RIM-binding protein 2 regulates release probability by fine-tuning calcium channel localization at murine hippocampal synapses

M. Katharina Grauel; Marta Maglione; Suneel Reddy-Alla; Claudia G. Willmes; Marisa M. Brockmann; Thorsten Trimbuch; Tanja Rosenmund; Maria Pangalos; Gülçin Vardar; Alexander Stumpf; Alexander M. Walter; Benjamin R. Rost; Britta J. Eickholt; Volker Haucke; Dietmar Schmitz; Stephan J. Sigrist; Christian Rosenmund

doi:10.1073/pnas.1605256113

New Research In

Edited by Thomas C. Südhof, Stanford University School of Medicine, Stanford, CA, and approved August 15, 2016 (received for review March 31, 2016)

Significance

Highly regulated and precise positioning of Ca²⁺ channels at the active zone (AZ) controls Ca²⁺ nanodomains at release sites. Their exact localization affects vesicular release probability (P_VR) and is important for proper synaptic transmission during repetitive stimulation. We provide a detailed analysis of synaptic transmission combined with superresolution imaging of the AZ organization in mouse hippocampal synapses lacking Rab-interacting molecule-binding protein 2 (RIM-BP2). By dual- and triple-channel time-gated stimulated emission depletion (gSTED) microscopy, we directly show that RIM-BP2 fine-tunes voltage-gated Ca²⁺ channel 2.1 (Ca_V2.1) localization at the AZ. We reveal that RIM-BP2 likely regulates the Ca²⁺ nanodomain by positioning Ca_V2.1 channels close to synaptic vesicle release sites. Loss of RIM-BP2 reduces P_VR and alters short-term plasticity.

Abstract

The tight spatial coupling of synaptic vesicles and voltage-gated Ca²⁺ channels (Ca_Vs) ensures efficient action potential-triggered neurotransmitter release from presynaptic active zones (AZs). Rab-interacting molecule-binding proteins (RIM-BPs) interact with Ca²⁺ channels and via RIM with other components of the release machinery. Although human RIM-BPs have been implicated in autism spectrum disorders, little is known about the role of mammalian RIM-BPs in synaptic transmission. We investigated RIM-BP2–deficient murine hippocampal neurons in cultures and slices. Short-term facilitation is significantly enhanced in both model systems. Detailed analysis in culture revealed a reduction in initial release probability, which presumably underlies the increased short-term facilitation. Superresolution microscopy revealed an impairment in Ca_V2.1 clustering at AZs, which likely alters Ca²⁺ nanodomains at release sites and thereby affects release probability. Additional deletion of RIM-BP1 does not exacerbate the phenotype, indicating that RIM-BP2 is the dominating RIM-BP isoform at these synapses.

Footnotes

↵¹M.K.G., M.M., and S.R.-A. contributed equally to this work.
↵²To whom correspondence may be addressed. Email: christian.rosenmund{at}charite.de, dietmar.schmitz{at}charite.de, or stephan.sigrist{at}fu-berlin.de.

Author contributions: M.K.G., M.M., S.R.-A., B.J.E., V.H., D.S., S.J.S., and C.R. designed research; M.K.G., M.M., S.R.-A., C.G.W., M.M.B., T.T., T.R., M.P., G.V., A.S., and B.R.R. performed research; A.M.W. contributed new reagents/analytic tools; M.K.G., M.M., S.R.-A., C.G.W., M.M.B., T.T., T.R., M.P., and B.R.R. analyzed data; A.M.W. provided data discussion; and M.K.G., M.M., S.R.-A., V.H., D.S., S.J.S., and C.R. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1605256113/-/DCSupplemental.

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