Rab3-interacting molecules 2α and 2β promote the abundance of voltage-gated Ca _V 1.3 Ca ²⁺ channels at hair cell active zones

Abstract

Ca2+ influx triggers the fusion of synaptic vesicles at the presynaptic active zone (AZ). Here we demonstrate a role of Ras-related in brain 3 (Rab3)-interacting molecules 2α and β (RIM2α and RIM2β) in clustering voltage-gated Ca V 1.3 Ca 2+ channels at the AZs of sensory inner hair cells (IHCs). We show that IHCs of hearing mice express mainly RIM2α, but also RIM2β and RIM3γ, which all localize to the AZs, as shown by immunofluorescence microscopy. Immunohistochemistry, patch-clamp, fluctuation analysis, and confocal Ca 2+ imaging demonstrate that AZs of RIM2α-deficient IHCs cluster fewer synaptic Ca V 1.3 Ca 2+ channels, resulting in reduced synaptic Ca 2+ influx. Using superresolution microscopy, we found that Ca 2+ channels remained clustered in stripes underneath anchored ribbons. Electron tomography of high-pressure frozen synapses revealed a reduced fraction of membrane-tethered vesicles, whereas the total number of membrane-proximal vesicles was unaltered. Membrane capacitance measurements revealed a reduction of exocytosis largely in proportion with the Ca 2+ current, whereas the apparent Ca 2+ dependence of exocytosis was unchanged. Hair cell-specific deletion of all RIM2 isoforms caused a stronger reduction of Ca 2+ influx and exocytosis and significantly impaired the encoding of sound onset in the postsynaptic spiral ganglion neurons. Auditory brainstem responses indicated a mild hearing impairment on hair cell-specific deletion of all RIM2 isoforms or global inactivation of RIM2α. We conclude that RIM2α and RIM2β promote a large complement of synaptic Ca 2+ channels at IHC AZs and are required for normal hearing.ens of Ca V 1.3 Ca 2+ channels are thought to cluster within the active zone (AZ) membrane underneath the presynaptic density of inner hair cells (IHCs) (1-4). They make up the key signaling element, coupling the sound-driven receptor potential to vesicular glutamate release (5-7). The mechanisms governing the number of Ca 2+ channels at the AZ as well as their spatial organization relative to membrane-tethered vesicles are not well understood. Disrupting the presynaptic scaffold protein Bassoon diminishes the numbers of Ca 2+ channels and membrane-tethered vesicles at the AZ (2, 8). However, the loss of Bassoon is accompanied by the loss of the entire synaptic ribbon, which makes it challenging to distinguish the direct effects of gene disruption from secondary effects (9).Among the constituents of the cytomatrix of the AZ, RIM1 and RIM2 proteins are prime candidates for the regulation of Ca 2+ channel clustering and function (10, 11). The family of RIM proteins has seven identified members (RIM1α, RIM1β, RIM2α, RIM2β, RIM2γ, RIM3γ, and RIM4γ) encoded by four genes (RIM1-RIM4). All isoforms contain a C-terminal C 2 domain but differ in the presence of additional domains. RIM1 and RIM2 interact with Ca 2+ channels, most other proteins of the cytomatrix of the AZ, and synaptic vesicle proteins. They interact directly with the auxiliary β (Ca V β) subunits (12, 13) and poreforming Ca V α subun...

Hair cells use active zones with different voltage dependence of Ca 2+ influx to decompose sounds into complementary neural codes

“…The Ca 2+ -channel complex at IHC AZs is likely composed of splice variants of the pore-forming subunit Ca V 1.3α containing exons 43S or 43L (36), of the Ca V β2 (33), and less likely of other Ca V β subunits (32), and of a yet-to-be-identified Ca V α2δ subunit. Ca V β2, the synaptic ribbon, and the presynaptic scaffolds bassoon and RIM2α and β were previously shown to promote the abundance of Ca V 1.3 at IHC AZs (15,33,43), whereas harmonin seems to reduce the number of synaptic Ca V 1.3 (44). Work in zebrafish hair cells has revealed an intriguing molecular interplay between RIBEYE and Ca V 1.3 channels, whereby RIBEYE overexpression promotes the formation of Ca V 1.3-positive AZ-like specializations and synaptic Ca 2+ influx negatively regulates RIBEYE abundance at the AZ (63,64).…”

“…Future experiments simultaneously addressing presynaptic properties and postsynaptic firing will be required to further test our hypothesis. -channel subunits and splice variants (33,36,59) as well as by that of scaffold proteins that tether Ca 2+ channels to the AZ (15,42,43,60,61) and/or regulate their turnover (44) by direct or indirect interaction with the channels. Moreover, subunit composition as well as splice variants and interacting proteins also modulate functional properties of the specific Ca 2+ channel (62).…”

Ca 2+ -binding protein 2 inhibits Ca 2+ -channel inactivation in mouse inner hair cells

“…IHC Ca V 1.3 Ca 2+ channels stand out by reason of their hyperpolarized activation range and modest inactivation (10,18,19,22,45). These properties have been attributed to the molecular composition of the native Ca 2+ -channel complexes that, in addition to the specific pore-forming Ca V 1.3α1 splice variant (46), auxiliary Ca V β2 (6,47), and Ca V α 2 δ2 (7) subunit, also contain interactions partners, such as Rab3-interacting molecule (RIM) (48,49); harmonin (50, 51); calmodulin; and, notably, CaBPs (this study and refs. 18,19,34).…”

Molecularly and structurally distinct synapses mediate reliable encoding and processing of auditory information

“…The expression of Ca 2þ channels at active zones is partially regulated by the protein RIM in the calyx of Held (Han et al, 2015), RIM isoforms have been found at both calyceal synapses (Dulubova et al, 2005;Mendoza Schulz et al, 2014) and as well as at mature IHC ribbon synapses (Jung et al, 2015). The RIM interactor Rab3 seems to be present at the calyx of Held (Dulubova et al, 2005) and unpublished data (Susann Michanski, G€ ottingen) suggest the presence of Rab3 in IHCs.…”