I. Introduction

Hearing is the sensory process through which the auditory system detects, transduces, and interprets sound waves into neural signals, enabling individuals to perceive sounds and interact with their environment. This complex process involves the integration of the peripheral and central auditory systems. The peripheral auditory system, encompassing the outer ear, middle ear, cochlea and Spiral Ganglion Neurons (SGNs), is responsible for capturing, transducing, and transmitting sound information to the brainstem within the central auditory system. The sound waves collected by the outer ear are funneled toward the middle ear, where the tympanic membrane and ossicles amplify the mechanical vibrations generated by acoustic stimuli. These vibrations are transmitted to the inner ear, where the cochlea converts mechanical energy into electrical signals. These electrical signals are then relayed to the SGNs, which transform them into neural impulses.^[1]

The cochlea is a spirally coiled structure within the inner ear, comprising three distinct fluid-filled compartments. The scala vestibule and scala tympani are filled with perilymph, an extracellular-like fluid with high sodium (Na⁺) and low potassium (K⁺) concentrations. Perilymph is maintained by exchange with blood plasma across capillaries in the spiral ligament and adjacent cochlear structures. In contrast, the scala media contains endolymph, a fluid with high K⁺ and low Na⁺ levels, secreted actively by the stria vascularis using ion transport mechanisms such as the Na⁺/K⁺-ATPase pump and KCNQ1-KCNE1 K⁺ channels, maintaining the electrochemical gradients essential for generating endocochlear potential.^[3,4] The ion composition of endolymph differs from other extracellular fluids, which typically exhibit high Na⁺ and low K⁺. This distinct composition supports a unique depolarization process in Hair Cells (HCs), where, unlike Na⁺ driven depolarization in most cells, HCs rely on K⁺ influx. This process is facilitated by the electrochemical gradient maintained by the stria vascularis in the cochlea.^[5]

HCs, particularly Inner HCs (IHCs), are the core sensory elements of the cochlea, responsible for converting mechanical sound vibrations into electrical signals. Incoming sound waves create traveling waves along the basilar membrane, producing shear forces that deflect the stereocilia of HCs, triggering K⁺ influx and depolarization of HC. The resulting receptor potentials are transmitted to SGNs via specialized ribbon synapses, enabling rapid and precise neurotransmitter release. As the first neurons in the auditory pathway, SGNs encode these electrical signals into action potentials, transmitting them to the Cochlear Nucleus (CN) in the brainstem, which then passes them on to higher auditory centers along the central auditory pathway.^[6,7]

The mechanoelectrical mechanism of IHCs relies on precise regulation of membrane potentials by ion channels. Disruptions in these processes contribute to a variety of auditory pathologies including noise-induced hearing loss, ototoxicity, and presbycusis.^[8] The present study aims to investigate the cellular and molecular mechanisms underlying auditory signal encoding in IHCs, with a particular emphasis on the functional roles of ion channels and ribbon synapses. By examining the dynamic regulation of ionic homeostasis and synaptic transmission, this study seeks to provide a comprehensive understanding of how IHCs perform MechanoElectrical Transduction (MET), maintain ionic conductance, and regulate neurotransmitter release for precise encoding of sound information.

II. Structure and function of HCs

2.1 Structure of HCs

HCs are the sensory receptor cells in the auditory and vestibular systems. These epithelial cells, located within the organ of Corti in the cochlea, are classified into two distinct types: IHCs and Outer HCs (OHCs). In the human cochlea, IHCs are flask-shaped and arranged in a single row along the cochlear spiral, totaling approximately 3,500 per ear. In contrast, OHCs are cylindrical and organized in three rows, occasionally four or even five rows, with a total of approximately 12,000 per ear.^[9,10,11]

Each HC has bundles of stereocilia, which are actin-filled protrusions located on its apical surface. These stereocilia are arranged in rows of increasing height, with the tallest stereocilium positioned toward the lateral wall of the cochlea. IHCs have shorter and fewer stereocilia (approximately 50 to 100 per cell), arranged in a shallow U shape, which is specialized for sensory signal encoding, allowing precise transduction of mechanical stimuli into graded receptor potentials. On the other hand, OHCs possess longer and more numerous stereocilia (approximately 100 to 150 per cell), arranged in V or W shape, reflecting their role in mechanical amplification.^[9,12,13] These stereocilia are interconnected by tip links, protein filaments composed of cadherin-23 and protocadherin-15, which mechanically couple their movements and play a crucial role in gating mechanosensitive ion channels.^[14]

2.2 Function of HCs

IHCs serve as the primary sensory transducer of sound in the cochlea, converting mechanical energy from incoming sound waves into electrical signals, which are transmitted to Type I SGNs via ribbon synapses This process occurs when basilar membrane vibration deflects the stereocilia of IHCs. The stereocilia deflection opens MET ion channels located at the tips of the stereocilia, allowing K⁺ and calcium (Ca²⁺) from the endolymph to enter the cell, resulting in depolarization of IHCs.^[15,16]

IHCs encode essential auditory information, including frequency, intensity and timing, and relay it to SGNs for transmission to the central auditory system. Place encoding relies on the tonotopic arrangement of the cochlea where high frequency sounds stimulate IHCs near the base and while low frequency sound activate IHCs closer to the apex. Intensity encoding depends on the amplitude of receptor potentials with greater stereocilia deflection causing increased glutamate release and higher firing rates in SGNs. Temporal encoding involves phase-locked firing of SGNs, driven by synchronized glutamate release from IHCs.^[16,17,18] When stereocilia oscillate in sync with incoming sound waves, IHCs generate alternating receptor potentials via depolarization-hyperpolarization cycles, leading to synchronous glutamate release. This enables phase locked firing in SGNs, particularly for frequencies below approximately 4 kHz to 5 kHz, preserving temporal precision.^[19,20] Thus, IHCs serve as an essential link between mechanical sound stimuli and the neural encoding of auditory information, contributing to the accurate signal transmission to the central auditory system.

OHCs, on the other hand, actively serve as mechanical amplifiers through electromotility, enhancing basilar membrane motion. The motor protein prestin, localized in the lateral membrane of OHCs, derives rapid cell length changes in response to voltage fluctuations. During depolarization, prestin molecules contracts, causing the OHCs to shorten, while during hyperpolarization, prestin relaxes, allowing the OHCs to elongate.^[21,22] This active mechanical mechanism boosts sound sensitivity and frequency resolution, increasing the cochlea’s sensitivity by up to 50 dB for low to moderate intensity sounds and sharpening its tonotopicity.^[23] Also, efferent fibers from the brainstem synapse onto OHCs and release Acetylcholine (ACh). This efferent control modulates the electromotility of OHCs, reducing cochlear gain during sustained or loud sounds and thereby protecting HCs from overstimulation.^[24]

III. Cellular and molecular mechanisms of IHCs

3.1 Depolarization

The apical surface of IHCs, where the stereocilia are located, is exposed to the endolymph in the scala media while the basolateral membrane of IHCs is bathed in the perilymph. The lipid bilayer of IHCs maintains a negative Resting Membrane Potential (RMP), resulting from ion gradients across the membrane and the selective permeability of ion channels. At rest, the RMPs of IHCs range approximately from -45 mV to -60 mV relative to the surrounding extracellular fluid, which is less negative compared to many other excitable cells in the body.^[3,25] The perilymph has an electrical potential close to 0 mV relative to the surrounding extracellular fluid whereas the endolymph in the scala media has a positive potential of +80 mV to +100 mV, termed endocochlear potential, which is primarily maintained by the high K⁺ concentration and low Na⁺ and Ca²⁺ levels.^[3,26]

The basilar membrane induced by sound waves causes deflection of the stereocilia. When the stereocilia deflect toward the tallest row, stretched tip links connecting adjacent stereocilia open mechanically gated ion channels, whereas these channels close when stereocilia bend away from the tallest row. The opening of MET channels allows K⁺, abundant in the endolymph, to flow into IHCs. When IHCs depolarize, their membrane potential shifts from RMP to a more positive value, depending on the intensity of sound stimulus and the amplitude of stereocilia deflection. This intracellular charge, known as depolarization, generates an electrical signal essential for auditory transduction.^{[9,26,27,28,29]}

3.2 Graded receptor potential

The receptor potential of IHCs refers to a graded change in the membrane potential of the cells that arises due to stereocilia deflection. It takes place before synaptic transmission, representing localized changes in RMP of IHCs. The graded receptor potential develops simultaneously with depolarization when MET ion channels open, allowing K⁺ ion into IHCs.^[29]

The influx of K⁺ into IHCs enables them to convert mechanical stimuli into graded receptor potentials, which are electrical signals, not neural signals (i.e. action potentials). The graded receptor potential triggers the opening of Voltage-Gated Ca²⁺ Channels (VGCCs) on the basolateral membrane of the IHCs, allowing extracellular Ca²⁺ to enter IHCs. Specifically, CaV1.3 channels, a subtype of L-type Ca²⁺ channels, mediate sustained Ca²⁺ entry into IHCs due to their low activation threshold and slow inactivation.^[30] These VGCCs begin to activate from near -70 mV, with maximal opening occurring at around -20 mV.^[31] The influx of extracellular Ca²⁺ from the perilymph into IHCs plays a critical role in facilitating synaptic vesicle fusion and neurotransmitter release, which are indispensable for signal transmission to the SGNs.

3.3 Neurotransmitter release

As intracellular Ca²⁺ concentration increases, synaptic vesicles containing glutamate, the primary excitatory neurotransmitter, fuse with the presynaptic membrane. This presynaptic membrane located at the base of IHCs contains the active zone where synaptic vesicles are docked and neurotransmitters are released. This process occurs at the ribbon synapse, which forms the junction between the presynaptic IHC and the postsynaptic SGN.^[7,32,33] Ribbon synapses are optimized for rapid, continuous, and precise neurotransmitter release, ensuring accurate encoding of auditory signal. Once glutamate diffuses into the synaptic cleft between the IHC and the SGN, it binds to α-Amino-3-hydroxy-5-methyl-4- isoxazolepropionic Acid (AMPA) receptors located on the postsynaptic membrane of SGNs, generating Excitatory Postsynaptic Potentials (EPSPs) in SGNs. While IHCs serve as the primary source of glutamate release, OHCs also release small amounts of glutamate, showing their less prominent role in synaptic transmission.^[7,32,34,35]

Neurotransmitter release occurs at ribbon synapses, highly specialized synaptic structures found in IHCs. Ribbon synapses are also present in the vestibular HCs, retinal photoreceptors, and bipolar cells. Ribbon synapses are electron-dense, proteinaceous structure with a plate- or crescent- shaped appearance. They serve as the presynaptic terminal between IHCs and SGNs, providing a platform for synaptic vesicle docking, priming and neurotransmitter release.^[32,33,36]

The ribbon structure is primarily composed of the protein ribeye, supported by other proteins such as Bassoon and Piccolo. Ribeye is a unique protein with two domains: A-Domain which provides structural support for the ribbon and B-Domain which regulates synaptic vesicle tethering. Bassoon and Piccolo, presynaptic scaffolding proteins, anchor the ribbon to the active zone and facilitate tight coupling between VGCCs and docked synaptic vesicles on the ribbon.^{[37,38,39,40,41]} The presynaptic membrane of IHCs contains VGCCs positioned near the ribbon synapse in the basolateral region of IHCs. Upon Ca²⁺ influx into IHCs, the Ca²⁺ sensor protein Otoferlin detects rising intracellular Ca²⁺ levels and triggers synaptic vesicle fusion through interaction with the Soluble N-ethylmaleimide- sensitive factor Attachment protein REceptor (SNARE) protein complex.^[29,42]

Synaptic vesicles are arranged along the ribbon in a gradient, enabling efficient neurotransmitter release. Unlike conventional synapses that fire in an all-or-nothing manner, ribbon synapses release neurotransmitters in a graded fashion, proportional to the strength of the receptor potentials.^[33] Thus, stronger depolarization induces greater Ca²⁺ influx and increased neurotransmitter release. Docked vesicles ready for immediate release are positioned closest to the ribbon, while reserve vesicles are tethered along the ribbon to replenish docked vesicles during sustained stimulation. This vesicle arrangement allows ribbon synapses to rapidly replenish synaptic vesicles, maintaining continuous neurotransmitter release even during prolonged auditory stimuli. Consequently, ribbon synapses support high speed synaptic transmission that enables precise encoding of auditory signals, particularly at high frequencies.^{[33,38,43,44,45]}

3.4 Returning to RMPs

Repolarization, the restoration of the RMP in IHCs, is often broadly described as encompassing the entire process following depolarization. This process is essential for maintaining the sensitivity of IHCs to subsequent sound stimuli. By restoring ionic gradients across the cell membrane, repolarization contributes to preserving cellular responsiveness to new stimuli and helps prevent excessive K⁺ accumulation that might disrupt cellular homeostasis.^[25,46]

More specifically, repolarization can represent a distinct phase immediately after depolarization and before subsequent hyperpolarization. Following depolarization, stereocilia begin returning toward their resting state due to the oscillatory motion of the basilar membrane, causing MET channels to close. At this stage, IHCs remain depolarized at approximately -20 mV with continued Ca²⁺ influx through VGCC alongside K⁺ efflux through voltage-gated, Ca²⁺ -activated, and passive leak channels. As the driving force for Ca²⁺ influx weakens due to the membrane potential becoming less negative, K⁺ efflux surpasses Ca²⁺ influx, causing the membrane potential to become more negative.^[25,31] Subsequently, VGCCs begin to deactivate, reducing neurotransmitter release at the ribbon synapse while voltage-gated K⁺ channels on the basolateral membrane of IHCs remain open, facilitating further K⁺ efflux. Thus, the combined activity of voltage- gated K⁺ channels, Ca^2+-activated K⁺channels, and passive K⁺ leak channels allows the IHC membrane potential to rapidly return to its resting state. The Kv7.4 (KCNQ4) channels on the basolateral membrane serve as the primary mediators of K⁺ efflux while BK channels may also contribute to this process.^[3,47] As K⁺ leak channels, TWIK-1 (KCNK1), TASK-3 (KCNK9), TASK-1 (KCNK3), and TREK-1 (KCNK2) provide passive K⁺ efflux.^[48]

To facilitate repolarization, intracellular Ca²⁺ is expelled through Ca²⁺ pumps and exchangers. The plasma membrane Ca²⁺ ATPase (PMCA), an ATPase enzyme located in the plasma membrane, serves as a Ca²⁺ pump actively expelling Ca²⁺ from IHCs by utilizing energy derived from ATP. IHCs lack Na⁺/Ca²⁺ exchangers (NCXs), which would typically exchange intracellular Ca²⁺ for extracellular Na⁺ to maintain ionic homeostasis.^[49]

Subsequently, hyperpolarization occurs when the stereocilia deflect away from the tallest stereocilium, fully closing MET channels and stopping the influx of K⁺ and Ca²⁺. Meanwhile, voltage-gated K⁺ channels on the basolateral membrane of IHCs remain open longer than physiologically required due to their slow deactivation kinetics. This prolonged opening results in continued K⁺ efflux into the perilymph, causing the membrane potential to drop below the RMP, inducing hyperpolarization. Hyperpolarization of IHCs inhibits synaptic vesicle release at the ribbon synapse by reducing presynaptic membrane potentials, thereby suppressing the neurotransmitter release.^[25,50,51]

Hyperpolarization contributes to temporal precision up to 3 kHz by defining the cycles of depolarization and hyperpolarization.^[18,25,52] This phase increases the electrochemical gradient across the IHC membrane, strengthening the electrochemical driving force for ensuing K⁺ influx when MET channel reopens. This facilitates rapid and robust depolarization in response to subsequent sound stimuli.^[25,50,51] Hyperpolarization also makes the activation of VGCC less probable, thereby reducing spontaneous neurotransmitter release, which decreases synaptic noise and enhances temporal resolution. This effect is mediated by a decrease in intracellular Ca²⁺ concentrations which limits unnecessary synaptic vesicle fusion.^[51,53] At the cellular level, the reduction of neurotransmitter release also occurs through an efferent mechanism, which prevents synaptic fatigue or glutamate excitotoxicity caused by overstimulation, thereby preserving the accurate encoding of auditory signals.^[54,55]

The refractory period refers to the time following an action potential during which excitable cells reset their ionic balance and restore their membrane potential, temporarily making them less responsive to subsequent stimuli. Though it is typically associated with neurons, IHCs also exhibit a comparable functional recovery phenomenon that involves replenishment of synaptic vesicles at the ribbon synapse, ensuring precise and sustained neurotransmitter release.^[25,56] To maintain precise signal transmission, IHCs located in the basal cochlea, which are responsible for encoding high-frequency sounds, may exhibit shorter synaptic recovery periods due to faster vesicle cycling and enhanced Ca²⁺ dynamics compared to apical IHCs.^[57]

When the membrane potential falls below the RMP, voltage-gated K⁺ channels gradually close, stopping further K⁺ efflux and enabling the membrane potential to return to RMP. The Na⁺/K⁺ ATPase pump also helps restore the ionic gradient by transporting three Na⁺ out of the cell and two K⁺ into the cell, driven by ATP hydrolysis.^[58,59] Additionally, leak K⁺ channels, which are not gaited by voltage, ligands or mechanical stimuli, remain open, contributing to the stabilization of the RMP by allowing passive K⁺ efflux into the perilymph. This constant ionic flow ensures that the IHCs remain electrically stable and ready for subsequent depolarization for continuous auditory signal transduction.^[60] Table 1 summarizes the sequential phases of IHC activity.

Normal sound transduction in IHCs also relies on the stria vascularis, which maintains high K⁺ levels and a +80 mV endocochlear potential. Stereocilia deflection allows K⁺ to enter IHCs via MET channels and subsequently exit into the perilymph, after which it is recycled through the spiral ligament and stria vascularis back into the endolymph. This recycling is sustained by a network of ion transport systems. Na⁺/K⁺/2Cl⁻ cotransporters and Na⁺/ K⁺-ATPase pumps on the basolateral membrane of marginal cells in the stria vascularis mediate K⁺ uptake from the intrastrial space into the cytoplasm of marginal cells, while Cl⁻ exits into the intrastrial space via Cl⁻ channels on the same membrane. K⁺ is then secreted into the endolymph via K⁺ channels on the apical membrane of marginal cells, contributing to ionic homeostasis and the maintenance of the endocochlear potential.^[5,53]

IV. Conclusions

The present study comprehensively reviews the major cellular and molecular mechanisms supporting auditory signal encoding in IHCs. IHCs play a critical role in converting mechanical sound waves into electrical signals through a complex interaction of diverse ion channels and ribbon synapses working synergistically to ensure precise auditory signal processing. These intricate mechanisms are essential for maintaining the high spectral and temporal sensitivity of the auditory system. Understanding these mechanisms would deepen our knowledge of auditory function and offer critical insights for developing therapeutic strategies for hearing disorders, including the advancement of cochlear implants.