Mammalian Inner Hair Cells Research Articles

Distribution and Functional Characteristics of Voltage-Gated Sodium Channels in Immature Cochlear Hair Cells.

Voltage-gated sodium channels (VGSCs) are transiently expressed in cochlear hair cells before hearing onset and play an indispensable role in shaping spontaneous activity. In this study, we showed that Na+ currents shaped the spontaneous action potentials in developing mouse inner hair cells (IHCs) by decreasing the time required for the membrane potential to reach the action-potential threshold. In immature IHCs, we identified 9 known VGSC subtypes (Nav1.1α-1.9α), among which Nav1.7α was the most highly expressed subtype and the main contributor to Na+ currents in developing hair cells. Electrophysiological recordings of two cochlea-specific Nav1.7 variants (CbmNav1.7a and CbmNav1.7b) revealed a novel loss-of-function mutation (C934R) at the extracellular linker between segments 5 and 6 of domain II. In addition, post-transcriptional modification events, such as alternative splicing and RNA editing, amended the gating properties and kinetic features of CbmNav1.7a(C934). These results provide molecular and functional characteristics of VGSCs in mammalian IHCs and their contributions to spontaneous physiological activity during cochlear maturation.

Activity-Dependent Phosphorylation by CaMKIIδ Alters the Ca2+ Affinity of the Multi-C2-Domain Protein Otoferlin.

Otoferlin is essential for fast Ca2+-triggered transmitter release from auditory inner hair cells (IHCs), playing key roles in synaptic vesicle release, replenishment and retrieval. Dysfunction of otoferlin results in profound prelingual deafness. Despite its crucial role in cochlear synaptic processes, mechanisms regulating otoferlin activity have not been studied to date. Here, we identified Ca2+/calmodulin-dependent serine/threonine kinase II delta (CaMKIIδ) as an otoferlin binding partner by pull-downs from chicken utricles and reassured interaction by a co-immunoprecipitation with heterologously expressed proteins in HEK cells. We confirmed the expression of CaMKIIδ in rodent IHCs by immunohistochemistry and real-time PCR. A proximity ligation assay indicates close proximity of the two proteins in rat IHCs, suggesting that otoferlin and CaMKIIδ also interact in mammalian IHCs. In vitro phosphorylation of otoferlin by CaMKIIδ revealed ten phosphorylation sites, five of which are located within C2-domains. Exchange of serines/threonines at phosphorylated sites into phosphomimetic aspartates reduces the Ca2+ affinity of the recombinant C2F domain 10-fold, and increases the Ca2+ affinity of the C2C domain. Concordantly, we show that phosphorylation of otoferlin and/or its interaction partners are enhanced upon hair cell depolarization and blocked by pharmacological CaMKII inhibition. We therefore propose that otoferlin activity is regulated by CaMKIIδ in IHCs.

Membrane properties specialize mammalian inner hair cells for frequency or intensity encoding.

The auditory pathway faithfully encodes and relays auditory information to the brain with remarkable speed and precision. The inner hair cells (IHCs) are the primary sensory receptors adapted for rapid auditory signaling, but they are not thought to be intrinsically tuned to encode particular sound frequencies. Here I found that under experimental conditions mimicking those in vivo, mammalian IHCs are intrinsically specialized. Low-frequency gerbil IHCs (~0.3 kHz) have significantly more depolarized resting membrane potentials, faster kinetics, and shorter membrane time constants than high-frequency cells (~30 kHz). The faster kinetics of low-frequency IHCs allow them to follow the phasic component of sound (frequency-following), which is not required for high-frequency cells that are instead optimally configured to encode sustained, graded responses (intensity-following). The intrinsic membrane filtering of IHCs ensures accurate encoding of the phasic or sustained components of the cell's in vivo receptor potential, crucial for sound localization and ultimately survival.

Tonotopic Variation in the Calcium Dependence of Neurotransmitter Release and Vesicle Pool Replenishment at Mammalian Auditory Ribbon Synapses

The mammalian cochlea is specialized to recognize and process complex auditory signals with remarkable acuity and temporal precision over a wide frequency range. The quality of the information relayed to the auditory afferent fibers mainly depends on the transfer characteristics of inner hair cell (IHC) ribbon synapses. To investigate the biophysical properties of the synaptic machinery, we measured changes in membrane capacitance (DeltaC(m)) in low-frequency (apical region, approximately 300 Hz) and high-frequency (basal, approximately 30 kHz) gerbil IHCs maintained in near physiological conditions (1.3 mm extracellular Ca(2+) and body temperature). With maturation, the Ca(2+) efficiency of exocytosis improved in both apical and basal IHCs and was more pronounced in the latter. Prehearing IHCs showed a similar Ca(2+) cooperativity of exocytosis despite the smaller DeltaC(m) in apical cells. After maturation, DeltaC(m) in high-frequency IHCs increased linearly with the Ca(2+) current, whereas, somewhat surprisingly, the relationship was significantly more nonlinear in low-frequency cells. This tonotopic difference seemed to be correlated with ribbon synapse morphology (spherical in apical and ellipsoid in basal IHCs) but not with the expression level of the proposed Ca(2+) sensor otoferlin or the spatial coupling between Ca(2+) channels and active zones. Repetitive stimulation of adult IHCs showed that vesicle pool refilling could become rate limiting for vesicle release, with high-frequency IHCs able to sustain greater release rates. Together, our findings provide the first evidence for a tonotopic difference in the properties of the synaptic machinery in mammalian IHCs, which could be essential for fine-tuning their receptor characteristics during sound stimulation.

Calcium- and Calmodulin-Dependent Inactivation of Calcium Channels in Inner Hair Cells of the Rat Cochlea

Modulation of voltage-gated calcium channels was studied in inner hair cells (IHCs) in an ex vivo preparation of the apical turn of the rat organ of Corti. Whole cell voltage clamp in the presence of potassium channel blockers showed inward calcium currents with millisecond activation and deactivation kinetics. When temperature was raised from 22 to 37 degrees C, the calcium currents of immature IHCs [<12 days postnatal (P12)] increased threefold in amplitude, and developed more pronounced inactivation. This was determined to be calcium-dependent inactivation (CDI) on the basis of its reliance on external calcium (substitution with barium), sensitivity to internal calcium-buffering, and voltage dependence (reflecting the calcium driving force). After the onset of hearing at P12, IHC calcium current amplitude and the extent of inactivation were greatly reduced. Although smaller than in prehearing IHCs, CDI remained significant in the mature IHC near the resting membrane potential. CDI in mature IHCs was enhanced by application of the endoplasmic calcium pump blocker, benzo-hydroquinone. Conversely, CDI in immature IHCs was reduced by calmodulin inhibitors. Thus voltage-gated calcium channels in mammalian IHCs are subject to a calmodulin-mediated process of CDI. The extent of CDI depends on the balance of calcium buffering mechanisms and may be regulated by calmodulin-specific processes. CDI provides a means for the rate of spontaneous transmitter release to be adjusted to variations in hair cell resting potential and steady state calcium influx.

Gerbils can tune in

Gerbils can tune in

Direct measurements of Ca(2+)-activated K+ currents in inner hair cells of the guinea-pig cochlea using photolabile Ca2+ chelators.

Intracellular photorelease of Ca2+ from caged Ca2+ (DM-nitrophen or nitr5) and the patch-clamp technique in the whole-cell configuration were used to investigate Ca(2+)-activated currents in inner hair cells (IHCs) of the mammalian cochlea. Photoliberation of intracellular Ca2+ activated outward currents with a mean amplitude of 260 +/- 110 pA when IHCs were voltage-clamped, near the resting membrane potential, at -50 mV. The photoactivated currents were reversibly blocked by extracellular application of tetraethylammonium (TEA, 10 mM), neomycin (1 mM) and charybdotoxin (1 microM), but not by apamin. The voltage dependence of membrane currents activated by photolysis of DM-nitrophen demonstrated a reversal potential near the K+ equilibrium potential (Ek) and saturation near 0 mV. The presence of Ca(2+)-activated currents was further confirmed by the effects of extracellular adenosine 5'-triphosphate (ATP, 10 microM) and the Ca2+ ionophore ionomycin (10 microM). Both agents raised intracellular Ca2+ and simultaneously activated outward currents when IHCs were voltage-clamped near the resting membrane potential. In experiments where currents were activated by depolarizing voltage steps, nifedipine (50 microM) and Cd2+ (1 mM) reduced significantly (20-50%) the whole-cell outward currents, suggesting the presence of L-type Ca2+ currents activating K+ currents. These results are the first direct evidence for Ca(2+)-activated K+ currents in mammalian IHCs, these currents being potentially important for cell repolarization during sound-induced depolarization and synaptic transmission.