Jaime García-Añoveros

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.

NOCICEPTOR AND HAIR CELL TRANSDUCER PROPERTIES OF TRPA1, A CHANNEL FOR PAIN AND HEARING

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 ∼100 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.

The C. elegans unc-104 4 gene encodes a putative kinesin heavy chain-like protein

Mutations in the unc-104 gene of the nematode C. elegans result in uncoordinated and slow movement. Transposon insertions in three unc-104 alleles (e2184, rh1016, and rh1017) were used as physical markers to clone the unc-104 gene. DNA sequence analysis of unc-104 cDNAs revealed an open reading frame capable of encoding a 1584 amino acid protein with similarities to kinesin heavy chain. The similarities are greatest in the amino-terminal ATPase and microtubule-binding domains. Although the primary sequence relatedness to kinesin is weak in the remainder of the molecule, the predicted secondary structure and regional isoelectric points are similar to kinesin heavy chain.

The PDZ Domain Protein PICK1 and the Sodium Channel BNaC1 Interact and Localize at Mechanosensory Terminals of Dorsal Root Ganglion Neurons and Dendrites of Central Neurons

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 BNaC1·PICK1 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 BNaC1α isoform, is present at their peripheral mechanosensory endings. Both PICK1 and BNaC1α 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.

Mechanosensation and the DEG/ENaC Ion Channels

Cloning of a new protein that participates in mechanosensation in Caenorhabditis elegans (see J. Liu et al., p. 361) prompts this Perspectives discussion of the superfamily of ion channels that underlie this receptive process and the parallels between mechanosensation in worms and vertebrates.

Regulation of Caenorhabditis elegans degenerin proteins by a putative extracellular domain

BACKGROUND Rare, dominant mutations in the degenerin genes of Caenorhabditis elegans (deg-1, mec-4 and mec-10) cause neuronal degeneration. The extensive sequence similarity between degenerins and mammalian genes that encode subunits of the amiloride-sensitive sodium channel from kidney, colon and lung suggests that the C. elegans degenerins form ion channels. As mec-4 and mec-10 are needed for the reception of gentle touch stimuli, they may contribute to a mechanosensory ion channel. All the dominant degeneration-causing mutations in the C. elegans degenerin genes affect equivalent residues in a hydrophobic region that is structurally similar to the H5 domain of several ion channels, and so could form the channel lining. Increased channel activity may underlie the resulting degeneration, in which the affected cells vacuolate and swell. RESULTS We now demonstrate that a missense change in a predicted extracellular region of the proteins encoded by deg-1 and mec-4 causes cell death similar to that caused by the dominant mutations. The missense mutation lies within a 22 amino-acid region found in all the C. elegans degenerins for which the sequences have been published, but not in the similar mammalian proteins. Deletion of nine amino acids surrounding the mutation site in mec-4 also causes neuronal degeneration. The degeneration-causing mutations in either the predicted pore-lining or the predicted extracellular regions of deg-1 are suppressed by additional, dominantly acting mutations that substitute larger for smaller residues within the channel lining. CONCLUSIONS Our data suggest that the putative extracellular domain negatively regulates degenerin activity, perhaps by gating the channel. As this region is only found in the C. elegans proteins, it may allow more rapid regulation of the nematode channels, which may be needed for them to function in mechanosensation. The suppressor mutations, by adding larger amino acids to the putative pore lining, could prevent degeneration by blocking the pore of a multisubunit channel.

Chromosome organization of the protozoan Trypanosoma brucei.

The genome of the protozoan Trypanosoma brucei is known to be diploid. Karyotype analysis has, however, failed to identify homologous chromosomes. Having refined the technique for separating trypanosome chromosomes (L. H. T. Van der Ploeg, C. L. Smith, R. I. Polvere, and K. Gottesdiener, Nucleic Acids Res. 17:3217-3227, 1989), we can now provide evidence for the presence of homologous chromosomes. By determining the chromosomal location of different genetic markers, most of the chromosomes (14, excluding the minichromosomes), could be organized into seven chromosome pairs. In most instances, the putative homologs of a pair differed in size by about 20%. Restriction enzyme analysis of chromosome-sized DNA showed that these chromosome pairs contained large stretches of homologous DNA sequences. From these data, we infer that the chromosome pairs represent homologs. The identification of homologous chromosomes gives valuable insight into the organization of the trypanosome genome, will facilitate the genetic analysis of T. brucei, and suggests the presence of haploid gametes.

The varitint-waddler (Va) deafness mutation in TRPML3 generates constitutive, inward rectifying currents and causes cell degeneration

Varitint-waddler (Va and VaJ) mice are deaf and have vestibular impairment, with inner ear defects that include the degeneration and loss of sensory hair cells. The semidominant Va mutation results in an alanine-to-proline substitution at residue 419 (A419P) of the presumed ion channel TRPML3. Another allele, VaJ, has the A419P mutation in addition to an I362T mutation. We found that hair cells, marginal cells of stria vascularis, and other cells lining the cochlear and vestibular endolymphatic compartments express TRPML3. When heterologously expressed in LLC-PK1-CL4 epithelial cells, a culture model for hair cells, TRPML3 accumulated in lysosomes and in espin-enlarged microvilli that resemble stereocilia. We also demonstrated that wild-type TRPML3 forms channels that are blocked by Gd3+, have a conductance of 50–70 pS and, like many other TRP channels, open at very positive potentials and thus rectify outwardly. In addition to this outward current, TRPML3(419P) and (I362T+A419P) generated a constitutive inwardly rectifying current that suggests a sensitivity to hyperpolarizing negative potentials and that depolarized the cells. Cells expressing TRPML3(A419P) or (I362T+A419P), but not wild-type TRPML3, died and were extruded from the epithelium in a manner reminiscent of degenerating hair cells in Va mice. The increased open probability of TRPML3(A419P) and (I362T+A419P) at physiological potentials likely underlies hair cell degeneration and deafness in Va and VaJ mice.

THE NEMATODE DEGENERIN UNC-105 FORMS ION CHANNELS THAT ARE ACTIVATED BY DEGENERATION-OR HYPERCONTRACTION-CAUSING MUTATIONS

Nematode degenerins have been implicated in touch sensitivity and other forms of mechanosensation. Certain mutations in several degenerin genes cause the swelling, vacuolation, and death of neurons, and other mutations in the muscle degenerin gene unc-105 cause hypercontraction. Here, we confirm that unc-105 encodes an ion channel and show that it is constitutively active when mutated. These mutations disrupt different regions of the channel and have different effects on its gating. The UNC-105 channels are permeable to small monovalent cations but show voltage-dependent block by Ca2+ and Mg2+. Amiloride also produces voltage-dependent block, consistent with a single binding site 65% into the electric field. Mammalian cells expressing the mutant channels accumulate membranous whorls and multicompartment vacuoles, hallmarks of degenerin-induced cell death across species.

A Non-canonical Pathway from Cochlea to Brain Signals Tissue-Damaging Noise

Intense noise damages the cochlear organ of Corti, particularly the outer hair cells (OHCs) [1]; however, this epithelium is not innervated by nociceptors of somatosensory ganglia, which detect damage elsewhere in the body. The only sensory neurons innervating the organ of Corti originate from the spiral ganglion, roughly 95% of which innervate exclusively inner hair cells (IHCs) [2-4]. Upon sound stimulation, IHCs release glutamate to activate AMPA-type receptors on these myelinated type-I neurons, which carry the neuronal signals to the cochlear nucleus. The remaining spiral ganglion cells (type IIs) are unmyelinated and contact OHCs [2-4]. Their function is unknown. Using immunoreactivity to cFos, we documented neuronal activation in the brainstem of Vglut3(-/-) mice, in which the canonical auditory pathway (activation of type-I afferents by glutamate released from inner hair cells) is silenced [5, 6]. In these deaf mice, we found responses to noxious noise, which damages hair cells, but not to innocuous noise, in neurons of the cochlear nucleus, but not in the vestibular or trigeminal nuclei. This response originates in the cochlea and not in other areas also stimulated by intense noise (middle ear and vestibule) as it was absent in CD1 mice with selective cochlear degeneration but normal vestibular and somatosensory function. These data imply the existence of an alternative neuronal pathway from cochlea to brainstem that is activated by tissue-damaging noise and does not require glutamate release from IHCs. This detection of noise-induced tissue damage, possibly by type-II cochlear afferents, represents a novel form of sensation that we term auditory nociception.