Ebenezer N. Yamoah

TRPA1 Mediates the Inflammatory Actions of Environmental Irritants and Proalgesic Agents

TRPA1 is an excitatory ion channel targeted by pungent irritants from mustard and garlic. TRPA1 has been proposed to function in diverse sensory processes, including thermal (cold) nociception, hearing, and inflammatory pain. Using TRPA1-deficient mice, we now show that this channel is the sole target through which mustard oil and garlic activate primary afferent nociceptors to produce inflammatory pain. TRPA1 is also targeted by environmental irritants, such as acrolein, that account for toxic and inflammatory actions of tear gas, vehicle exhaust, and metabolic byproducts of chemotherapeutic agents. TRPA1-deficient mice display normal cold sensitivity and unimpaired auditory function, suggesting that this channel is not required for the initial detection of noxious cold or sound. However, TRPA1-deficient mice exhibit pronounced deficits in bradykinin-evoked nociceptor excitation and pain hypersensitivity. Thus, TRPA1 is an important component of the transduction machinery through which environmental irritants and endogenous proalgesic agents depolarize nociceptors to elicit inflammatory pain.

Mice Lacking the Basolateral Na-K-2Cl Cotransporter Have Impaired Epithelial Chloride Secretion and Are Profoundly Deaf

In chloride-secretory epithelia, the basolateral Na-K-2Cl cotransporter (NKCC1) is thought to play a major role in transepithelial Cl− and fluid transport. Similarly, in marginal cells of the inner ear, NKCC1 has been proposed as a component of the entry pathway for K+ that is secreted into the endolymph, thus playing a critical role in hearing. To test these hypotheses, we generated and analyzed an NKCC1-deficient mouse. Homozygous mutant (Nkcc1−/− ) mice exhibited growth retardation, a 28% incidence of death around the time of weaning, and mild difficulties in maintaining their balance. Mean arterial blood pressure was significantly reduced in both heterozygous and homozygous mutants, indicating an important function for NKCC1 in the maintenance of blood pressure. cAMP-induced short circuit currents, which are dependent on the CFTR Cl− channel, were reduced in jejunum, cecum, and trachea of Nkcc1−/− mice, indicating that NKCC1 contributes to cAMP-induced Cl− secretion. In contrast, secretion of gastric acid in adult Nkcc1−/− stomachs and enterotoxin-stimulated fluid secretion in the intestine of sucklingNkcc1−/− mice were normal. Finally, homozygous mutants were deaf, and histological analysis of the inner ear revealed a collapse of the membranous labyrinth, consistent with a critical role for NKCC1 in transepithelial K+ movements involved in generation of the K+-rich endolymph and the endocochlear potential.

Balance and Hearing Deficits in Mice with a Null Mutation in the Gene Encoding Plasma Membrane Ca2+-ATPase Isoform 2

Plasma membrane Ca2+-ATPase isoform 2 (PMCA2) exhibits a highly restricted tissue distribution, suggesting that it serves more specialized physiological functions than some of the other isoforms. A unique role in hearing is indicated by the high levels of PMCA2 expression in cochlear outer hair cells and spiral ganglion cells. To analyze the physiological role of PMCA2 we used gene targeting to produce PMCA2-deficient mice. Breeding of heterozygous mice yielded live homozygous mutant offspring. PMCA2-null mice grow more slowly than heterozygous and wild-type mice and exhibit an unsteady gait and difficulties in maintaining balance. Histological analysis of the cerebellum and inner ear of mutant and wild-type mice revealed that null mutants had slightly increased numbers of Purkinje neurons (in which PMCA2 is highly expressed), a decreased thickness of the molecular layer, an absence of otoconia in the vestibular system, and a range of abnormalities of the organ of Corti. Analysis of auditory evoked brainstem responses revealed that homozygous mutants were deaf and that heterozygous mice had a significant hearing loss. These data demonstrate that PMCA2 is required for both balance and hearing and suggest that it may be a major source of the calcium used in the formation and maintenance of otoconia.

Null Mutation of α1D Ca2+ Channel Gene Results in Deafness but No Vestibular Defect in Mice

Multiple Ca2+ channels confer diverse functions to hair cells of the auditory and vestibular organs in the mammalian inner ear. We used gene-targeting technology to generate α1D Ca2+ channel-deficient mice to determine the physiological role of these Ca2+ channels in hearing and balance. Analyses of auditory-evoked brainstem recordings confirmed that α1D−/− mice were deaf and revealed that heterozygous (α1D+/−) mice have increased hearing thresholds. However, hearing deficits in α1D+/− mice were manifested mainly by the increase in threshold of low-frequency sounds. In contrast to impaired hearing, α1D−/− mice have balance performances equivalent to their wild-type littermates. Light and electron microscope analyses of the inner ear revealed outer hair cell loss at the apical cochlea, but no apparent abnormality at the basal cochlea and the vestibule. We determined the mechanisms underlying the auditory function defects and the normal vestibular functions by examining the Ba2+ currents in cochlear inner and outer hair cells versus utricular hair cells in α1D+/− mice. Whereas the whole-cell Ba2+ currents in inner hair cells consist mainly of the nimodipine-sensitive current (~85%), the utricular hair cells express only ~50% of this channel subtype. Thus, differential expression of α1D channels in the cochlear and utricular hair cells confers the phenotype of the α1D null mutant mice. Because vestibular and cochlear hair cells share common features and null deletion of several genes have yielded both deafness and imbalance in mice, α1D null mutant mice may serve as a model to disentangle vestibular from auditory-specific functions.

Direct measurement of single-channel Ca2+ currents in bullfrog hair cells reveals two distinct channel subtypes

1 To confer their acute sensitivity to mechanical stimuli, hair cells employ Ca2+ ions to mediate sharp electrical tuning and neurotransmitter release. We examined the diversity and properties of voltage‐gated Ca2+ channels in bullfrog saccular hair cells by means of perforated and cell‐attached patch‐clamp techniques. Whole‐cell Ca2+ current records provided hints that hair cells express L‐type as well as dihydropyridine‐insensitive Ca2+ currents. 2 Single Ca2+ channel records confirmed the presence of L‐type channels, and a distinct Ca2+ channel, which has sensitivity towards ω‐conotoxin GVIA. Despite its sensitivity towards ω‐conotoxin GVIA, the non‐L‐type channel cannot necessarily be considered as an N‐type channel because of its distinct voltage‐dependent gating properties. 3 Using 65 mm Ca2+ as the charge carrier, the L‐type channels were recruited at about –40 mV and showed a single‐channel conductance of 13 pS. Under similar recording conditions, the non‐L‐type channels were activated at ∼–60 mV and had a single‐channel conductance of ∼16 pS. 4 The non‐L‐type channel exhibited at least two fast open time constants (τo= 0.2 and 5 ms). In contrast, the L‐type channels showed long openings (τo=∼23 ms) that were enhanced by Bay K 8644, in addition to the brief openings (τo= 0.3 and 10 ms). 5 The number of functional channels observed in patches of similar sizes suggests that Ca2+ channels are expressed singly, in low‐density clusters (2–15 channels) and in high‐density clusters (20–80 channels). Co‐localization of the two channel subtypes was observed in patches containing low‐density clusters, but was rare in patches containing high‐density clusters. 6 Finally, we confirmed the existence of two distinct Ca2+ channel subtypes by using immunoblot and immunohistochemical techniques.

Differential expression of KCNQ4 in inner hair cells and sensory neurons is the basis of progressive high-frequency hearing loss.

Human KCNQ4 mutations known as DFNA2 cause non-syndromic, autosomal-dominant, progressive high-frequency hearing loss in which the cellular and molecular basis is unclear. We provide immunofluorescence data showing that Kcnq4 expression in the adult cochlea has both longitudinal (base to apex) and radial (inner to outer hair cells) gradients. The most intense labeling is in outer hair cells at the apex and in inner hair cells as well as spiral ganglion neurons at the base. Spatiotemporal expression studies show increasing intensity of KCNQ4 protein labeling from postnatal day 21 (P21) to P120 mice that is most apparent in inner hair cells of the middle turn. We have identified four alternative splice variants of Kcnq4 in mice. The alternative use of exons 9-11 produces three transcript variants (v1-v3), whereas the fourth variant (v4) skips all three exons; all variants have the same amino acid sequence at the C termini. Both reverse transcription-PCR and quantitative PCR analyses demonstrate that these variants have differential expression patterns along the length of the mouse organ of Corti and spiral ganglion neurons. Our expression data suggest that the primary defect leading to high-frequency loss in DFNA2 patients may be attributable to high levels of the dysfunctional Kcnq4_v3 variant in the spiral ganglion and inner hair cells in the basal hook region. Progressive hearing loss associated with aging may result from an increasing mutational load expansion toward the apex in inner hair cells and spiral ganglion neurons.

Cochlear Function in Mice Lacking the BK Channel α, β1, or β4 Subunits

Large conductance voltage- and calcium-activated potassium (BK) channels are important for regulating many essential cellular functions, from neuronal action potential shape and firing rate to smooth muscle contractility. In amphibians, reptiles, and birds, BK channels mediate the intrinsic frequency tuning of the cochlear hair cell by an electrical resonance mechanism. In contrast, inner hair cells of the mammalian cochlea are extrinsically tuned by accessory structures of the cochlea. Nevertheless, BK channels are present in inner hair cells and encode a fast activating outward current. To understand the role of the BK channel α and β subunits in mammalian inner hair cells, we analyzed the morphology, physiology, and function of these cells from mice lacking the BK channel α (Slo-/-) and also the β1 and β4 subunits (β1/4-/-). β1/4-/- mice showed normal subcellular localization, developmental acquisition, and expression of BK channels. β1/4-/- mice showed normal cochlear function as indicated by normal auditory brainstem responses and distortion product otoacoustic emissions. Slo-/- mice also showed normal cochlear function despite the absence of the BKα subunit and the absence of fast activating outward current from the inner hair cells. Moreover, microarray analyses revealed no compensatory changes in transcripts encoding ion channels or transporters in the cochlea from Slo-/- mice. Slo-/- mice did, however, show increased resistance to noise-induced hearing loss. These findings reveal the fundamentally different contribution of BK channels to nonmammalian and mammalian hearing and suggest that BK channels should be considered a target in the prevention of noise-induced hearing loss.

Phosphate Analogs Block Adaptation in Hair Cells by Inhibiting Adaptation-Motor Force Production

To ensure optimal sensitivity for mechanoelectrical transduction, hair cells adapt to prolonged stimuli using active motors. Adaptation motors are thought to employ myosin molecules as their force-producing components. We find that beryllium fluoride, vanadate, and sulfate, phosphate analogs that inhibit the ATPase activity of myosin, inhibit adaptation by abolishing motor force production. Phosphate analogs interact with a 120-kDa bundle protein, most likely myosin 1 beta, in a manner that coincides with their effects on adaptation. Features of transduction following inhibition of motor force production suggest that the gating and extent springs of the hair cell orient in parallel at rest and that the negative limit of adaptation arises when force in the stretched extent spring matches the force output of the adaptation motor.

Assembly of the otoconia complex to the macular sensory epithelium of the vestibule

In the inner ear, specificity of stimulus perception is achieved by associating the sensory epithelia of the three mechanoreceptor organs, the utricle/saccule, cristae, and cochlea, with distinct types of acellular matrices. Only the utricle and saccule have an extremely dense matrix, the otoconial complex, which overlies the sensory epithelium (macula) and provides inertial mass to generate shearing forces essential for the mechanoreceptors to sense gravity and linear acceleration. Such sensation is necessary for spatial orientation and balance. The importance of otoconia is clearly demonstrated by the impact of balance disorders upon the elderly population that involve otoconia degeneration, as well as by canalithiasis and cupulolithiasis, in which otoconia are dislocated. This underscores the need to understand how otoconia are formed and maintained and how to prevent their degeneration. To date, a number of otoconia-related proteins have been identified mostly in mice and bony fish. Although most of these proteins are also present in other structures of the inner ear, a distinct collection of proteins in the macula plus the unique ionic microenvironment of the endolymph near its epithelium likely contribute to the site-specific calcification of otoconia. Based on the current literature and ongoing research, this mini-review postulates a working model of how the otoconia complex is assembled specifically above the macular sensory epithelium of the vestibule. The central hypothesis of this model is that proteins are critical in sequestering calcium for crystallization in the calcium-poor endolymph. The review also sets forth some issues that need to be resolved in the future.

Ca2+ transport properties and determinants of anomalous mole fraction effects of single voltage-gated Ca2+ channels in hair cells from bullfrog saccule

We studied the permeation properties of two distinct single voltage‐gated Ca2+ channels in bullfrog saccular hair cells to assess the roles of the channels as physiological Ca2+ transporters and multi‐ion pores. By varying the permeant ions (Ba2+, Ca2+) and concentrations (2–70 mm), we estimated the affinity constant (KD) of the two channels as follows (mm): L‐type channel, KD,Ba= 7.4 ± 1.0, KD,Ca= 7.1 ± 2.2 (n= 7); non‐L‐type channel, KD,Ba= 5.3 ± 3.2, KD,Ca= 2.0 ± 1.0 (n= 8). Using ionic concentrations close to physiological conditions (2 mm Ca2+ and 1.0 mm Mg2+), the conductance of the L‐type channel was ∼2 pS. We determined the mechanisms by which ions traverse the pore of these single Ca2+ channels, using mixtures of Ba2+ and Ca2+ at total concentrations above (70 mm) or close to (5 mm) the KD of the channels. We found evidence for an anomalous mole fraction effect (AMFE) only when the total divalent ion concentration was 5 mm, consistent with a multi‐ion pore. We show that AMFE arises from the boundaries between the pore and bulk solution in the atria of the channel, which is derived from the presence of depletion zones that become apparent at low divalent cation concentrations. The present findings provide an explanation as to why previous whole‐cell Ca2+ currents that were recorded in quasi‐physiological Ca2+ concentrations (∼2–5 mm) showed clear AMFE, whereas single Ca2+ channel currents that were recorded routinely at high Ca2+ concentrations (20–110 mm) did not.