Dataset Information

RBP2 stabilizes slow Cav1.3 Ca2+ channel inactivation properties of cochlear inner hair cells.

ABSTRACT: Cav1.3 L-type Ca2+ channels (LTCCs) in cochlear inner hair cells (IHCs) are essential for hearing as they convert sound-induced graded receptor potentials into tonic postsynaptic glutamate release. To enable fast and indefatigable presynaptic Ca2+ signaling, IHC Cav1.3 channels exhibit a negative activation voltage range and uniquely slow inactivation kinetics. Interaction with CaM-like Ca2+-binding proteins inhibits Ca2+-dependent inactivation, while the mechanisms underlying slow voltage-dependent inactivation (VDI) are not completely understood. Here we studied if the complex formation of Cav1.3 LTCCs with the presynaptic active zone proteins RIM2? and RIM-binding protein 2 (RBP2) can stabilize slow VDI. We detected both RIM2? and RBP isoforms in adult mouse IHCs, where they co-localized with Cav1.3 and synaptic ribbons. Using whole-cell patch-clamp recordings (tsA-201 cells), we assessed their effect on the VDI of the C-terminal full-length Cav1.3 (Cav1.3L) and a short splice variant (Cav1.342A) that lacks the C-terminal RBP2 interaction site. When co-expressed with the auxiliary ?3 subunit, RIM2? alone (Cav1.342A) or RIM2?/RBP2 (Cav1.3L) reduced Cav1.3 VDI to a similar extent as observed in IHCs. Membrane-anchored ?2 variants (?2a, ?2e) that inhibit inactivation on their own allowed no further modulation of inactivation kinetics by RIM2?/RBP2. Moreover, association with RIM2? and/or RBP2 consolidated the negative Cav1.3 voltage operating range by shifting the channel's activation threshold toward more hyperpolarized potentials. Taken together, the association with "slow" ? subunits (?2a, ?2e) or presynaptic scaffolding proteins such as RIM2? and RBP2 stabilizes physiological gating properties of IHC Cav1.3 LTCCs in a splice variant-dependent manner ensuring proper IHC function.

SUBMITTER: Ortner NJ

PROVIDER: S-EPMC6960213 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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RBP2 stabilizes slow Cav1.3 Ca2+ channel inactivation properties of cochlear inner hair cells.

Ortner Nadine J NJ Pinggera Alexandra A Hofer Nadja T NT Siller Anita A Brandt Niels N Raffeiner Andrea A Vilusic Kristina K Lang Isabelle I Blum Kerstin K Obermair Gerald J GJ Stefan Eduard E Engel Jutta J Striessnig Jörg J

Pflugers Archiv : European journal of physiology 20191217 1

Cav1.3 L-type Ca2+ channels (LTCCs) in cochlear inner hair cells (IHCs) are essential for hearing as they convert sound-induced graded receptor potentials into tonic postsynaptic glutamate release. To enable fast and indefatigable presynaptic Ca2+ signaling, IHC Cav1.3 channels exhibit a negative activation voltage range and uniquely slow inactivation kinetics. Interaction with CaM-like Ca2+-binding proteins inhibits Ca2+-dependent inactivation, while ...[more]

PMID: 31848688

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Project description:Noise-induced hearing loss has gained relevance as one of the most common forms of hearing impairment. The anatomical correlates of hearing loss, principally cell damage and/or death, are relatively well-understood histologically. However, much less is known about the physiological aspects of damaged, surviving cells. Here we addressed the functional consequences of noise exposure on the capacity of inner hair cells (IHCs) to release synaptic vesicles at synapses with spiral ganglion neurons (SGNs). Mice of either sex at postnatal day (P) 15-16 were exposed to 1-12 kHz noise at 120 dB sound pressure level (SPL), for 1 h. Exocytosis was measured by tracking changes in membrane capacitance (ΔCm) from IHCs of the apical cochlea. Upon IHC depolarization to different membrane potentials, ΔC m showed the typical bell-shaped curve that mirrors the voltage dependence of Ca2+ influx, in both exposed and unexposed cells. Surprisingly, from IHCs at 1-day after exposure (d.a.e.), we found potentiation of exocytosis at the peak of the bell-shaped curve. The increase in exocytosis was not accompanied by changes in whole-cell Ca2+ influx, suggesting a modification in coupling between Ca2+ channels and synaptic vesicles. Consistent with this notion, noise exposure also changed the Ca2+-dependence of exocytosis from linear to supralinear. Noise exposure did not cause loss of IHCs, but did result in a small reduction in the number of IHC-SGN synapses at 1-d.a.e. which recovered by 14-d.a.e. In contrast, a strong reduction in auditory brainstem response wave-I amplitude (representing synchronous firing of SGNs) and distortion product otoacoustic emissions (reflecting outer hair cell function) indicated a profound hearing loss at 1- and 14-d.a.e. To determine the role of glutamate release in the noise-induced potentiation of exocytosis, we evaluated vesicular glutamate transporter-3 (Vglut3) knock-out (KO) mice. Unlike WT, IHCs from Vglut3 KO mice showed a noise-induced reduction in ΔC m and Ca2+ influx with no change in the Ca2+-dependence of exocytosis. Together, these results indicate that traumatic noise exposure triggers changes of IHC synaptic function including a Vglut3-dependent potentiation of exocytosis.

Project description:The early auditory pathway processes information at high rates and with utmost temporal fidelity. Consequently, the synapses in the auditory pathway are highly specialized to meet the extraordinary requirements on signal transmission. The calyceal synapses in the auditory brainstem feature more than a hundred active zones (AZs) with thousands of releasable synaptic vesicles (SVs). In contrast, the first auditory synapse, the afferent synapse of inner hair cells (IHCs) and type I spiral ganglion neurons (SGNs), typically exhibits a single ribbon-type AZ tethering only tens of SVs resulting in a highly stochastic pattern of transmitter release. Spontaneous excitatory postsynaptic currents (sEPSCs), besides more conventional EPSCs with a single peak, fast rise and decay (compact), also include EPSCs with multiple peaks, variable rise and decay times (non-compact). The strong heterogeneity in size and shape of spontaneous EPSCs has led to the hypothesis of multivesicular release (MVR) that is more (compact) or less (non-compact) synchronized by coordination of release sites. Alternatively, univesicular release (UVR), potentially involving glutamate release through a flickering fusion pore for non-compact EPSCs, has been suggested to underlie IHC exocytosis. Here, we further investigated the mode of release by recording sEPSCs from SGNs of hearing rats while manipulating presynaptic IHC Ca2+ influx by changes in extracellular [Ca2+] ([Ca2+]e) and by application of the Ca2+ channel antagonist, isradipine, or the Ca2+ channel agonist, BayK8644 (BayK). Our data reveal that Ca2+ influx manipulation leaves the distributions of sEPSC amplitude and charge largely unchanged. Regardless the type of manipulation, the rate of sEPSC decreased with the reduction in Ca2+ influx. The fraction of compact sEPSCs was increased in the presence of BayK, an effect that was abolished when combined with decreased [Ca2+]e. In conclusion, we propose that UVR is the prevailing mode of exocytosis at cochlear IHCs of hearing rats, whereby the rate of exocytosis and the kinetics of SV fusion are regulated by Ca2+ influx.

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RBP2 stabilizes slow Cav1.3 Ca2+ channel inactivation properties of cochlear inner hair cells.

Publications

RBP2 stabilizes slow Cav1.3 Ca<sup>2+</sup> channel inactivation properties of cochlear inner hair cells.

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