Single particle cryo-EM structure of the outer hair cell motor protein prestin

The mammalian outer hair cell (OHC) protein prestin (Slc26a5) differs from other Slc26 family members due to its unique piezoelectric-like property that drives OHC electromotility, the putative mechanism for cochlear amplification. Here, we use cryo-electron microscopy to determine prestin’s structure at 3.6 Å resolution. Prestin is structurally similar to the anion transporter Slc26a9. It is captured in an inward-open state which may reflect prestin’s contracted state. Two well-separated transmembrane (TM) domains and two cytoplasmic sulfate transporter and anti-sigma factor antagonist (STAS) domains form a swapped dimer. The transmembrane domains consist of 14 transmembrane segments organized in two 7+7 inverted repeats, an architecture first observed in the bacterial symporter UraA. Mutation of prestin’s chloride binding site removes salicylate competition with anions while retaining the prestin characteristic displacement currents (Nonlinear Capacitance), undermining the extrinsic voltage sensor hypothesis for prestin function.

Efferent neurons control hearing sensitivity and protect hearing from noise through the regulation of gap junctions between cochlear supporting cells

It is critical for hearing that the descending cochlear efferent system provide a negative feedback to hair cells to regulate hearing sensitivity and provide the protection of hearing from noise. Here, we report that the medial olivocochlear (MOC) efferent nerves, which project to outer hair cells (OHCs), also could innervate OHC surrounding supporting cells (SCs) to regulate hearing sensitivity. MOC nerve fibers are cholinergic and acetylcholine (ACh) is a primary neurotransmitter. MOC nerve endings, presynaptic vesicular acetylcholine transporters (VAChT), and postsynaptic ACh receptors were visible in SCs and the SC area. Application of ACh in the SC could evoke a typical inward current, which reduced gap junctions (GJs) between SCs and consequently declined OHC electromotility, which is an active cochlear amplification and can increase hearing sensitivity. This indirect, GJ-mediated inhibition enhanced the direct inhibition of ACh on OHC electromotility but had long-lasting influence. In vivo experiments further demonstrated that deficiency of this GJ-mediated efferent pathway declined the regulation of active cochlear amplification and compromised the protection against noise. In particular, distortion production otoacoustic emission (DPOAE) showed a delayed reduction after noise exposure. Our findings reveal a new pathway for the MOC efferent system via innervating SCs to control active cochlear amplification and hearing sensitivity. These data also suggest that this GJ-mediated efferent pathway may play a critical role in the long-term efferent inhibition and is required for protecting hearing from noise trauma.

Measurements From Ears With Endolymphatic Hydrops and 2-Hydroxypropyl-Beta-Cyclodextrin Provide Evidence That Loudness Recruitment Can Have a Cochlear Origin

Background: Loudness recruitment is commonly experienced by patients with putative endolymphatic hydrops. Loudness recruitment is abnormal loudness growth with high-level sounds being perceived as having normal loudness even though hearing thresholds are elevated. The traditional interpretation of recruitment is that cochlear amplification has been reduced. Since the cochlear amplifier acts primarily at low sound levels, an ear with elevated thresholds from reduced cochlear amplification can have normal processing at high sound levels. In humans, recruitment can be studied using perceptual loudness but in animals physiological measurements are used. Recruitment in animal auditory-nerve responses has never been unequivocally demonstrated because the animals used had damage to sensory and neural cells, not solely a reduction of cochlear amplification. Investigators have thus looked for, and found, evidence of recruitment in the auditory central nervous system (CNS). While studies on CNS recruitment are informative, they cannot rule out the traditional interpretation of recruitment originating in the cochlea.Design: We used techniques that could assess hearing function throughout entire frequency- and dynamic-range of hearing. Measurements were made from two animal models: guinea-pig ears with endolymphatic-sac-ablation surgery to produce endolymphatic hydrops, and naïve guinea-pig ears with cochlear perfusions of 13 mM 2-Hydroxypropyl-Beta-Cyclodextrin (HPBCD) in artificial perilymph. Endolymphatic sac ablation caused low-frequency loss. Animals treated with HPBCD had hearing loss at all frequencies. None of these animals had loss of hair cells or synapses on auditory nerve fibers.Results: In ears with endolymphatic hydrops and those perfused with HPBCD, auditory-nerve based measurements at low frequencies showed recruitment compared to controls. Recruitment was not found at high frequencies (> 4 kHz) where hearing thresholds were normal in ears with endolymphatic hydrops and elevated in ears treated with HPBCD.Conclusions: We found compelling evidence of recruitment in auditory-nerve data. Such clear evidence has never been shown before. Our findings suggest that, in patients suspected of having endolymphatic hydrops, loudness recruitment may be a good indication that the associated low-frequency hearing loss originates from a reduction of cochlear amplification, and that measurements of recruitment could be used in differential diagnosis and treatment monitoring of Ménière's disease.

Cryo-EM structures reveal the electromotility mechanism of prestin, the cochlear amplifier

Outer hair cell electromotility, driven by prestin, is essential for mammalian cochlear amplification. Here, we report the cryo-EM structures of thermostabilized human prestin (hPresTS), complexed with chloride, sulfate, or salicylate at 3.52–3.61 Å resolutions, revealing a crossed dimeric arrangement. The central positively-charged cavity allows flexible binding of various anion species, resulting in distinct modulations of nonlinear capacitance (NLC), playing an important role in electromotility. Comparisons of these hPresTS structures suggest rigid-body movement between the core and gate domains, and provide mechanistic insight into prestin inhibition by salicylate. Mutations at the dimeric interface severely diminished NLC, suggesting that stabilization of the gate domain facilitates core domain movement, thereby contributing to the expression of NLC. These findings advance our understanding of the molecular mechanism underlying mammalian cochlear amplification.

A gap-junction mutation in the mouse cochlea reveals cochlear amplification is driven by outer hair cell extracellular receptor potentials

Cochlear amplification, whereby cochlear responses to low-to-moderate sound levels are 31 amplified and compressed to loud sounds, is attributed to outer hair cell (OHC) electromotility 32 driven by voltage changes across the OHC basolateral membranes due to sound-induced 33 receptor-current modulation. Cochlear operation at high acoustic frequencies is enigmatic 34 because the OHC intracellular receptor potential (RP) is severely attenuated at these 35 frequencies. Clues to understanding the voltage control of OHC electromotility at different 36 frequencies was provided by measurements from CD-1 mice with an A88V mutation of the 37 gap-junction (GJ) protein connexin 30 (Cx30), which with Cx26, form heterogeneous GJs 38 between supporting cells in the organ of Corti (OoC) and stria vascularis. The A88V mutation 39 results in a smaller GJ conductance which may explain why the resistance across the OoC in 40 CD-1Cx30A88V/A88V mutants is higher compared with wild-type mice. The endocochlear 41 potential, which drives the OHC receptor current and, consequently, the OHC RPs, is smaller 42 in CD-1Cx30A88V/A88V mutants. Even so, their high-frequency hearing sensitivity equals that of 43 wild-type mice. Preservation of high-frequency hearing correlates with similar amplitude of 44 extracellular receptor potentials (ERPs), measured immediately adjacent to the OHCs. ERPs 45 are generated through OHC receptor current flow across the OoC electrical resistance, which 46 is larger in CD-1Cx30A88V/A88V than in wild-type mice. Thus, smaller OHC receptor currents 47 flowing across a larger OoC resistance in CD-1Cx30A88V/A88V mice may explain why their ERP 48 magnitudes are similar to wild-type mice. It is proposed that the ERPs, which are not subject 49 to low-pass electrical filtering, drive high-frequency cochlear amplification.

Efferent control of hearing sensitivity and protection via inner ear supporting cells

It is critical for hearing that the descending cochlear efferent system provide negative feedback to hair cells to regulate hearing sensitivity and provide protection from noise. The medial olivocochlear (MOC) efferent nerves project to outer hair cells (OHCs) and inhibit OHC electromotility, which is an active cochlear amplification and can increase hearing sensitivity. Here, we report that the MOC efferent nerves also have functional innervation with the cochlear supporting cells to regulate hearing sensitivity. The MOC efferent nerve fibers and the corresponding MOC neurotransmitter acetylcholine (ACh) receptors were visible in the cochlear supporting cells. Application of ACh in the cochlear supporting cells could also evoke inward currents in a dose-dependent manner and reduced gap junctional (GJ) coupling between the cochlear supporting cells, which consequently declined electromotility in OHCs. This indirect inhibitory effect through the mediated GJs between the cochlear supporting cells on OHC electromotility was consistent and enhanced the direct inhibition of ACh on OHC electromotility but had long-lasting influence. In vivo experiments further demonstrated that deficiency of this GJ-mediated efferent control pathway declined the regulation of active cochlear amplification and impaired the protection from noise trauma. Our findings reveal a new pathway for the cochlear efferent system to control hearing sensitivity, and also demonstrate that this supporting cell GJ-mediated efferent pathway is critical for control of hearing sensitivity and the protection of hearing from noise trauma.