Genki Ogata

The mechanism underlying maintenance of the endocochlear potential by the K+ transport system in fibrocytes of the inner ear

•  The endocochlear potential (EP) of +80 mV in cochlear endolymph is essential for audition and controlled by K+ transport across the lateral cochlear wall composed of two epithelial barrier layers, the syncytium containing the fibrocytes and the marginal cells. •  The EP depends upon the diffusion potential elicited by a large K+ gradient across the apical surface of the syncytium. •  We examined by electrophysiological approaches an involvement of Na+,K+‐ATPase, which occurs at the syncytiums basolateral surface comprising the fibrocytes’ membranes and would mediate K+ transport across the lateral wall, in maintenance of the EP. •  We show that the Na+,K+‐ATPase sustains the syncytiums high [K+] that is crucial for the K+ gradient across the apical surface of the syncytium. •  The results help us better understand the mechanism underlying the establishment of the EP as well as the pathophysiological process for deafness induced by dysfunction of the ion transport apparatus.

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 Ca2+ 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 Ca2+ 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.

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.Sensory neuroscience: let ion run through a cochlear labyrinthIn in vivo mammalian cochlea, ionic current constantly and unidirectionally flows—this unique “circulation current”, which contributes to high sensitivity of sensory cells transducing atomic scale acoustic vibrations to electrical signals, likely depends upon ion transports across a multiple-layered epithelial tissue. To determine how the circulation current is established, a team conducted by Hiroshi Hibino at Niigata University in Japan used a theoretical approach, because ionic currents are unmeasurable in vivo. A conceptual computational model they previously developed lacked involvement of an epithelial tissue membrane recently found to show unusual ion transport profile; integration and coupling of this element to other membrane transport machineries resulted in reproducing experimental measurements. This work defines the comprehensive system driving the circulation current, which remains uncertain for nearly 20 years, and helps us to understand the mechanism for hearing.

A microsensing system for the in vivo real-time detection of local drug kinetics

Real-time recording of the kinetics of systemically administered drugs in in vivo microenvironments may accelerate the development of effective medical therapies. However, conventional methods require considerable analyte quantities, have low sampling rates and do not address how drug kinetics correlate with target function over time. Here, we describe the development and application of a drug-sensing system consisting of a glass microelectrode and a microsensor composed of boron-doped diamond with a tip of around 40 μm in diameter. We show that, in the guinea pig cochlea, the system can measure—simultaneously and in real time—changes in the concentration of bumetanide (a diuretic that is ototoxic but applicable to epilepsy treatment) and the endocochlear potential underlying hearing. In the rat brain, we tracked the kinetics of the drug and the local field potentials representing neuronal activity. We also show that the actions of the antiepileptic drug lamotrigine and the anticancer reagent doxorubicin can be monitored in vivo. Our microsensing system offers the potential to detect pharmacological and physiological responses that might otherwise remain undetected.A system consisting of a glass microelectrode and a boron-doped diamond microsensor can simultaneously track, in rat brains and in the guinea pig cochlea, the local real-time kinetics of injected drugs and the resulting electrophysiological activity.

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 β-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.

Theoretical and experimental analysis of ototoxic mechanism in the spiral ligament fibrocytes by multi-level simulation with ion transports in the cochlea

生 体 機 能 の 多 階 層 的 理 解 と 創 薬 研 究 へ の 応 用 2 要約:空気の振動である音は,内耳蝸牛に存在する 音の感覚細胞である有毛細胞を振動させる.この時, 有毛細胞の毛に存在するイオンチャネルが開口し, 常時+80 mVを示す特殊な内リンパ液からイオンが流 入する.この「内リンパ液高電位」は,有毛細胞の興 奮に不可欠であり,蝸牛側壁の血管条が成立させる. Na,K,2Cl共輸送体(NKCC)や Na,K-ATPaseの 阻害薬は,内リンパ液高電位を低下させることで,薬剤 性難聴を惹起することが報告されているが,その電位 低下のメカニズムは明らかにされていなかった.我々 はこれまでに,血管条に発現する NKCCと Na,KATPaseが制御する内リンパ液高電位成立機構を電気 生理学的手法により示し,さらに蝸牛内の多階層イオ ン輸送モデル「Nin-Hibino-Kurachi(NHK)model」の 構築とコンピュータシミュレーションによって,阻害 薬を経動脈的に投与した時に起こる内リンパ液高電 位低下のメカニズムを説明した.血管条に隣接し,そ の一部と一体化しているらせん靭帯を構成する線維 細胞にも,NKCCと Na,K-ATPaseが発現している ことが知られているが,薬剤性難聴時のこれらの関与 は不明である.近年我々は,らせん靭帯ではおもに Na,K-ATPaseが,K輸送と K濃度バランスに寄与 すること,そしてNKCCはほとんど機能していないこ とを明らかにした.これらに基づいて,NHKモデル を改訂した.今後シミュレーションを行うことにより, 輸送体阻害薬の経動脈投与の実験結果を正確に再現す ることが期待される. 1. はじめに