Characterisation of N-glycans in the epithelial-like tissue of the rat cochlea.

Membrane proteins (such as ion channels, transporters, and receptors) and secreted proteins are essential for cellular activities. N-linked glycosylation is involved in stability and function of these proteins and occurs at Asn residues. In several organs, profiles of N-glycans have been determined by comprehensive analyses. Nevertheless, the cochlea of the mammalian inner ear, a tiny organ mediating hearing, has yet to be examined. Here, we focused on the stria vascularis, an epithelial-like tissue in the cochlea, and characterised N-glycans by liquid chromatography with mass spectrometry. This hypervascular tissue not only expresses several ion transporters and channels to control the electrochemical balance in the cochlea but also harbours different transporters and receptors that maintain structure and activity of the organ. Seventy-nine N-linked glycans were identified in the rat stria vascularis. Among these, in 55 glycans, the complete structures were determined; in the other 24 species, partial glycosidic linkage patterns and full profiles of the monosaccharide composition were identified. In the process of characterisation, several sialylated glycans were subjected sequentially to two different alkylamidation reactions; this derivatisation helped to distinguish α2,3-linkage and α2,6-linkage sialyl isomers with mass spectrometry. These data should accelerate elucidation of the molecular architecture of the cochlea.

Hearing Loss Controlled by Optogenetic Stimulation of Nonexcitable Nonglial Cells in the Cochlea of the Inner Ear.

Light-gated ion channels and transporters have been applied to a broad array of excitable cells including neurons, cardiac myocytes, skeletal muscle cells and pancreatic ß-cells in an organism to clarify their physiological and pathological roles. Nonetheless, among nonexcitable cells, only glial cells have been studied in vivo by this approach. Here, by optogenetic stimulation of a different nonexcitable cell type in the cochlea of the inner ear, we induce and control hearing loss. To our knowledge, deafness animal models using optogenetics have not yet been established. Analysis of transgenic mice expressing channelrhodopsin-2 (ChR2) induced by an oligodendrocyte-specific promoter identified this channel in nonglial cells-melanocytes-of an epithelial-like tissue in the cochlea. The membrane potential of these cells underlies a highly positive potential in a K+-rich extracellular solution, endolymph; this electrical property is essential for hearing. Illumination of the cochlea to activate ChR2 and depolarize the melanocytes significantly impaired hearing within a few minutes, accompanied by a reduction in the endolymphatic potential. After cessation of the illumination, the hearing thresholds and potential returned to baseline during several minutes. These responses were replicable multiple times. ChR2 was also expressed in cochlear glial cells surrounding the neuronal components, but slight neural activation caused by the optical stimulation was unlikely to be involved in the hearing impairment. The acute-onset, reversible and repeatable phenotype, which is inaccessible to conventional gene-targeting and pharmacological approaches, seems to at least partially resemble the symptom in a population of patients with sensorineural hearing loss. Taken together, this mouse line may not only broaden applications of optogenetics but also contribute to the progress of translational research on deafness.

Computer modeling defines the system driving a constant current crucial for homeostasis in the mammalian cochlea by integrating unique ion transports.

The cochlear lateral wall-an epithelial-like tissue comprising inner and outer layers-maintains +80 mV in endolymph. This endocochlear potential supports hearing and represents the sum of all membrane potentials across apical and basolateral surfaces of both layers. The apical surfaces are governed by K+ equilibrium potentials. Underlying extracellular and intracellular [K+] is likely controlled by the "circulation current," which crosses the two layers and unidirectionally flows throughout the cochlea. This idea was conceptually reinforced by our computational model integrating ion channels and transporters; however, contribution of the outer layer's basolateral surface remains unclear. Recent experiments showed that this basolateral surface transports K+ using Na+, K+-ATPases and an unusual characteristic of greater permeability to Na+ than to other ions. To determine whether and how these machineries are involved in the circulation current, we used an in silico approach. In our updated model, the outer layer's basolateral surface was provided with only Na+, K+-ATPases, Na+ conductance, and leak conductance. Under normal conditions, the circulation current was assumed to consist of K+ and be driven predominantly by Na+, K+-ATPases. The model replicated the experimentally measured electrochemical properties in all compartments of the lateral wall, and endocochlear potential, under normal conditions and during blocking of Na+, K+-ATPases. Therefore, the circulation current across the outer layer's basolateral surface depends primarily on the three ion transport mechanisms. During the blockage, the reduced circulation current partially consisted of transiently evoked Na+ flow via the two conductances. This work defines the comprehensive system driving the circulation current.

The unique ion permeability profile of cochlear fibrocytes and its contribution to establishing their positive resting membrane potential.

Eukaryotic cells exhibit negative resting membrane potential (RMP) owing to the high K(+) permeability of the plasma membrane and the asymmetric [K(+)] between the extracellular and intracellular compartments. However, cochlear fibrocytes, which comprise the basolateral surface of a multilayer epithelial-like tissue, exhibit a RMP of +5 to +12 mV in vivo. This positive RMP is critical for the formation of an endocochlear potential (EP) of +80 mV in a K(+)-rich extracellular fluid, endolymph. The epithelial-like tissue bathes fibrocytes in a regular extracellular fluid, perilymph, and apically faces the endolymph. The EP, which is essential for hearing, represents the potential difference across the tissue. Using in vivo electrophysiological approaches, we describe a potential mechanism underlying the unusual RMP of guinea pig fibrocytes. The RMP was +9.0 ± 3.7 mV when fibrocytes were exposed to an artificial control perilymph (n = 28 cochleae). Perilymphatic perfusion of a solution containing low [Na(+)] (1 mM) markedly hyperpolarized the RMP to -31.1 ± 11.2 mV (n = 10; p < 0.0001 versus the control, Tukey-Kramer test after one-way ANOVA). Accordingly, the EP decreased. Little change in RMP was observed when the cells were treated with a high [K(+)] of 30 mM (+10.4 ± 2.3 mV; n = 7; p = 0.942 versus the control). During the infusion of a low [Cl(-)] solution (2.4 mM), the RMP moderately hyperpolarized to -0.9 ± 3.4 mV (n = 5; p < 0.01 versus the control), although the membranes, if governed by Cl(-) permeability, should be depolarized. These observations imply that the fibrocyte membranes are more permeable to Na(+) than K(+) and Cl(-), and this unique profile and [Na(+)] gradient across the membranes contribute to the positive RMP.

Molecular architecture of the stria vascularis membrane transport system, which is essential for physiological functions of the mammalian cochlea.

Stria vascularis of the mammalian cochlea transports K(+) to establish the electrochemical property in the endolymph crucial for hearing. This epithelial tissue also transports various small molecules. To clarify the profile of proteins participating in the transport system in the stria vascularis, membrane components purified from the stria of adult rats were analysed by liquid chromatography tandem mass spectrometry. Of the 3236 proteins detected in the analysis, 1807 were membrane proteins. Ingenuity Knowledge Base and literature data identified 513 proteins as being expressed on the 'plasma membrane', these included 25 ion channels and 79 transporters. Sixteen of the former and 62 of the latter had not yet been identified in the stria. Unexpectedly, many Cl(-) and Ca(2+) transport systems were found, suggesting that the dynamics of these ions play multiple roles. Several transporters for organic substances were also detected. Network analysis demonstrated that a few kinases, including protein kinase A, and Ca(2+) were key regulators for the strial transports. In the library of channels and transporters, 19 new candidates for uncloned deafness-related genes were identified. These resources provide a platform for understanding the molecular mechanisms underlying the epithelial transport essential for cochlear function and the pathophysiological processes involved in hearing disorders.

NKCCs in the fibrocytes of the spiral ligament are silent on the unidirectional K⁺ transport that controls the electrochemical properties in the mammalian cochlea.

Unidirectional K(+) transport across the lateral cochlear wall contributes to the endocochlear potential (EP) of +80 mV in the endolymph, a property essential for hearing. The wall comprises two epithelial layers, the syncytium and the marginal cells. The basolateral surface of the former and the apical membranes of the latter face the perilymph and the endolymph, respectively. Intrastrial space (IS), an extracellular compartment between the two layers, exhibits low [K(+)] and a potential similar to the EP. This IS potential (ISP) dominates the EP and represents a K(+) diffusion potential elicited by a large K(+) gradient across the syncytial apical surface. The K(+) gradient depends on the unidirectional K(+) transport driven by Na(+),K(+)-ATPases on the basolateral surface of each layer and the concomitant Na(+),K(+),2Cl(-)-cotransporters (NKCCs) in the marginal cell layer. The NKCCs coexpressed with the Na(+),K(+)-ATPases in the syncytial layer also seem to participate in the K(+) transport. To test this hypothesis, we examined the electrochemical properties of the lateral wall with electrodes measuring [K(+)] and potential. Blocking NKCCs by perilymphatic perfusion of bumetanide suppressed the ISP. Unexpectedly and unlike the inhibition of the syncytial Na(+),K(+)-ATPases, the perfusion barely altered the electrochemical properties of the syncytium but markedly augmented [K(+)] of the IS. Consequently, the K(+) gradient decreased and the ISP declined. These observations resembled those when the marginal cells' Na(+),K(+)-ATPases or NKCCs were blocked with vascularly applied inhibitors. It is plausible that NKCCs in the marginal cells are affected by the perilymphatically perfused bumetanide, and these transporters, but not those in the syncytium, mediate the unidirectional K(+) transport.

Transient low-tone air-bone gaps during convalescence immediately after canal plugging surgery for BPPV.

OBJECTIVES: The aim of the present study was to elucidate the time course and frequency patterns of transient low-tone air-bone gaps (ABGs) after canal plugging for intractable BPPV. METHODS: We investigated eight patients with intractable BPPV who underwent canal plugging. Four were cases with posterior type (pBPPV) and the other four were those with horizontal type (hBPPV). Pure-tone audiometries (PTAs) were performed before and 7 days, 1 month and 6 months after surgery. ABGs (+) were defined as the three-tone-average ≥20dB formulated by (a+b+c)/3, where a, b, and c are ABGs at 0.25, 0.5, and 1kHz, respectively. RESULTS: The ratio of the number of patients with ABGs (+) at the post-operative 7th day and 1st month was 100.0% (8/8). The ratio at the post-operative 6th month was 0.0% (0/8). There were no significant differences in the time course or frequency patterns of the ABGs between pBPPV and hBPPV. CONCLUSIONS: We clearly demonstrated eight cases with intractable BPPV showing transient low-tone ABGs during convalescence immediately after canal plugging. During that period, patients also complained of motion-evoked dizziness. All these findings suggest that, during such a convalescence period, the plugged area might not be fixed yet and could still induce the dizziness and low-tone ABGs, as enlarged vestibular aqueduct syndrome and superior semicircular canal deficiency syndrome exhibit low-tone ABGs due to the third mobile inner ear window. More than one month after surgery, both the ABGs and dizziness could disappear according to fixation of the plugged area.