Tight coupling of Ca 2+ channels to the presynaptic active zone is critical for fast synchronous neurotransmitter release. RIMs are multidomain proteins that tether Ca 2+ channels to active zones, dock and prime synaptic vesicles for release, and mediate presynaptic plasticity. Here, we use conditional knockout mice targeting all RIM isoforms expressed by the Rims1 and Rims2 genes to examine the contributions and mechanism of action of different RIMs in neurotransmitter release. We show that acute single deletions of each Rims gene decreased release and impaired vesicle priming but did not alter the extracellular Ca 2+ -responsiveness of release (which for Rims gene mutants is a measure of presynaptic Ca 2+ influx). Moreover, single deletions did not affect the synchronization of release (which depends on the close proximity of Ca 2+ channels to release sites). In contrast, deletion of both Rims genes severely impaired the Ca 2+ responsiveness and synchronization of release. RIM proteins may act on Ca 2+ channels in two modes: They tether Ca 2+ channels to active zones, and they directly modulate Ca 2+ -channel inactivation. The first mechanism is essential for localizing presynaptic Ca 2+ influx to nerve terminals, but the role of the second mechanism remains unknown. Strikingly, we find that although the RIM2 C 2 B domain by itself significantly decreased Ca 2+ -channel inactivation in transfected HEK293 cells, it did not rescue any aspect of the RIM knockout phenotype in cultured neurons. Thus, RIMs primarily act in release as physical Ca 2+ -channel tethers and not as Ca 2+ -channel modulators. Different RIM proteins compensate for each other in recruiting Ca 2+ channels to active zones, but contribute independently and incrementally to vesicle priming.I n a presynaptic nerve terminal, Ca 2+ triggers synaptic vesicle exocytosis at specialized sites called active zones. Among the major active zone proteins (e.g., RIMs, α-liprins, ELKS's, RIMBPs, Piccolo/Bassoon, and Munc13's), RIMs stand out because they bind to all other components of active zones and are involved in all central aspects of neurotransmitter release (1, 2). In vertebrates, two RIM genes (Rims1 and Rims2) synthesize five principal RIM isoforms from independent promoters (RIM1α, RIM1β, RIM2α, RIM2β, and RIM2γ; Fig. 1A); these isoforms are further diversified by alternative splicing (3-5). Moreover, two additional RIM genes (Rims3 and Rims4) produce only γ-isoforms, which are not further considered here. Gene deletion experiments (Table S1) showed that RIMs are essential for multiple aspects of neurotransmitter release (4, 6-10) and for presynaptic short-and long-term plasticity (4, 6, 11-13). However, how different RIM isoforms contribute to neurotransmitter release is unclear.Recent studies revealed that RIMs regulate presynaptic Ca 2+ channels via two independent mechanisms, namely by recruiting Ca 2+ channels to active zones (14) and by modulating Ca 2+ -channel opening times (15,16). The first activity is mediated by a tripartite complex of ...

The Rem, Rem2, Rad, and Gem/Kir (RGK) family of small GTP-binding proteins potently inhibits high voltage-activated (HVA) Ca 2+ channels, providing a powerful means of modulating neural, endocrine, and muscle functions. The molecular mechanisms of this inhibition are controversial and remain largely unclear. RGK proteins associate directly with Ca 2+ channel β subunits (Ca v β), and this interaction is widely thought to be essential for their inhibitory action. In this study, we investigate the molecular underpinnings of Gem inhibition of P/Q-type Ca 2+ channels. We find that a purified Gem protein markedly and acutely suppresses P/Q channel activity in inside-out membrane patches, that this action requires Ca v β but not the Gem/Ca v β interaction, and that Gem coimmunoprecipitates with the P/Q channel α 1 subunit (Ca v α 1 ) in a Ca v β-independent manner. By constructing chimeras between P/Q channels and Gem-insensitive low voltage-activated T-type channels, we identify a region encompassing transmembrane segments S1, S2, and S3 in the second homologous repeat of Ca v α 1 critical for Gem inhibition. Exchanging this region between P/Q and T channel Ca v α 1 abolishes Gem inhibition of P/Q channels and confers Ca v β-dependent Gem inhibition to a chimeric T channel that also carries the P/Q I-II loop (a cytoplasmic region of Ca v α 1 that binds Ca v β). Our results challenge the prevailing view regarding the role of Ca v β in RGK inhibition of high voltage-activated Ca 2+ channels and prompt a paradigm in which Gem directly binds and inhibits Ca v β-primed Ca v α 1 on the plasma membrane.

Ca2؉ channel ␤ subunits (Ca v ␤s) are essential for regulating the surface expression and gating of high voltage-activated Ca 2؉ channels through their interaction with Ca 2؉ channel ␣ 1 subunits. In efforts to uncover new interacting partners and new functions for Ca v ␤, we identified a new splicing isoform of Pax6, a transcription factor crucial for the development of the eye, nose, brain, and pancreas. Pax6 contains two DNA binding domains (paired domain and homeodomain), a glycine-rich linker connecting these two domains and a C-terminal proline-, serine-, and threonine-rich transactivation domain. The protein sequence and function of Pax6 are highly conserved from invertebrate to human. The newly isolated isoform, named Pax6(S), retains the paired domain, linker, and homeodomain of Pax6, but its C terminus is composed of a truncated classic proline, serine, and threonine domain and a unique S tail. Pax6(S) shows a similar level of transcriptional activity in vitro as does Pax6, but only in primates is the protein sequence highly conserved. Its spatial-temporal expression profiles are also different from those of Pax6. These divergences suggest a noncanonical role of Pax6(S) during development. The interaction between Pax6(S) and Ca v ␤ is mainly endowed by the S tail. Co-expression of Pax6(S) with a Ca 2؉ channel complex containing the ␤ 3 subunit in Xenopus oocytes does not affect channel properties. Conversely, however, ␤ 3 is able to suppress the transcriptional activity of Pax6(S). Furthermore, in the presence of Pax6(S), ␤ 3 is translocated from the cytoplasm to the nucleus. These results suggest that full-length Ca v ␤ may act directly as a transcription regulator independent of its role in regulating Ca 2؉ channel activity.4 is a cytosolic auxiliary protein of multimeric high voltage-activated (HVA) Ca 2ϩ channel complexes, which include L-, N-, P/Q-, and R-type Ca 2ϩ channels. It plays an essential role in chaperoning the channel complex to the plasma membrane and normalizing its gating properties (1-4). Crystal structures of Ca v ␤ in complex with its high affinity binding site in the principal poreforming ␣ 1 subunit (Ca v ␣ 1 ) show that much of the exposed surface of Ca v ␤ is unoccupied and is available to engage in interactions with other regions of Ca v ␣ 1 or with other proteins (5-7). Indeed, an increasing number of proteins has been shown to directly interact with Ca v ␤, including the Rem/Rad/Gem/Kir (RGK) family of small monomeric GTPases (8, 9), RIM1 (10), ryanodine receptors (11), Ahnak (12, 13), bestrophin-1 (14), and dynamin (15). Many of these proteins have been reported to regulate the activity of HVA Ca 2ϩ channels. To search for other potential Ca v ␤-interacting proteins, we carried out yeast two-hybrid screens using the ␤ 3 subunit as bait. Among the candidate target proteins we isolated, one was related to Pax6.Pax6 is a transcription factor that belongs to the paired box (Pax) family (16 -25). It is widely expressed in the eye, nose, pancreas, and the central nervous system ...

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