear undifferentiated or degenerate ( Fig. 1i and SI Fig. 6i); (ii) cells of the spiral limbus that produce the tectorial membrane, which in Va mice develops abnormally and fails to attach to the reticular lamina ( Fig. 1i and SI Fig. 6i...

Members of the BNaC/ASIC family of ion channels have been implicated in mechanotransduction and nociception mediated by dorsal root ganglion (DRG) neurons. These ion channels are also expressed in the CNS. We identified the PDZ domain protein PICK1 as an interactor of BNaC1(ASIC2) in a yeast two-hybrid screen. We show by two-hybrid assays, glutathione S-transferase pull-down assays, and coimmunoprecipitations that the BNaC1⅐PICK1 interaction is specific, and that coexpression of both proteins leads to their clustering in intracellular compartments. The interaction between BNaC1 and PICK1 requires the PDZ domain of PICK1 and the last four amino acids of BNaC1. BNaC1 is similar to two other BNaC/ASIC family members, BNaC2 (ASIC1) and ASIC4, at its extreme C terminus, and we show that PICK1 also interacts with BNaC2. We found that PICK1, like BNaC1 and BNaC2, is expressed by DRG neurons and, like the BNaC1␣ isoform, is present at their peripheral mechanosensory endings. Both PICK1 and BNaC1␣ are also coexpressed by some pyramidal neurons of the cortex, by pyramidal neurons of the CA3 region of hippocampus, and by cerebellar Purkinje neurons, localizing to their dendrites and cell bodies. Therefore, PICK1 interacts with BNaC/ASIC channels and may regulate their subcellular distribution or function in both peripheral and central neurons. BNaC1 and BNaC21 are members of the DEG/ENaC superfamily of ion channel subunits (1-4). Members of this family have two transmembrane domains separated by a large extracellular loop, with cytoplasmic amino and C termini (5-8). They form homomeric and heteromeric channels that are permeable to sodium but are not gated by voltage. DEG/ENaC channels have been implicated in several forms of mechanosensation. For example, certain members of the degenerin branch are necessary for the sensation of touch in Caenorhabditis elegans (9). Members of the ENaC branch in mammals control Na ϩ and fluid absorption in the kidney, colon, and lung (10 -12), but they have also been found at baroreceptor (13) and somatic touch receptor endings (14, 15). The Pickpocket channel of Drosophila melanogaster also localizes to putative mechanosensory nerve endings (16).The mammalian BNaC/ASIC branch of the superfamily contains four genes, encoding at least six isoforms: BNaC1␣ (also known as BNC1, MDEG, and ASIC2) (2-4) and its differentially spliced isoform, BNaC1␤ (MDEG2) (17); BNaC2␣ (ASIC␣ or ASIC1) (4, 18) and its differentially spliced isoform, BNaC2␤ (ASIC␤) (19); DRASIC (ASIC3 or TNaC) (20 -23); and ASIC4 (SPASIC) (24, 25). These genes are expressed in both central and peripheral neurons and form channels that can be activated by extracellular protons (26). Because tissue acidosis is a source of pain (27), it has been proposed that these ion channels may play a role in acid-induced nociception (26,28).In addition, recent work has demonstrated that BNaC1 is required for normal mechanosensation. Mice with a targeted deletion of the BNaC1 gene show reduced sensitivity of rapidly and slowly adapting mechanor...