Binding to Rab3A-interacting Molecule RIM Regulates the Presynaptic Recruitment of Munc13-1 and ubMunc13-2

Abstract

Transmitter release at synapses between nerve cells is spatially restricted to active zones, where synaptic vesicle docking, priming, and Ca2+-dependent fusion take place in a temporally highly coordinated manner. Munc13s are essential for priming synaptic vesicles to a fusion competent state, and their specific active zone localization contributes to the active zone restriction of transmitter release and the speed of excitation-secretion coupling. However, the molecular mechanism of the active zone recruitment of Munc13s is not known. We show here that the active zone recruitment of Munc13 isoforms Munc13-1 and ubMunc13-2 is regulated by their binding to the Rab3A-interacting molecule RIM1α, a key determinant of long term potentiation of synaptic transmission at mossy fiber synapses in the hippocampus. We identify a single point mutation in Munc13-1 and ubMunc13-2 (I121N) that, depending on the type of assay used, strongly perturbs or abolishes RIM1α binding in vitro and in cultured fibroblasts, and we demonstrate that RIM1α binding-deficient ubMunc13-2I121 is not efficiently recruited to synapses. Moreover, the levels of Munc13-1 and ubMunc13-2 levels are decreased in RIM1α-deficient brain, and Munc13-1 is not properly enriched at active zones of mossy fiber terminals of the mouse hippocampus if RIM1α is absent. We conclude that one function of the Munc13/RIM1α interaction is the active zone recruitment of Munc13-1 and ubMunc13-2. Transmitter release at synapses between nerve cells is spatially restricted to active zones, where synaptic vesicle docking, priming, and Ca2+-dependent fusion take place in a temporally highly coordinated manner. Munc13s are essential for priming synaptic vesicles to a fusion competent state, and their specific active zone localization contributes to the active zone restriction of transmitter release and the speed of excitation-secretion coupling. However, the molecular mechanism of the active zone recruitment of Munc13s is not known. We show here that the active zone recruitment of Munc13 isoforms Munc13-1 and ubMunc13-2 is regulated by their binding to the Rab3A-interacting molecule RIM1α, a key determinant of long term potentiation of synaptic transmission at mossy fiber synapses in the hippocampus. We identify a single point mutation in Munc13-1 and ubMunc13-2 (I121N) that, depending on the type of assay used, strongly perturbs or abolishes RIM1α binding in vitro and in cultured fibroblasts, and we demonstrate that RIM1α binding-deficient ubMunc13-2I121 is not efficiently recruited to synapses. Moreover, the levels of Munc13-1 and ubMunc13-2 levels are decreased in RIM1α-deficient brain, and Munc13-1 is not properly enriched at active zones of mossy fiber terminals of the mouse hippocampus if RIM1α is absent. We conclude that one function of the Munc13/RIM1α interaction is the active zone recruitment of Munc13-1 and ubMunc13-2. The release of neurotransmitters at chemical synapses between nerve cells is spatially restricted to specialized compartments of the axonal plasma membrane. These so called active zones, at which exocytosis takes place in a temporally highly coordinated manner, are typically localized in axon terminals, face the postsynaptic signal reception apparatus, and consist of electron-dense, insoluble proteinaceous material (1Rosenmund C. Rettig J. Brose N. Curr. Opin. Neurobiol. 2003; 13: 509-519Crossref PubMed Scopus (108) Google Scholar, 2Dresbach T. Qualmann B. Kessels M.M. Garner C.C. Gundelfinger E.D. Cell. Mol. Life Sci. 2001; 58: 94-116Crossref PubMed Scopus (163) Google Scholar, 3Zhai R.G. Vardinon-Friedman H. Cases-Langhoff C. Becker B. Gundelfinger E.D. Ziv N.E. Garner C.C. Neuron. 2001; 29: 131-143Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). Most presynaptic axon terminals contain hundreds of vesicles that are clustered in close proximity of the active zone. Usually, only a fraction of vesicles docked at the active zone are in a fusion-competent primed state, and only these primed vesicles, which are referred to as the readily releasable pool, can fuse with the plasma membrane in response to an elevation of the intracellular Ca2+ concentration. The readily releasable vesicle pool represents a reservoir that allows a synapse to repetitively release neurotransmitters during bursts of action potentials, and its size determines synaptic release probability and signaling capacity (1Rosenmund C. Rettig J. Brose N. Curr. Opin. 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EMBO J. 2005; PubMed Scopus Google Scholar), the of X. J. A. R. Südhof T.C. Rizo J. EMBO J. 2005; PubMed Scopus Google Scholar). RIM1α a role in the of synaptic transmitter release and presynaptic of synaptic long term RIM1α synaptic vesicle priming, term synaptic and in transmitter release N. S. Südhof T.C. Neuron. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), a that is to that of A. C. T. Brose N. J. 1999; PubMed Scopus Google Scholar, Sigler A. Brose N. C. K. C. Sci. U. S. A. PubMed Scopus Google Scholar). with the interaction between Munc13-1 and synaptic vesicle priming A. U. C. Rettig J. Brose N. Neuron. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, X. J. A. R. Südhof T.C. Rizo J. EMBO J. 2005; PubMed Scopus Google Scholar), that interaction a function in synaptic vesicle priming in and that the two proteins in the presynaptic is by the that Munc13-1 levels are in the of RIM1α-deficient S. T. K. M. Y. Südhof T.C. PubMed Scopus Google Scholar), and that in C. the of of function in the and are by the of a that an 2001; PubMed Scopus Google Scholar, Neurosci. 2001; PubMed Scopus Google Scholar). to its role in synaptic transmitter RIM1α is essential for long term potentiation long term Rab3A-interacting of synaptic transmission at mossy fiber synapses in the hippocampus and at fiber synapses in the S. Südhof T.C. PubMed Scopus Google Scholar, S. Südhof T.C. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). of are and the of protein and protein of RIM1α is for the of mossy fiber S. Südhof T.C. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). role of RIM1α in mossy fiber to for as RIM1α-deficient and S. L. M. N. Südhof T.C. Neuron. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). the for a interaction of Munc13s and in is the role of interaction is not known. The are with a role of in and of Munc13-1 and ubMunc13-2. the we the role of RIM1α in the synaptic recruitment of Munc13-1 and ubMunc13-2. show that RIM1α the active zone recruitment of Munc13-1 and ubMunc13-2. of ubMunc13-2 binding-deficient ubMunc13-2 by and a with as the A. U. C. Rettig J. Brose N. Neuron. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, Cell. Full Text PDF PubMed Scopus Google Scholar). that the which with of ubMunc13-2 and represents the binding of ubMunc13-2 A. U. C. Rettig J. Brose N. Neuron. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), as a for to a of with in a with which with of RIM1α and the binding for Munc13-1 and ubMunc13-2 A. U. C. Rettig J. Brose N. Neuron. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). binding-deficient by of that which to binding-deficient and we RIM1α binding-deficient of which to these we binding-deficient that one or several of and for their on RIM1α binding in we a of and that binding of to and are in the binding of ubMunc13-2 and Munc13-1 the binding Munc13 a A. U. C. Rettig J. Brose N. Neuron. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) RIM1α binding of Munc13-1 and the with the ubMunc13-2 these to in The Munc13-1 and and the ubMunc13-2 and in binding the of the mutation in the of Munc13-1 and ubMunc13-2 by of with the RIM1α binding of Munc13-1 and ubMunc13-2 in and PubMed Scopus Google Scholar). and The Munc13 binding of RIM1α as a fusion protein a and with protein in a of N. K. Y. Südhof T.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, A. U. C. Rettig J. Brose N. 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Full Text PDF PubMed Google Scholar) and by or for The by and with the of RIM1α Munc13-1 and ubMunc13-2 in Munc13-1 that its the binding priming in A. U. C. Rettig J. Brose N. Neuron. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). that binding active zone or priming of Munc13-1 and of ubMunc13-2. However, the of the of Munc13-1 that are not to the molecular for the of we a for the of in Munc13-1 and ubMunc13-2 that their interaction with The of ubMunc13-2 which is highly to the of Munc13-1 and the binding and as a protein in in with the RIM1α binding-deficient the that of the binding not which that of ubMunc13-2 the binding binding A. U. C. Rettig J. Brose N. Neuron. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). to these we binding-deficient that one or several of and for their on RIM1α binding in we a of and that binding of to and are in the binding of ubMunc13-2 and Munc13-1 the binding Munc13 a A. U. C. Rettig J. Brose N. Neuron. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) binding of Munc13-1 and the with the ubMunc13-2 these to in of of Munc13-1 and ubMunc13-2 in in and the binding-deficient of ubMunc13-2 and which we in in we fusion proteins of and of ubMunc13-2 and Munc13-1 and in on and for binding of RIM1α interaction with RIM1α or the binding However, in the of only binding to RIM1α in to RIM1α the point mutation the only that and with RIM1α binding of the Munc13-1 and ubMunc13-2 and We on mutation and for a of in vitro type and and for binding to the of RIM1α these the Munc13 to the not at these that the mutation abolishes binding of Munc13-1 and at only proteins are for the binding and can as a to the interaction in the of the mutation on binding in the of of and Munc13-1 with a in and These Munc13-1 and RIM1α in cells or in in and large in to a pool of protein RIM1α in the as as in the and RIM1α Munc13-1 large and RIM1α with Munc13-1 in these the and RIM1α in RIM1α recruited the RIM1α and as the in the of to the of a Munc13-1 as as and and and to the plasma membrane of and cells in response to binding A. U. M. E. Südhof T.C. Rettig J. Brose N. Neuron. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, U. A. T. Brose N. Rettig J. J. Cell Biol. 1999; PubMed Scopus Google Scholar). We of of Munc13-1 and if the binding to Munc13-1 the of RIM1α in We that and RIM1α to the plasma membrane of cells with RIM1α or in with not to the plasma membrane in response to in cells show that Munc13-1 RIM1α in a and that the mutation is to the and of the Munc13-1 Given the and very RIM1α binding of Munc13-1 and ubMunc13-2 and ubMunc13-2 is to of of ubMunc13-2 and in the role of binding in of we and as proteins in Sigler A. Brose N. C. K. C. Sci. U. S. A. PubMed Scopus Google Scholar) and their a that with Bassoon We the of presynaptic active zone of and by the of active zones that contain or in of We that in of the of active zones in of of the active zones These show that, Munc13-1 A. S. K. D. Südhof T.C. M. C. Brose N. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar), the fusion protein is recruited to presynaptic active zones, is that to the recruitment of ubMunc13-2 by of Munc13-1 to active zones or to their the proteinaceous at active of the of Munc13-1 and ubMunc13-2 in RIM1α the role of in the presynaptic and of RIM1α S. T. K. M. Y. Südhof T.C. PubMed Scopus Google Scholar) on these that Munc13-1 levels are strongly in the of RIM1α S. T. K. M. Y. Südhof T.C. PubMed Scopus Google Scholar). We the levels of Munc13-1 and of which not and which to RIM1α A. U. C. Rettig J. Brose N. Neuron. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, X. J. A. R. Südhof T.C. Rizo J. EMBO J. 2005; PubMed Scopus Google Scholar), in and RIM1α-deficient the levels of as a We that the levels of Munc13-1 and to by in RIM1α the levels of not not We the Munc13-1 and ubMunc13-2 that in the of RIM1α are that we on and RIM1α-deficient and the of Munc13-1 and ubMunc13-2 in the with A. C. T. Brose N. J. 1999; PubMed Scopus Google Scholar), we Munc13-1 to enriched in and synaptic membrane in and for of Munc13-1 and ubMunc13-2 not in RIM1α-deficient with the of ubMunc13-2 levels in fraction which of The levels of ubMunc13-2 in fraction to in the of RIM1α-deficient as with that ubMunc13-2 is efficiently the presynaptic protein in the of the of RIM1α on the of Munc13-1 and in and protein by and The levels of and Munc13 proteins by and levels as and the between insoluble and protein levels We that in the of RIM1α, the of Munc13-1 and ubMunc13-2 are as with the of Munc13 protein levels in and RIM1α-deficient show that RIM1α binding Munc13-1 and are efficiently to synaptic as a a and levels in the of These the that binding of Munc13-1 and ubMunc13-2 to is for their recruitment to and at active of Munc13-1 in the of and RIM1α that are in the presynaptic of Munc13-1 and ubMunc13-2. a role of in the brain, we the of Munc13-1 in and RIM1α-deficient hippocampus by on ubMunc13-2 not of the of Munc13-1 to strongly enriched in the mossy fiber terminals, which the of the and in the and RIM1α-deficient of a of Munc13-1 in mossy fiber terminals of the the presynaptic of Munc13-1 in RIM1α-deficient hippocampus of the Munc13-1 the and of the Munc13-1 at the between the and and decreased at the between and the of the RIM1α-deficient on the Munc13-1 only at the between the and and at the between and We the of of Munc13-1 and in mossy fiber terminals of and RIM1α-deficient by the Munc13-1 and in the and of and the to The for in for and RIM1α-deficient which that is enriched in mossy fiber terminals of the of an for Munc13-1 in only in not in RIM1α-deficient that mossy fiber of Munc13-1 on the of these in for a role of in presynaptic of Munc13-1 at active to the of RIM1α on the and localization of Munc13-1 and the Sigler A. Brose N. C. K. C. Sci. U. S. A. PubMed Scopus Google Scholar) and of RIM1α are not in the of Munc13-1 and ubMunc13-2. RIM1α and Bassoon to in and proteins contain two that The mammalian and and the C. and are very in their which a two Munc13 and a A. U. C. Rettig J. Brose N. Neuron. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The an that is highly to that of Munc13-1 and the of and are These Munc13 The is and for vesicle priming A. U. C. Rettig J. Brose N. Neuron. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, U. C. U. Brose N. Rettig J. Curr. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Curr. Biol. 2005; Full Text Full Text PDF PubMed Scopus (108) Google Scholar, U. T. A. H. E. Brose N. Rettig J. EMBO J. 2000; PubMed Scopus Google Scholar, J. N. J. R. O. C. Rizo J. Mol. Biol. 2005; PubMed Scopus Google Scholar). The binding of Munc13-1 and on the to a function that is not in the priming A. U. C. Rettig J. Brose N. Neuron. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, X. J. A. R. Südhof T.C. Rizo J. EMBO J. 2005; PubMed Scopus Google Scholar). binding to active zone recruitment or of Munc13-1 and ubMunc13-2 or to in these Munc13s that their priming The central of the is that one function of the interaction is the active zone recruitment of Munc13-1 and ubMunc13-2. is by the (i) Munc13-1 and ubMunc13-2 RIM1α, and in binding is strongly or by the mutation the binding-deficient of is not to synapses of cultured the levels of Munc13-1 and are by in RIM1α-deficient in RIM1α-deficient the of insoluble Munc13-1 and ubMunc13-2 are decreased and Munc13-1 is enriched in mossy fiber terminals of RIM1α-deficient The that RIM1α is not the only active zone protein for Munc13-1 and ubMunc13-2. The Munc13-1 proteins that in RIM1α-deficient are localized in the synaptic and large of Munc13-1 and with membrane in the of RIM1α that or active zone to the of Munc13-1 and ubMunc13-2 to the active zone protein However, RIM1α to the active zone recruitment protein for Munc13-1 and ubMunc13-2 Munc13-1 and ubMunc13-2 levels are by in RIM1α-deficient and the synaptic recruitment of the binding-deficient is The levels of Munc13-1 and ubMunc13-2 in RIM1α-deficient are to and of the proteins in the as with the active on the interaction are with a role of in the of Munc13-1 and ubMunc13-2 at active A. U. C. Rettig J. Brose N. Neuron. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) and in the of X. J. A. R. Südhof T.C. Rizo J. EMBO J. 2005; PubMed Scopus Google Scholar), of the interaction to decreased synaptic vesicle These can by active zone recruitment of the essential priming proteins Munc13-1 and ubMunc13-2 of their interaction with their for of the of synaptic the are of with to the of the role of in mossy fiber contain binding for and Munc13s X. J. A. R. Südhof T.C. Rizo J. EMBO J. 2005; PubMed Scopus Google Scholar), and RIM1α and are essential for mossy fiber S. Südhof T.C. PubMed Scopus Google Scholar, R. Südhof T.C. T. PubMed Scopus Google Scholar). Given the central and essential role of Munc13s in the transmitter release of is that Munc13-1 and ubMunc13-2 are in the of mossy fiber is by the that the Munc13-1 Munc13-1 are by show mossy fiber in RIM1α in which Munc13-1 levels are by mossy fiber is S. Südhof T.C. PubMed Scopus Google Scholar). However, the of RIM1α not only Munc13-1 a of ubMunc13-2 levels and a of Munc13-1 with strongly synaptic in mossy fiber terminals the of RIM1α is to on the Munc13 levels in mossy fiber terminals the of a single Munc13-1 where active zone recruitment of Munc13s is not The in Munc13 at mossy fiber terminals in RIM1α one of the mossy fiber are to as to Munc13-1 and ubMunc13-2 are of mossy fiber to of RIM1α, which is essential for mossy fiber S. Südhof T.C. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), the interaction and the levels or of Munc13-1 and ubMunc13-2 in mossy fiber mouse that binding-deficient Munc13-1 and ubMunc13-2 or Munc13 binding-deficient RIM1α of the proteins are to the of in mossy fiber the an Given that only on the is X. J. A. R. Südhof T.C. Rizo J. EMBO J. 2005; PubMed Scopus Google Scholar), we to to and to identify in Munc13-1 and ubMunc13-2 that with binding to the of a point that depending on the assay used, strongly perturbs or abolishes binding of Munc13-1 and ubMunc13-2 their or that the mutation not with protein and of the proteins as for the of the molecular that mossy fiber We are to T. C. Südhof of for the mouse in the We and the of the and the at the of for