Hair Cell Exocytosis Research Articles

Ca2+ binding to the C2E domain of otoferlin is required for hair cell exocytosis and hearing.

Ca2+ binding to the C2E domain of otoferlin is required for hair cell exocytosis and hearing.

Identification of Binding Partners of the Hair‐Cell Protein Otoferlin Using Co‐Immunoprecipitation and Proximity‐Dependent Labelling Techniques

Otoferlin is a calcium-sensitive multi-C2-domain transmembrane protein which localizes to auditory hair cell synapses and is associated with congenital deafness. Although the role of otoferlin in synaptic vesicle exocytosis in cochlear hair cells is widely accepted, the exact function(s) of the protein are poorly understood. This lack of understanding both prohibits formation of mechanistic models describing the encoding of sound, and prevents development of therapeutic treatments for deafness. Further, a lack of traditional synaptic vesicle proteins in hair cells suggests a fundamentally different method for synaptic vesicle exocytosis in these cells. In this study, we used a combination of co-immunoprecipitation and proximity-labelling methods to identify otoferlin binding partners that may participate in hair cell exocytosis. As co-immunoprecipitation involves lysing the cell, and lacks the ability to capture weak and transient interactions, an in-cell proximity-dependent labelling assay was used to find protein partners. Analysis of samples using mass spectroscopy allowed us to comprehensively characterize a molecular interaction network for otoferlin. This otoferlin binding network leads us to propose a novel molecular-level mechanism for neurotransmission at hair cell synapses.

Phase-Locking Requires Efficient Ca2+ Extrusion at the Auditory Hair Cell Ribbon Synapse.

Proper perception of sounds in the environment requires auditory signals to be encoded with extraordinary temporal precision up to tens of microseconds, but how it originates from the hearing organs in the periphery is poorly understood. In particular, sound-evoked spikes in auditory afferent fibers in vivo are phase-locked to sound frequencies up to 5 kHz, but it is not clear how hair cells can handle intracellular Ca2+ changes with such high speed and efficiency. In this study, we combined patch-clamp recording and two-photon Ca2+ imaging to examine Ca2+ dynamics in hair cell ribbon synapses in the bullfrog amphibian papilla of both sexes. We found that Ca2+ clearance from single synaptic ribbons followed a double exponential function, and the weight of the fast component, but not the two time constants, was significantly reduced for prolonged stimulation, and during inhibition of the plasma membrane Ca2+ ATPase (PMCA), the mitochondrial Ca2+ uptake (MCU), or the sarcolemma/endoplasmic reticulum Ca2+ ATPase (SERCA), but not the Na+/Ca2+ exchanger (NCX). Furthermore, we found that both the basal Ca2+ level and the Ca2+ rise during sinusoidal stimulation were significantly increased by inhibition of PMCA, MCU, or SERCA. Consistently, phase-locking of synaptic vesicle releases from hair cells was also significantly reduced by blocking PMCA, MCU, or SERCA, but not NCX. We conclude that, in addition to fast diffusion mediated by mobile Ca2+ buffer, multiple Ca2+ extrusion pumps are required for phase-locking at the auditory hair cell ribbon synapse.SIGNIFICANCE STATEMENT Hair cell synapses can transmit sound-driven signals precisely in the kHz range. However, previous studies of Ca2+ handling in auditory hair cells have often been conducted in immature hair cells, with elevated extracellular Ca2+ concentration, or through steady-state stimulation that may not be physiologically relevant. Here we examine Ca2+ clearance from hair cell synaptic ribbons in a fully mature preparation at physiological concentration of external Ca2+ and at physiological temperature. By stimulating hair cells with sinusoidal voltage commands that mimic pure sound tones, we recapitulated the phase-locking of hair cell exocytosis with an in vitro approach. This allowed us to reveal the Ca2+ extrusion mechanisms that are required for phase-locking at auditory hair cell ribbon synapses.

Identification of Proteins that Interact with Otoferlin to build a ‘Otoferlin Interactome’

Otoferlin is a C2‐domain transmembrane vesicular protein that has been associated with congenital deafness. It is majorly responsible for facilitating synaptic vesicle exocytosis in cochlear hair cells. Synaptotagmin and Otoferlin have previously been compared to one another for their calcium sensor functions, but hair cells exocytosis is not as well‐understood as neuronal exocytosis. In case of hair cell synapses, we still lack complete knowledge of the SNARE proteins or their equivalent that aid in the fusion of vesicle membranes to the presynaptic membranes, thereby leading to exocytosis. Currently, a major bottleneck in our understanding of Otoferlin’s role in hearing and deafness can be attributed to the lack of knowledge about its mechanism of functioning. Using brain extracts from mouse, we conducted coimmunoprecipitation experiments to find interaction partners for Otoferlin so as to build a Otoferlin interactome, that can shed some light on the mechanism by which hair cell exocytosis actually occurs. The proteins interacting with Otoferlin were identified by Mass spectroscopy and consisted largely of neuronal proteins that share functions with SNARE proteins or had other neurotransmission‐related functions. We use Human Embryonic Kidney (HEK) cells and a zebrafish model to validate the direct interactors of Otoferlin and conduct further assays to determine their role in hair cell exocytosis and/or endocytosis. This knowledge of Otoferlin interactome leads us to a better understanding of the mechanism whereby neurotransmission is carried out in the inner hair cells.Support or Funding Information1R01DC014588 (C.P. Johnson) 4/2016‐3/2021 NIDCD (NIH). In vivo and in vitro studies of the deafness associated protein otoferlin

Tryptophan-rich basic protein (WRB) mediates insertion of the tail-anchored protein otoferlin and is required for hair cell exocytosis and hearing.

The transmembrane recognition complex (TRC40) pathway mediates the insertion of tail-anchored (TA) proteins into membranes. Here, we demonstrate that otoferlin, a TA protein essential for hair cell exocytosis, is inserted into the endoplasmic reticulum (ER) via the TRC40 pathway. We mutated the TRC40 receptor tryptophan-rich basic protein (Wrb) in hair cells of zebrafish and mice and studied the impact of defective TA protein insertion. Wrb disruption reduced otoferlin levels in hair cells and impaired hearing, which could be restored in zebrafish by transgenic Wrb rescue and otoferlin overexpression. Wrb-deficient mouse inner hair cells (IHCs) displayed normal numbers of afferent synapses, Ca2+ channels, and membrane-proximal vesicles, but contained fewer ribbon-associated vesicles. Patch-clamp of IHCs revealed impaired synaptic vesicle replenishment. In vivo recordings from postsynaptic spiral ganglion neurons showed a use-dependent reduction in sound-evoked spiking, corroborating the notion of impaired IHC vesicle replenishment. A human mutation affecting the transmembrane domain of otoferlin impaired its ER targeting and caused an auditory synaptopathy. We conclude that the TRC40 pathway is critical for hearing and propose that otoferlin is an essential substrate of this pathway in hair cells.

Uniquantal Release through a Dynamic Fusion Pore Is a Candidate Mechanism of Hair Cell Exocytosis

The mechanisms underlying the large amplitudes and heterogeneity of excitatory postsynaptic currents (EPSCs) at inner hair cell (IHC) ribbon synapses are unknown. Based on electrophysiology, electron and superresolution light microscopy, and modeling, we propose that uniquantal exocytosis shaped by a dynamic fusion pore is a candidate neurotransmitter release mechanism in IHCs. Modeling indicated that the extended postsynaptic AMPA receptor clusters enable large uniquantal EPSCs. Recorded multiphasic EPSCs were triggered by similar glutamate amounts as monophasic ones and were consistent with progressive vesicle emptying during pore flickering. The fraction of multiphasic EPSCs decreased in absence of Ca(2+) influx and upon application of the Ca(2+) channel modulator BayK8644. Our experiments and modeling did not support the two most popular multiquantal release interpretations of EPSC heterogeneity: (1) Ca(2+)-synchronized exocytosis of multiple vesicles and (2) compound exocytosis fueled by vesicle-to-vesicle fusion. We propose that IHC synapses efficiently use uniquantal glutamate release for achieving high information transmission rates.

Calcium Regulates Molecular Interactions of Otoferlin with Soluble NSF Attachment Protein Receptor (SNARE) Proteins Required for Hair Cell Exocytosis

Mutations in otoferlin, a C2 domain-containing ferlin family protein, cause non-syndromic hearing loss in humans (DFNB9 deafness). Furthermore, transmitter secretion of cochlear inner hair cells is compromised in mice lacking otoferlin. In the present study, we show that the C2F domain of otoferlin directly binds calcium (KD = 267 μM) with diminished binding in a pachanga (D1767G) C2F mouse mutation. Calcium was found to differentially regulate binding of otoferlin C2 domains to target SNARE (t-SNARE) proteins and phospholipids. C2D-F domains interact with the syntaxin-1 t-SNARE motif with maximum binding within the range of 20-50 μM Ca(2+). At 20 μM Ca(2+), the dissociation rate was substantially lower, indicating increased binding (KD = ∼10(-9)) compared with 0 μM Ca(2+) (KD = ∼10(-8)), suggesting a calcium-mediated stabilization of the C2 domain·t-SNARE complex. C2A and C2B interactions with t-SNAREs were insensitive to calcium. The C2F domain directly binds the t-SNARE SNAP-25 maximally at 100 μM and with reduction at 0 μM Ca(2+), a pattern repeated for C2F domain interactions with phosphatidylinositol 4,5-bisphosphate. In contrast, C2F did not bind the vesicle SNARE protein synaptobrevin-1 (VAMP-1). Moreover, an antibody targeting otoferlin immunoprecipitated syntaxin-1 and SNAP-25 but not synaptobrevin-1. As opposed to an increase in binding with increased calcium, interactions between otoferlin C2F domain and intramolecular C2 domains occurred in the absence of calcium, consistent with intra-C2 domain interactions forming a "closed" tertiary structure at low calcium that "opens" as calcium increases. These results suggest a direct role for otoferlin in exocytosis and modulation of calcium-dependent membrane fusion.

Exocytosis in the Frog Amphibian Papilla

Using whole-cell patch-clamp recordings, we measured changes in membrane capacitance (ΔC (m)) in two subsets of hair cells from the leopard frog amphibian papilla (AP): the low-frequency (100-500 Hz), rostral hair cells and the high-frequency (500-1200 Hz), caudal hair cells, in order to investigate tonotopic differences in exocytosis. Depolarizations of both rostral and caudal hair cells evoked robust ΔC (m) responses of similar amplitude. However, the calcium dependence of release, i.e., the relationship between ΔC (m) relative to the amount of calcium influx (Q (Ca) (2+)), was found to be linear in rostral hair cells but supra-linear in caudal hair cells. In addition, the higher numbers of vesicles released at caudal hair cell active zones suggests increased temporal precision of caudal hair cell exocytosis. ΔC (m) responses were also obtained in response to sinusoidal stimuli of varying frequency, but neither rostral nor caudal hair cell ΔC (m) revealed any frequency selectivity. While all AP hair cells express both otoferlin and synaptotagmin IV (SytIV), we obtained evidence of a tonotopic distribution of the calcium buffer calretinin which may further increase temporal resolution at the level of the hair cell synapse. Our findings suggest that the low (rostral) and high (caudal) frequency hair cells apply different mechanisms for fine-tuning exocytosis.

Otoferlin interacts with myosin VI: implications for maintenance of the basolateral synaptic structure of the inner hair cell

Otoferlin has been proposed to be the Ca(2+) sensor in hair cell exocytosis, compensating for the classical synaptic fusion proteins synaptotagmin-1 and synaptotagmin-2. In the present study, yeast two-hybrid assays reveal myosin VI as a novel otoferlin binding partner. Co-immunoprecipitation assay and co-expression suggest an interaction of both proteins within the basolateral part of inner hair cells (IHCs). Comparison of otoferlin mutants and myosin VI mutant mice indicates non-complementary and complementary roles of myosin VI and otoferlin for synaptic maturation: (i) IHCs from otoferlin mutant mice exhibited a decoupling of CtBP2/RIBEYE and Ca(V)1.3 and severe reduction of exocytosis. (ii) Myosin VI mutant IHCs failed to transport BK channels to the membrane of the apical cell regions, and the exocytotic Ca(2+) efficiency did not mature. (iii) Otoferlin and myosin VI mutant IHCs showed a reduced basolateral synaptic surface area and altered active zone topography. Membrane infoldings in otoferlin mutant IHCs indicated disturbed transport of endocytotic membranes and link the above morphological changes to a complementary role of otoferlin and myosin VI in transport of intracellular compartments to the basolateral IHC membrane.

Probing the Mechanism of Exocytosis at the Hair Cell Ribbon Synapse

Hearing relies on faithful synaptic transmission at the ribbon synapse of cochlear inner hair cells (IHCs). Postsynaptic recordings from this synapse in prehearing animals had delivered strong indications for synchronized release of several vesicles. The underlying mechanism, however, remains unclear. Here, we used presynaptic membrane capacitance measurements to test whether IHCs release vesicles in a statistically independent or dependent (coordinated) manner. Exocytic changes of membrane capacitance (deltaC(m)) were repeatedly stimulated in IHCs of prehearing and hearing mice by short depolarizations to preferentially recruit the readily releasable pool of synaptic vesicles. A compound Poisson model was devised to describe hair cell exocytosis and to test the analysis. From the trial-to-trial fluctuations of the deltaC(m) we were able to estimate the apparent size of the elementary fusion event (C(app)) at the hair cell synapse to be 96-223 aF in immature and 55-149 aF in mature IHCs. We also approximated the single vesicle capacitance in IHCs by measurements of synaptic vesicle diameters in electron micrographs. The results (immature, 48 aF; mature, 45 aF) were lower than the respective C(app) estimates. This indicates that coordinated exocytosis of synaptic vesicles occurs at both immature and mature hair cell synapses. Approximately 35% of the release events in mature IHCs and approximately 50% in immature IHCs were predicted to involve coordinated fusion, when assuming a geometric distribution of elementary sizes. In summary, our presynaptic measurements indicate coordinated exocytosis but argue for a lesser degree of coordination than suggested by postsynaptic recordings.

Temperature insensitivity of short‐term adaptation in single‐units of the chick cochlear nerve

Short-term adaptation in acoustically stimulated chick cochlear nerve fibers has recently been shown to have similar kinetics as the readily-releasable vesicle pool in patch-clamped chick hair cells, suggesting that short-term adaptation depends on the dynamics of hair cell exocytosis. Our understanding of the relationship between these two phenomena has been hampered by differences in the temperatures at which the two types of data have been collected. In this report, the effect of temperature on short-term adaptation was studied in single-units of the chick cochlear nerve. Compared to units recorded at 38-41 degrees C, spontaneous and evoked firing rates were markedly decreased when the temperature was lowered to 28-32 degrees C, but the rate of short-term adaptation during 100 ms tone bursts was relatively unchanged, with a temperature Q(10), of approximately 1.2. The continued similarity of the adaptation time-constant of cooled units to vesicle depletion kinetics in chick hair cells measured at room temperature suggests that comparison of in vitro hair cell exocytosis and in vivo cochlear nerve firing properties may not be confounded by temperature differences between the two approaches.

Large releasable pool of synaptic vesicles in chick cochlear hair cells.

Hearing requires the hair cell synapse to maintain notable temporal fidelity (< or =1 ms) while sustaining neurotransmitter release for prolonged periods of time (minutes). Here we probed the properties and possible anatomical substrate of prolonged neurotransmitter release by using electrical measures of cell surface area as a proxy for neurotransmitter release to study hair cell exocytosis evoked by repetitive stimuli. We observed marked depression of exocytosis by chick tall hair cells. This exocytic depression cannot be explained by calcium current inactivation, presynaptic autoinhibition by metabotropic glutamate receptors, or postsynaptic receptor desensitization. Rather, cochlear hair cell exocytic depression resulted from the exhaustion of a functional vesicle pool. This releasable vesicle pool is large, totaling approximately 8,000 vesicles, and is nearly 10 times greater than the number of vesicles tethered to synaptic ribbons. Such a large functional pool suggests the recruitment of cytoplasmic vesicles to sustain exocytosis, important for maintaining prolonged, high rates of neural activity needed to encode sound.

Chick cochlear hair cell exocytosis mediated by dihydropyridine-sensitive calcium channels.

1. A semi-intact preparation of the chick basilar papilla was developed to study calcium-dependent neurotransmitter release by tall hair cells (avian equivalent of cochlear inner hair cells). 2. Tall hair cell depolarization resulted in changes in cell membrane capacitance (DeltaC(m)) that reflected cell surface area increases following synaptic vesicle exocytosis and provided a surrogate measure of neurotransmitter release. Both calcium current (I(Ca)) and DeltaC(m) were reversibly blocked by cobalt, and exhibited a similar bell-shaped dependency on voltage with a peak response around -10 mV. 3. Pharmacological agents selective for L-type calcium channels were employed to assess the role of this channel type in neurotransmitter exocytosis. Nimodipine, a dihydropyridine (DHP) antagonist, suppressed I(Ca) and blocked DeltaC(m). Conversely, the DHP agonist Bay K 8644 increased both I(Ca) and DeltaC(m) amplitude nearly 3-fold. These findings suggest that chick tall hair cell neurotransmitter release is mediated by calcium influx through L-type calcium channels.