Unravelling the role of RIM proteins in synaptic transmission: RIM1α tunes Ca²⁺ channel subtype contribution to neurotransmitter release

Abstract

Synaptic transmission is extremely fast and tightly regulated due to the rapid and spatially restricted Ca²⁺ signal at the presynaptic active zone (AZ). At the AZ, voltage-gated Ca²⁺ channels (VGCC) are kept in close proximity to synaptic vesicles (SV) allowing fast and reliable neurotransmitter release. The composition and state of the presynaptic AZ determine release probability (P_r), by influencing vesicular release probability (P_ves) and the availability of SVs for release, determinants of the synaptic strength. The large RIM proteins, such as RIM1α, are considered the main scaffolding components of the AZ due to their multiple interactions with other AZ proteins as well as with VGCCs and SVs. RIM1α plays a crucial role in the regulation of synaptic transmission by maintaining sufficient and localized Ca²⁺ entry and by maintaining SVs in a release-ready state. The ablation of RIM1α leads to a strong reduction of synaptic output (~40%). The mechanisms by which RIM1α regulates synaptic strength are still not precisely known. It remains unclear whether the alterations in release observed in RIM1α KO mice are caused by a lack of docked SVs or by a decrease in the local Ca²⁺ concentration operating the Ca²⁺ sensor, which would impact directly P_ves. To evaluate the role of RIM1α in synaptic transmission is essential to get a better understanding on its function regulating different aspects of the release-inducing Ca²⁺ signal, such as VGCC trafficking, tethering, or VGCC subtype specificity. Apart from the large RIM isoforms, the small RIM isoforms RIM3 and RIM4, which consist only of the RIM C-terminal C₂B domain, may play a role in synaptic transmission due to their broader subcellular localization pattern, the high homology of the RIM C₂B domain, and the partial overlap of binding partners with those of RIM1α.<br /> Through electrophysiological recordings and Ca²⁺ imaging data, we investigated the role of RIM1α, RIM3, and RIM4 regulating neurotransmitter release properties and its impact on short-term synaptic plasticity. We developed a mathematical approach, based on a Hill equation describing the cooperative Ca²⁺-binding scheme, to quantitatively analyze the Ca²⁺-dependence of the inhibitory effect on Schaffer-collateral fEPSPs caused by specific VGCC blockers, w-conotoxin GVIA (Cgtx) and w-agatoxin IVA (Agatx), and infer the degree of Ca²⁺ binding to the sensor in the presence and absence of the RIM proteins. This analysis revealed for the first time that the local Ca²⁺ concentration and the P_ves are reduced by ~20% in RIM1α KO mice indicating that the remaining reduction in synaptic output is caused by a lack of releasable vesicles. Interestingly, the subtype contribution of the VGCCs to activate the Ca²⁺ sensor also differed: while N- and P/Q-type VGCCs equally contributed to the activation of the Ca²⁺ sensor (52%, 48%) in WT mice, the fractional contribution was strongly shifted towards P/Q-type VGCCs in KO mice (65%, 29%). This scenario favor the view that RIM1α deletion decreases synaptic strength by reducing the number of releasable SVs and altering the release-inducing Ca²⁺ signal by a distorted channel-vesicle geometry or by an altered specific N-type VGCC regulation, which causes a decrease in Pr, affecting synaptic strength. The removal of the y-RIMs affected release properties in a lesser extent as RIM1α ablation. We observed in RIM4 KO data a tendency towards an opposite scenario to the one observed in RIM1α KO mice. The local Ca²⁺ concentration and the P_ves were slightly increased in concert with a shift towards N-type VGCC contribution to the local Ca²⁺ concentration. Our data pointed out to a novel function for RIM1α specifically enhancing the contribution of N-type VGCC to synaptic transmission, and a possible small presynaptic role for RIM4 regulating synaptic transmission by an antagonistic mechanism with RIM1α.