• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of hhmipaAbout Author manuscriptsSubmit a manuscriptHHMI Howard Hughes Medical Institute; Author Manuscript; Accepted for publication in peer reviewed journal
Cell. Author manuscript; available in PMC Jul 21, 2011.
Published in final edited form as:
PMCID: PMC3063406
HHMIMSID: HHMIMS268767

RIM proteins tether Ca2+-channels to presynaptic active zones via a direct PDZ-domain interaction

SUMMARY

At a synapse, fast synchronous neurotransmitter release requires localization of Ca2+-channels to presynaptic active zones. How Ca2+-channels are recruited to active zones, however, remains unknown. Using unbiased yeast two-hybrid screens, we here identify a direct interaction of the central PDZ-domain of the active-zone protein RIM with the C-termini of presynaptic N- and P/Q-type Ca2+-channels, but not L-type Ca2+-channels. To test the physiological significance of this interaction, we generated conditional knockout mice lacking all presynaptic RIM isoforms. Deletion of all RIMs ablated most neurotransmitter release by simultaneously impairing the priming of synaptic vesicles and by decreasing the presynaptic localization of Ca2+-channels. Strikingly, rescue of the decreased Ca2+-channel localization required the RIM PDZ-domain, whereas rescue of vesicle priming required the RIM N-terminus. We propose that RIMs tether N- and P/Q-type Ca2+-channels to presynaptic active zones via a direct PDZ-domain mediated interaction, thereby enabling fast, synchronous triggering of neurotransmitter release at a synapse.

INTRODUCTION

At a synapse, action potentials induce Ca2+-influx into a presynaptic terminal, which triggers rapid synchronous neurotransmitter release, thereby initiating synaptic transmission (Katz, 1969). Release is mediated by synaptic vesicle exocytosis at the active zone, a specialized region of the presynaptic plasma membrane that docks and primes vesicles for exocytosis (Wojcik and Brose, 2007). Fast synchronous release requires co-localization of Ca2+-channels with the release machinery at the active zone (Llinas et al., 1992; Meinrenken et al., 2002). Voltage-gated Ca2+-channels consist of a pore-forming α1-subunit and accessory β- and α2δ-subunits (Catterall et al., 2005). Presynaptic neurotransmitter release almost exclusively depends on N- and P/Q-type Ca2+-channels containing CaV2.1 and CaV2.2 α-subunits, respectively; R-type Ca2+-channels containing CaV2.3 α1-subunits may also contribute, whereas L-type and T-type Ca2+-channels containing CaV1 and CaV3 α1-subunits do not (Castillo et al., 1994; Dietrich et al., 2003; Luebke et al., 1993; Poncer et al., 1997; Regehr and Mintz, 1994; Takahashi and Momiyama, 1993; Wu et al., 1999). However, how N- and P/Q-type Ca2+-channels are specifically localized to active zones and coupled to the release machinery is unknown.

Active zones are composed of evolutionarily conserved proteins, including Munc13s, RIMs, RIM-BPs (RIM-binding proteins), ELKS’s, and α-liprins (Wojcik and Brose, 2007). Of these proteins, RIMs are likely the central organizers because they directly or indirectly interact with all other known active zone proteins and with synaptic vesicles (Mittelstaedt et al., 2010). RIM proteins are expressed in three principal isoforms (Kaeser et al., 2008; Wang et al., 1997; Wang et al., 2000): RIM1α and RIM2α that contain all RIM domains (i.e., N-terminal Rab3- and Munc13-binding sequences, central PDZ-domains, and C-terminal C2A- and C2B-domains with an intercalated PxxP sequence that binds to RIM-BPs); RIM1β and RIM2β that are identical to RIM1α and RIM2α but lack the N-terminal Rab3-binding sequences; and RIM2γ, RIM3γ, and RIM4γ that are composed only of C2B-domains, and are not considered here further. Genetic experiments in C. elegans and mice revealed that RIMs are essential for synaptic vesicle docking and priming and for presynaptic plasticity (Castillo et al., 2002; Fourcaudot et al., 2008; Gracheva et al., 2008; Kaeser et al., 2008; Koushika et al., 2001; Schoch et al., 2002; Schoch et al., 2006), but their mechanism of action remains unclear.

Several presynaptic proteins were shown to interact with Ca2+-channels. However, none of the reported interactions is selective for N- and P/Q-type Ca2+-channels. For example, (i) the RIM C2A- and C2B-domains bind to α1-subunits of L- and N-type Ca2+-channels (Coppola et al., 2001), (ii) the RIM C2B-domain interacts with the β4 Ca2+-channel subunit (Kiyonaka et al., 2007) that associates with all Ca2+-channels subtypes but is not required for neurotransmitter release (Qian and Noebels, 2000), and (iii) the proline-rich sequences of RIMs bind to RIM-BPs (Wang et al., 2000) that in turn bind to L-, N- and P/Q-type Ca2+-channels (Hibino et al., 2002). Thus, no plausible hypothesis at present suggests how N- and P/Q-type Ca2+-channels are selectively recruited to presynaptic active zones. More importantly, no presynaptic protein has been identified that is essential for recruiting N- and P/Q-type Ca2+-channels to presynaptic terminals. Only α-neurexins, which are presynaptic cell-adhesion molecules, were found to be required for presynaptic Ca2+-channel function (Missler et al., 2003). α-Neurexins, however, are also essential for the organization of other components of the presynaptic release machinery, and no molecular mechanism is known that links α-neurexins to active zones or Ca2+-channels.

Using an unbiased yeast two-hybrid screen, we here identify a direct interaction of P/Q- and N- type Ca2+-channels with RIM PDZ-domains. To test whether RIMs act to localize Ca2+-channel to active zones, and whether this function requires the PDZ-domains of RIMs, we generated conditional double knockout (KO) mice of all RIM isoforms that contain PDZ-domains. Using these mice, we show by electrophysiological recordings, Ca2+-imaging, and quantitative immunostaining of Ca2+-channels that RIMs are essential for localizing Ca2+-channels to release sites. Moreover, we show that only two RIM sequences are required for localization of Ca2+-influx to active zones, their PDZ-domains that bind to Ca2+-channels, and their proline-rich sequences that bind to RIM-BPs which in turn bind to Ca2+-channels. Thus, we propose that RIMs tether Ca2+-channels to active zones via two parallel but essential interactions, direct binding of Ca2+-channels to the RIM PDZ-domains that is specific for N- and P/Q-type Ca2+-channels, and indirect binding of Ca2+-channels to RIMs via RIM-BPs that is shared among different types of Ca2+-channels.

RESULTS

A screen for synaptic proteins binding to Ca2+-channels

We performed yeast-two hybrid screens for proteins that interact with the cytoplasmic C-terminal sequences of P/Q- and N-type Ca2+-channels. We chose these Ca2+-channel baits because P/Q- and N-type Ca2+-channels mediate nearly all presynaptic Ca2+-influx, and their C-termini have been implicated in targeting Ca2+-channels to the active zone (Catterall et al., 2005). The C-termini of N- and P/Q-type Ca2+-channels contain three conserved sequence motifs (Fig. 1A and Fig. S1A): an SH3-domain binding motif (RQLPGTP), a PNGY motif, and a C-terminal sequence motif (DxWC). Among 84 and 134 independent prey clones obtained in P/Q- and N-type Ca2+-channel screens, 33 and 16 prey clones, respectively, represented RIM-BPs (Fig. 1B), consistent with earlier studies (Hibino et al., 2002). In addition, 2 and 3 independent prey clones, respectively, contained RIM1 fragments, whose only overlapping sequence encoded the PDZ-domain (Fig. 1C).

Figure 1
Direct interaction of P/Q- and N-type Ca2+-channels with RIM PDZ-domains

The direct interaction of the RIM1 PDZ-domain with P/Q- and N-type Ca2+-channels was unexpected, prompting us to quantify it using liquid yeast two-hybrid assays. We found that the RIM1 PDZ-domain strongly and specifically bound to P/Q- and N-type Ca2+-channels, whereas no other RIM1 domain tested exhibited Ca2+-channel binding activity (Figs. 1D-1F). Mutations in the three conserved motifs of the cytoplasmic sequences of N- and P/Q-type Ca2+-channels showed that only the C-terminal sequence motif was essential for binding to RIM1 PDZ-domains, as predicted for a PDZ-domain interaction (Figs. 1E and 1F).

To validate the interaction of RIM PDZ-domains with Ca2+-channels by an independent method, we employed NMR spectroscopy. We produced recombinant 15N-labeled RIM1 PDZ-domain, and acquired 1H-15N heteronuclear single quantum coherence (HSQC) spectra in the absence or presence of unlabeled peptide from the C-terminus of the P/Q-type Ca2+-channel (residues RHDAYSESEDDWC). Previous studies of the 1H-15N HSQC spectrum of the RIM1 PDZ-domain (Lu et al., 2005) allowed us to assign cross-peak shifts induced by the Ca2+-channel peptide to specific residues in the RIM PDZ-domain (see supplemental methods). We found that the C-terminus of P/Q-type Ca2+-channels directly bound to the ligand-binding pocket of the RIM1 PDZ-domain (Figs. (Figs.1G1G and S1B). Additional 1H-15N HSQC experiments revealed analogous binding of the C-terminus of N-type Ca2+-channels to the RIM1 PDZ-domain, and of Ca2+-channels to the RIM2 PDZ-domain (Figs. S1C and S1D).

An atomic model suggests that the Ca2+-channel sequence fits well into the PDZ-domain binding pocket (Figs. 1H and 1I). The binding envisioned in this model agrees with the shifts we observed in the HSQC spectra in cross-peaks from residues such as K651, K653 and K694, which contribute to the binding pocket of the RIM1 PDZ-domain, and move upon P/Q-type Ca2+-channel peptide binding (Fig. 1G). Furthermore, we confirmed these interactions by isothermal calorimetry with the RIM1 PDZ-domain (Figs. S1E and S1F), yielding dissociation constants similar to those of other PDZ-domain interactions (P/Q-type Ca2+-channel, 10.3 ± 0.6 μM (N = 0.93); N-type Ca2+-channel, 23.4 ± 1.7 μM (N =0.93); Wiedemann et al., 2004). Thus, RIM PDZ-domains stoichiometrically interact with the C-terminal sequences of N- and P/Q-type Ca2+-channels, which are not found in L- and T-type Ca2+-channels.

Conditional KO mice for presynaptic RIM isoforms

The binding of RIM PDZ-domains to Ca2+-channels was unexpected because RIM PDZ-domains interact with the active-zone protein ELKS (a.k.a. Rab6-binding protein, CAST, or ERC; Ohtsuka et al., 2002; Wang et al., 2002). To test whether the RIM PDZ-domain physiologically binds to presynaptic Ca2+-channels, we generated conditional double KO mice in which all RIM isoforms containing PDZ-domains (RIM1α, 1β, 2α, and 2β) can be deleted by cre-recombinase (Figs. 2A-2C and S2), thereby enabling us to avoid the lethality of RIM-deficient mice (Kaeser et al., 2008; Schoch et al., 2006). We cultured neurons from newborn conditional double KO mice, and infected these neurons with lentiviruses expressing EGFP-tagged active or inactive cre-recombinases. Rescue experiments were performed by co-expressing various RIM proteins from the same lentiviruses via an IRES sequence (Kaeser et al., 2009).

Figure 2
Conditional deletion of RIM proteins in mice

Immunoblotting demonstrated that after 10 days in vitro (DIV10), neurons expressing active cre-recombinase (referred to as cDKO neurons) lack RIM proteins, whereas neurons expressing inactive cre-recombinase (referred to as controls) retain RIM expression (Fig. 2D). Despite lacking RIM proteins, cDKO neurons exhibited an overall normal morphology with unchanged synapse size and density (Fig. 2E, 2F). Electron microscopy revealed that in cDKO neurons, the number of docked synaptic vesicles per active zone was decreased nearly two-fold (Fig. 2G, 2H), consistent with a role for RIM in vesicle docking (Gracheva et al., 2008). All other measured parameters were unchanged, and removal of RIMs did not alter the structure of presynaptic dense projections visualized by phosphotungstic acid staining (Fig. 2G).

RIM deletion severely impairs neurotransmitter release

Electrophysiologically, deletion of RIM proteins caused a 3-4 fold decrease in the frequency of spontaneous ‘minis’, in the amplitude of post-synaptic currents evoked by isolated action potentials, and in the size of the readily releasable pool measured by application of hypertonic sucrose (Figs. 3A-3D, and L.D., P.S.K & T.C.S., unpublished data). Moreover, deletion of RIMs significantly decelerated and desynchronized release, as evidenced by an increase in rise times and in rise-time variability (Figs. 3E-3G), but did not change the relative contributions of P/Q- and N-type Ca2+-channels to evoked synaptic responses (Figs. S3A and S3B). Similarly, the RIM deletion massively decreased release induced by short stimulus trains (30 stimuli at 10 Hz; Figs. 3H and 3I), decelerated release as manifested by a relative increase in delayed release (Figs. 3J and 3K), and again strongly desynchronized release (Figs. 3L and 3M). These data suggest that RIMs are not only essential for vesicle priming, a previously identified RIM function that partly accounts for the decrease in release (Koushika et al., 2001; Schoch et al., 2002), but also for the synchronous timing of fast release that is not accounted for by a priming deficit. Since RIMs directly bind to Ca2+-channels (Fig. 1), and a loss of Ca2+-channels from presynaptic terminals would explain the impaired synchronous timing of release, we hypothesized that RIM binding to N- and P/Q-type Ca2+-channels tethers Ca2+-channels to the active zone, thereby increasing the efficiency, speed, and synchrony of release.

Figure 3
RIM deletion decreases, decelerates, and desynchronizes neurotransmitter release

RIM deletion alters the Ca2+-dependence of release

To explore the hypothesis that RIMs localize Ca2+-channels to active zones, we first examined the speed with which addition of a membrane-permeable Ca2+-buffer (EGTA-AM) decreases Ca2+-triggered release, measured as the IPSC amplitude (see supplemental methods). EGTA-AM caused a significantly faster rate of decline in IPSC amplitude in RIM-deficient cDKO neurons than in control neurons (Figs. 3N-3P). Thus, EGTA-dependent chelation of Ca2+ inhibits release more effectively in RIM-deficient than in control neurons, consistent with a longer distance between Ca2+-channels and release sites in RIM-deficient synapses.

We next examined the dependence of release in RIM-deficient synapses on the extracellular Ca2+-concentration ([Ca2+]ex; Fig. 4). If RIM-deficient presynaptic terminals contain fewer tethered Ca2+-channels, presynaptic Ca2+-influx should be decreased, and more [Ca2+]exshould be required for equivalent amounts of release – i.e., release should exhibit a higher [Ca2+]ex-dependence without a change in apparent Ca2+-cooperativity (see supplemental methods). However, since Ca2+-influx through Ca2+-channels saturates at high [Ca2+]ex (Church and Stanley, 1996; Schneggenburger et al., 1999), [Ca2+]ex-titrations underestimate the change in Ca2+-dependence of release in mutant synapses, and only relative changes are interpretable.

Figure 4
Mutational dissection of RIM KO phenotype

RIM-deficient cDKO neurons exhibited a large reduction in neurotransmitter release at all [Ca2+]ex (Figs. 4B and 4C), and a major increase in the [Ca2+]ex-dependence of neurotransmitter release (Fig. 4D). Both phenotypes were equally observed in inhibitory and excitatory synapses, and fully rescued by full-length wild-type RIM1α (Figs. 4A-4D and S4J-S4N). Fitting the [Ca2+]ex-response curve of individual experiments to a Hill function showed that the RIM deletion increased the [Ca2+]ex-requirement for release almost 2-fold, without changing the apparent Ca2+-cooperativity of release (Figs. (Figs.4E4E and S4H). Note that in the Hill function fits, the experimentally measured amplitudes suggest near-saturation at 10 mM [Ca2+]ex, allowing direct comparison of the fitted parameters (Fig. S4I).

The [Ca2+]ex-titration provided us with a facile assay to examine which RIM sequences determine vesicle priming and the [Ca2+]ex-dependence of release. We first tested rescue of the RIM double KO phenotype by the N-terminal RIM-RZ fragment that contains the Rab3-binding (“R”) and Zn2+-finger domains (“Z”) of RIM1α, and that had been previously implicated in vesicle priming (Dulubova et al., 2005), and the C-terminal RIM-PASB fragment that contains its PDZ- (“P”), C2A- (“A”), PxxP- (“S” for SH3-domain binding) and C2B-domains (“B”)(Fig. 4A). At 2 mM [Ca2+]ex, the RIM-RZ and RIM-PASB fragments each partially rescued the decrease in release, and alleviated the previously described decrease in Munc13 protein levels in cDKO neurons (Figs. 4F and 4I, S4D, Schoch et al, 2002). However, whereas the RIM-PASB fragment completely reversed the impairment of the [Ca2+]ex-dependence of release in cDKO neurons, the RIM-RZ fragment did not (Figs. 4F-4K). RIM-RZ, conversely, completely rescued the readily-releasable pool, whereas RIM-PASB did not (L.D., P.S.K. & T.C.S., unpublished). Note that different from synaptotagmin mutations (Shin et al., 2009), none of the changes in RIMs changed the apparent Ca2+-cooperativity of release (Fig. S4H; see also Table S3).

We next tested whether RIM-RZ or RIM-PASB rescued the speed and synchrony of release. Consistent with the [Ca2+]ex-titrations, the RIM-PASB fragment fully reversed the deceleration and desynchronization of release in cDKO neurons, as assessed at 2 mM [Ca2+]ex, whereas the RIM-RZ fragment did not (Figs. 4L, 4M , and S4O-S4Q). Together, these data indicate that the N-terminal RIM domains function in vesicle docking and priming (Betz et al., 2001; Dulubova et al., 2005; Gracheva et al., 2008; Koushika et al., 2001; Schoch et al., 2002), whereas the C-terminal RIM domains function in the Ca2+-dependence and synchrony of release.

RIM deletion impairs presynaptic Ca2+-influx

The increased [Ca2+]ex-dependence, decreased synchrony, and lowered speed of release in RIM-deficient neurons supports the hypothesis that RIMs localize Ca2+-channels to the active zone. However, these measurements are indirect. Their results could be explained by other hypotheses, for example that RIM directly regulates Ca2+-triggering of release by binding to synaptotagmin (Coppola et al., 2001; Schoch et al., 2002). To address this issue with an independent approach, we monitored action-potential induced Ca2+-transients by Ca2+-imaging in presynaptic boutons and postsynaptic dendrites.

We engineered new active and inactive EGFP-tagged cre-recombinase proteins that exhibit a tight nuclear localization (Figs. S5A-S5C), and expressed them in neurons from conditional KO mice. We then filled single neurons via a patch pipette with Alexa594 and the Ca2+-indicator Fluo5F, identified presynaptic axonal boutons and second-order dendrites by imaging Alexa594, elicited isolated action potentials by brief somatic current injections, and monitored the resulting Ca2+-transients in boutons and dendrites by imaging Fluo5F in line scans (at 333 Hz, ~100-150 μm from the cell body). To ensure that the observed Ca2+-transients were not due to passive depolarizations in response to the somatic current injections, we decreased in control experiments the injected current to a threshold level that only sometimes evoked an action potential. In these experiments, Ca2+-transients in boutons and dendrites strictly depended on the induction of action potentials, confirming that we were monitoring action potential-induced Ca2+-transients (Figs. S5D and S5E).

In RIM-containing control boutons, isolated action potentials induced a brief, ~100% increase in Fluo5F Ca2+-indicator fluorescence, whereas in RIM-deficient cDKO boutons, action potentials induced only a ~50% increase (Figs. 5A-5D). Similar to the impaired [Ca2+]ex-dependence of release (Fig. 4), the decreased Ca2+-influx in RIM-deficient cDKO neurons was fully rescued by the C-terminal RIM-PASB fragment (Figs. 5A-5D). Deletion of RIM proteins did not alter dendritic Ca2+-transients (Fig. 5C, inset, and Figs. S5F-S5H), suggesting that the RIM deletion did not generally impair Ca2+-channel function or Ca2+-buffering. Moreover, we detected no change in expression levels of various Ca2+-channel subunits in cDKO neurons (Figs. (Figs.5E5E and S5I). Thus, RIM deletions selectively decreased presynaptic Ca2+-influx in hippocampal neurons.

Figure 5
RIM deletion decreases presynaptic Ca2+-transients

RIM PDZ-domain is required for localizing Ca2+-influx

We next tested whether the RIM PDZ-domain mediates the RIM-dependent presynaptic localization of Ca2+-influx, using systematic rescue experiments combined with [Ca2+]ex-titrations as an assay.

Deletions of either the PDZ-domain or the PxxP-motif from the RIM-PASB fragment (Figs. 6A, top) blocked rescue of the impaired [Ca2+]ex-dependence in RIM-deficient cDKO neurons, whereas deletion of the C2A- or the C2B-domain had no effect (Figs. 6A-6E and S6A-S6C). Moreover, expression of a RIM fragment composed only of the PDZ-domain and PxxP motif (‘RIM-PS’) in cDKO neurons fully rescued the impaired [Ca2+]ex-dependence of release (Figs. 6F-6H and S6D-S6F). In contrast, expression of a RIM fragment composed of its two C2-domains (‘RIM-AB’) did not rescue the [Ca2+]ex-dependence of release, but partially reversed the decrease in IPSC size in RIM-deficient cDKO neurons, suggesting that the C2-domains are dispensable for localizing presynaptic Ca2+-influx, but enhance the efficacy of release.

Figure 6
RIM PDZ-domain and PxxP-motif confer normal Ca2+-dependence to RIM-deficient synapses

To directly probe whether the RIM PDZ-domain is actually essential for localizing presynaptic Ca2+-influx to active zones, we next performed rescue experiments with full-length wild-type RIM1α or RIM1α lacking the PDZ-domain (RIM-ΔPDZ). Whereas the former rescued all RIM cDKO phenotypes (Figs. 4C-4E), the latter was unable to reverse the impairment in [Ca2+]ex-dependence of release in RIM-deficient neurons, although it completely rescued the decrease in evoked IPSC amplitude at high [Ca2+]ex-concentrations (Figs 7A-7E, S7A-S7C). Moreover, full-length but not PDZ-domain deleted RIM1α rescued the deceleration and desynchronization of release in RIM-deficient neurons (Figs. 4L and 4M, 7F-7H). Possibly most importantly, RIM1α lacking the PDZ-domain did not restore normal Ca2+-influx into presynaptic nerve terminals in RIM-deficient neurons as measured by Ca2+-imaging, whereas full-length RIM1α fully rescued Ca2+-influx (Figs. 7I and 7J, S7D and S7E). Thus, the RIM-PDZ domain is critical for localizing Ca2+-influx to presynaptic active zones, thereby promoting the normal Ca2+-dependence, speed, and precision of neurotransmitter release.

Figure 7
RIM function in localizing presynaptic Ca2+-influx requires its PDZ-domain

The RIM PDZ-domain localizes P/Q-type Ca2+-channels to presynaptic termianls

The decrease in presynaptic Ca2+-influx in RIM-deficient terminals could be due to a loss of presynaptic Ca2+-channels, or to a decrease in their activity. To address this question, we measured by quantitative immunofluorescence presynaptic levels of P/Q-type Ca2+-channels, which mediate >80% of the synaptic responses we measure (Fig S3A-S3C). Strikingly, the RIM deletion reduced presynaptic P/Q-type Ca2+-channel levels ~40%, but had no effect on the active-zone protein bassoon (Figs. 8A, 8B, S8A and S8B).

Figure 8
RIM-dependent Ca2+-channel tethering linked to synaptic vesicle docking and priming

Finally, to test whether the presynaptic localization of P/Q-type Ca2+-channels depends on the RIM PDZ-domain, we examined rescue of the Ca2+-channel localization deficit in RIM-deficient cDKO neurons with full-length wild-type RIM1α, or RIM1α lacking the PDZ-domain (Figs. 8A and 8B). In agreement with the electrophysiological and Ca2+-imaging results described above (Fig. 7), deletion of the PDZ-domain rendered RIM1α unable to rescue the decrease in Ca2+-channel levels in presynaptic terminals (Figs. 8A and 8B), suggesting that the RIM PDZ-domain is essential for tethering of Ca2+-channels to presynaptic terminals.

DISCUSSION

Based on protein/protein interaction studies (Fig. 1), generation of conditional KO mice (Fig. 2), electrophysiological recordings (Figs. (Figs.3,3, ,4,4, ,6,6, and and7),7), Ca2+-imaging (Figs. (Figs.55 and and7),7), and quantitative immunofluorescence (Fig. 8), we here propose that the PDZ-domains of RIM proteins stoichiometrically interact with N- and P/Q-type Ca2+-channels in vitro, that RIM proteins are essential for tethering Ca2+-channels to presynaptic terminals, and that the RIM PDZ-domain is required for this function. In addition, RIMs indirectly interact with Ca2+-channels via their RIM-BP binding sequence (Hibino et al., 2002), which we show is also essential. Thus, RIMs perform two parallel interactions with Ca2+-channels: a direct interaction via their PDZ-domains that is specific for N- and P/Q-type Ca2+-channels, and an indirect interaction via RIM-BPs that is not specific for N- and P/Q-type Ca2+-channels. Our results suggest a physiologically validated mechanism by which Ca2+-influx is localized to the active zone, as required for fast, synchronous triggering of neurotransmitter release (Fig. 8C).

The RIM PDZ-domain/Ca2+-channel interaction

We found that the C-terminal sequences of N- and P/Q-type Ca2+-channels specifically bind to RIM PDZ-domains. R-type Ca2+-channels have a similar C-terminal sequence, and may also interact, whereas L- and T-type Ca2+-channels do not (Figs. (Figs.11 and S1). The PDZ-domain/Ca2+-channel interaction was surprising because the PDZ-domain proteins Mints and CASK were previously shown to bind to Ca2+-channels (Maximov et al., 1999), and because the RIM PDZ-domain is known to bind to ELKS proteins (Ohtsuka et al., 2002; Wang et al., 2002). However, it remains unclear whether these previously described interactions are physiologically important; in fact, Mint- and CASK-deficient synapses exhibit multiple abnormalities that do not resemble a Ca2+-influx impairment (Atasoy et al., 2007; Ho et al., 2006), whereas ELKS2-deficient synapses display increased release at inhibitory synapses (Kaeser et al., 2009), and ELKS levels are not detectably changed in RIM cDKO neurons (data not shown). Thus, it seems unlikely that the effects we observe here are indirectly mediated via Mints, CASK, or ELKS. It is possible, however, that different PDZ-domain binding reactions compete with each other at the active zone. For example, ELKS-binding to RIM PDZ-domains may inhibit Ca2+-channel binding, and thereby attenuate neurotransmitter release; this inhibitory role of ELKS binding could be regulated during plasticity, which might account for the central role of RIM in short- and long-term plasticity (Castillo et al., 2002; Fourcaudot et al., 2008; Kaeser et al., 2008; Schoch et al., 2002).

The RIM PDZ-domain is essential for localizing Ca2+-channels to the active zone

Using rescue experiments in RIM-deficient neurons, we found that the RIM PDZ-domain was invariably required in various RIM rescue constructs to reverse the impairment in presynaptic Ca2+-influx in RIM-deficient neurons (Figs. (Figs.44--7),7), and for localizing P/Q-type Ca2+-channels to presynaptic boutons (Fig. 8). In addition, loss of RIM-BP binding sequences blocked rescue of Ca2+-influx (Fig. 6). These experiments suggest that RIMs tether Ca2+-channels to the active zone via two parallel interactions: directly by binding to Ca2+-channels via their PDZ-domains, and indirectly by binding to RIM-BPs which in turn bind to Ca2+-channels (see model in Fig. 8C, and reconstitution of the tripartite complex in Fig. S8C). Our data also account for the specificity of N- and P/Q-type Ca2+-channels in release, since the RIM PDZ-domain/Ca2+-channel interaction is specific for these Ca2+-channel types (Fig. 1).

Active zone functions of RIMs

Our findings corroborate the notion that RIM proteins are central organizers of active zones by showing that besides their role in vesicle docking and priming (Betz et al., 2001; Gracheva et al., 2008; Kaeser et al., 2008; Koushika et al., 2001; Schoch et al., 2002; Schoch et al., 2006), RIMs are essential for tethering Ca2+-channels to active zones (Figs. (Figs.33--8).8). Moreover, RIM proteins perform additional functions, as indicated by the fact that although the RIM C2-domains had no detectable role in Ca2+-influx, they boosted neurotransmitter release, possibly by binding to α-liprins, the β4 Ca2+-channel subunit, or other proteins (Kiyonaka et al., 2007; Schoch et al., 2002). Thus, RIMs occupy the center of an interaction network in the molecular anatomy of the active zone, and influence all aspects of neurotransmitter release (Fig. 8C). However, our data also raise new questions. How do RIM proteins function in priming – only via their interaction with Munc13’s (Betz et al., 2001; Schoch et al., 2002), or via other effectors? How do RIM C2-domains boost release without altering Ca2+-influx? Why does deletion of just one RIM isoform, RIM1α, which has only a partial effect on neurotransmitter release due to its redundancy with other RIM isoforms, block multiple forms of presynaptic long-term synaptic plasticity (Castillo et al., 2002; Fourcaudot et al., 2008; Kaeser et al., 2008)? With the availability of double conditional KO mice described here, these questions can now be addressed.

EXPERIMENTAL PROCEDURES

In vitro protein binding assays

Two yeast two-hybrid screens of a rat brain cDNA library using bait vectors encoding the CaV2.2 N-type Ca2+-channel C-terminus (residues 2163-2339) or CaV2.1 P/Q-type Ca2+-channel C-terminus (residues 2213-2368) and liquid yeast to hybrid essays were performed as described (Wang et al., 1997). Of the 134/84 isolates with the N-type/P/Q-type bait, 8/16 clones corresponded to RIM-BP1, 8/17 to RIM-BP2, and 3/2 to RIM1. For mapping of the interaction region and the relative strength, yeast strain L40 was co-transformed with the various Ca2+-channel or RIM1 bait vectors and the Ca2+-channel or RIM1 prey vectors. HSQC spectroscopy was performed with rat RIM1 (residues 596-704, expressed as described (Lu et al., 2005)), and non-labeled P/Q-type Ca2+-channel peptides were synthesized. 1H–15N HSQC spectra were acquired in a Varian Inova500 spectrometer at 40-200 μM protein.

Generation of double conditional RIM KO mice

The RIM2αβγ targeting vector was constructed from a λ-phage DNA clone isolated from a genomic library, and conditional RIM2αβγ KO mice were generated by homologous recombination in R1 embryonic stem cells. The recombined stem cells were used for blastocyst injections to obtain chimeric mice. After germline transmission of the mutant allele, the newly generated conditional RIM2αβγ KO mice were crossed to conditional RIM1αβ KO mice (Kaeser et al., 2008).

Electrophysiology

Whole-cell patch-clamp recordings were performed in cultured hippocampal neurons at DIV13-15. Synaptic responses were elicited by a local stimulation electrode, and were acquired with a multiclamp 700B amplifier. The extracellular solution contained (in mM) 140 NaCl, 4 KCl, 2 CaCl2, 2 MgCl2, 10 HEPES-NaOH pH 7.3, and 10 glucose, with 315 mOsm, and either 50 μM picrotoxin (EPSCs) or 10 μM CNQX and 50 μM D-APV (IPSCs). For all electrophysiological experiments, the experimenter was blind to the condition/genotype of the cultures analyzed.

Ca2+-imaging

All experiments were performed in a Zeiss LSM 510 confocal microscope. Cultured hippocampal neurons were examined at DIV14-18 in whole-cell patch-clamp configuration after filling with Fluo5F and Alexa594 dyes for 10 min. Action potentials were induced by short, somatic current injections through the patch pipette (typically 5 ms, 600 pA); Ca2+-transients were measured with line scans through presynaptic boutons and second order dendrites at a frequency of 333 Hz, typically 100-150 μm away from the neuronal cell body. Fluorescent signals were quantified as mean region of interest and plotted as G-G0/G0 (G = average green emission in a given line; G0 = average of 20 line scans before action potential induction). The experimenter was blind to the condition/genotype until all recordings and analyses were completed.

Immunofluorescence staining of cultured neurons

Cultured neurons were fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100/3% bovine serum albumin and incubated overnight with anti-CaV2.1 rabbit polyclonal antibodies (Alomone labs, 1:100), or anti-bassoon rabbit polyclonal antibodies (Synaptic Systems, 1:250), and anti-synapsin mouse monoclonal antibodies (Synaptic Systems, 1:1000). Alexa-Fluor 546 anti-mouse and Alexa-Fluor 633 anti-rabbit secondary antibodies were used for detection with a confocal microscope. Single sections were acquired with identical settings applied to all samples in an experiment, and were used to quantify levels of P/Q-type Ca2+-channels and bassoon in ImageJ (NIH); the data were normalized to synapsin staining, and expressed relative to control cultures. The experimenter was blind to the condition/genotype in all experiments.

Data anlaysis

All data are shown as means ± SEM. Statistical significance was determined by one-way ANOVA (some electrophysiological recordings), two-way ANOVA (Ca2+-imaging peak amplitude), Kolmogorov-Smirnov test (cumulative distribution of peak amplitudes in Ca2+-imaging), χ-test (mouse survival analysis), or Student’s t-test (all other experiments). 95% confidence intervals for [Ca2+]ex-titration data and fitting parameters were calculated based on the covariance matrix. All numerical and statistical values and the tests used can be found in the Suppl. Tables 1-7.

Miscellaneous

Mixed hippocampal cultures and lentiviruses generated in transfected HEK293T cells expressing EGFP-tagged active or inactive cre recombinases followed by an IRES sequence for expression of rescue constructs were produced as described (Kaeser et al., 2009). SDS/PAGE gels, immunoblotting, and electronmicroscopic analyses were done according to standard methods described in the supplementary materials. All animal experiments were performed according to institutional guidelines. A detailed methods section can be found in the supplemental materials.

RESEARCH HIGHLIGHTS

  • At presynaptic active zones, Ca2+-channels bind to PDZ-domains of RIM proteins
  • Deletion of RIM proteins decreases presynaptic Ca2+-influx and vesicle priming
  • RIMs tether Ca2+-channels to presynaptic active zones for fast, synchronous release
  • RIM PDZ- but not C2-domains are essential for tethering presynaptic Ca2+-channels

Supplementary Material

01

ACKNOWLEDGEMENTS

We thank E. Borowicz, I. Kornblum, L. Fan, J. Mitchell, H. Ly and I. Huryeva for technical assistance, Dr. R.E. Hammer for blastocyst injections, Dr. N. Brose for Munc13-1 antibodies, Dr. C. Acuna-Goycolea for advice on Ca2+-imaging experiments, Dr. Z. Ma for assistance with yeast two-hybrid screening, Drs. Z. Pang, T. Bacaj and C. Földy for help with data analysis, and Dr. R. Schneggenburger for comments. This work was supported by grants from the NIH (NINDS 33564 to T.C.S., NS37200 to J.R., DA029044 to P.S.K.), a Swiss National Science Foundation Postdoctoral Fellowship (to P.S.K.), and a NARSAD Young Investigator Award (to P.S.K.).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Current address: Reata Pharmaceuticals, 2801 Gateway Dr., Irving, TX 75063

REFERENCES

  • Atasoy D, Schoch S, Ho A, Nadasy KA, Liu X, Zhang W, Mukherjee K, Nosyreva ED, Fernandez-Chacon R, Missler M, et al. Deletion of CASK in mice is lethal and impairs synaptic function. Proc Natl Acad Sci U S A. 2007;104:2525–2530. [PMC free article] [PubMed]
  • Betz A, Thakur P, Junge HJ, Ashery U, Rhee JS, Scheuss V, Rosenmund C, Rettig J, Brose N. Functional interaction of the active zone proteins Munc13-1 and RIM1 in synaptic vesicle priming. Neuron. 2001;30:183–196. [PubMed]
  • Castillo PE, Schoch S, Schmitz F, Sudhof TC, Malenka RC. RIM1alpha is required for presynaptic long-term potentiation. Nature. 2002;415:327–330. [PubMed]
  • Castillo PE, Weisskopf MG, Nicoll RA. The role of Ca2+ channels in hippocampal mossy fiber synaptic transmission and long-term potentiation. Neuron. 1994;12:261–269. [PubMed]
  • Catterall WA, Perez-Reyes E, Snutch TP, Striessnig J. International Union of Pharmacology. XLVIII. Nomenclature and structure-function relationships of voltage-gated calcium channels. Pharmacol Rev. 2005;57:411–425. [PubMed]
  • Church PJ, Stanley EF. Single L-type calcium channel conductance with physiological levels of calcium in chick ciliary ganglion neurons. J Physiol. 1996;496(Pt 1):59–68. [PMC free article] [PubMed]
  • Coppola T, Magnin-Luthi S, Perret-Menoud V, Gattesco S, Schiavo G, Regazzi R. Direct interaction of the Rab3 effector RIM with Ca2+ channels, SNAP-25, and synaptotagmin. J Biol Chem. 2001;276:32756–32762. [PubMed]
  • Dietrich D, Kirschstein T, Kukley M, Pereverzev A, von der Brelie C, Schneider T, Beck H. Functional specialization of presynaptic Cav2.3 Ca2+ channels. Neuron. 2003;39:483–496. [PubMed]
  • Dulubova I, Lou X, Lu J, Huryeva I, Alam A, Schneggenburger R, Sudhof TC, Rizo J. A Munc13/RIM/Rab3 tripartite complex: from priming to plasticity? Embo J. 2005 [PMC free article] [PubMed]
  • Fourcaudot E, Gambino F, Humeau Y, Casassus G, Shaban H, Poulain B, Luthi A. cAMP/PKA signaling and RIM1alpha mediate presynaptic LTP in the lateral amygdala. Proc Natl Acad Sci U S A. 2008;105:15130–15135. [PMC free article] [PubMed]
  • Gracheva EO, Hadwiger G, Nonet ML, Richmond JE. Direct interactions between C. elegans RAB-3 and Rim provide a mechanism to target vesicles to the presynaptic density. Neurosci Lett. 2008;444:137–142. [PMC free article] [PubMed]
  • Hibino H, Pironkova R, Onwumere O, Vologodskaia M, Hudspeth AJ, Lesage F. RIM binding proteins (RBPs) couple Rab3-interacting molecules (RIMs) to voltage-gated Ca(2+) channels. Neuron. 2002;34:411–423. [PMC free article] [PubMed]
  • Ho A, Morishita W, Atasoy D, Liu X, Tabuchi K, Hammer RE, Malenka RC, Sudhof TC. Genetic analysis of Mint/X11 proteins: essential presynaptic functions of a neuronal adaptor protein family. J Neurosci. 2006;26:13089–13101. [PubMed]
  • Kaeser PS, Deng L, Chavez AE, Liu X, Castillo PE, Sudhof TC. ELKS2alpha/CAST deletion selectively increases neurotransmitter release at inhibitory synapses. Neuron. 2009;64:227–239. [PMC free article] [PubMed]
  • Kaeser PS, Kwon HB, Chiu CQ, Deng L, Castillo PE, Sudhof TC. RIM1alpha and RIM1beta are synthesized from distinct promoters of the RIM1 gene to mediate differential but overlapping synaptic functions. J Neurosci. 2008;28:13435–13447. [PMC free article] [PubMed]
  • Katz B. The Release of Neural Transmitter Substances. Liverpool Univ Press; Liverpool: 1969.
  • Kiyonaka S, Wakamori M, Miki T, Uriu Y, Nonaka M, Bito H, Beedle AM, Mori E, Hara Y, De Waard M, et al. RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels. Nat Neurosci. 2007;10:691–701. [PMC free article] [PubMed]
  • Koushika SP, Richmond JE, Hadwiger G, Weimer RM, Jorgensen EM, Nonet ML. A post-docking role for active zone protein Rim. Nat Neurosci. 2001;4:997–1005. [PMC free article] [PubMed]
  • Llinas R, Sugimori M, Silver RB. Microdomains of high calcium concentration in a presynaptic terminal. Science. 1992;256:677–679. [PubMed]
  • Lu J, Li H, Wang Y, Sudhof TC, Rizo J. Solution structure of the RIM1alpha PDZ domain in complex with an ELKS1b C-terminal peptide. Journal of molecular biology. 2005;352:455–466. [PubMed]
  • Luebke JI, Dunlap K, Turner TJ. Multiple calcium channel types control glutamatergic synaptic transmission in the hippocampus. Neuron. 1993;11:895–902. [PubMed]
  • Maximov A, Sudhof TC, Bezprozvanny I. Association of neuronal calcium channels with modular adaptor proteins. J Biol Chem. 1999;274:24453–24456. [PubMed]
  • Meinrenken CJ, Borst JG, Sakmann B. Calcium secretion coupling at calyx of held governed by nonuniform channel-vesicle topography. J Neurosci. 2002;22:1648–1667. [PubMed]
  • Missler M, Zhang W, Rohlmann A, Kattenstroth G, Hammer RE, Gottmann K, Sudhof TC. Alpha-neurexins couple Ca2+ channels to synaptic vesicle exocytosis. Nature. 2003;424:939–948. [PubMed]
  • Mittelstaedt T, Alvarez-Baron E, Schoch S. RIM proteins and their role in synapse function. Biol Chem. 2010;391:599–606. [PubMed]
  • Ohtsuka T, Takao-Rikitsu E, Inoue E, Inoue M, Takeuchi M, Matsubara K, Deguchi-Tawarada M, Satoh K, Morimoto K, Nakanishi H, et al. Cast: a novel protein of the cytomatrix at the active zone of synapses that forms a ternary complex with RIM1 and munc13-1. J Cell Biol. 2002;158:577–590. [PMC free article] [PubMed]
  • Poncer JC, McKinney RA, Gahwiler BH, Thompson SM. Either N- or P-type calcium channels mediate GABA release at distinct hippocampal inhibitory synapses. Neuron. 1997;18:463–472. [PubMed]
  • Qian J, Noebels JL. Presynaptic Ca(2+) influx at a mouse central synapse with Ca(2+) channel subunit mutations. J Neurosci. 2000;20:163–170. [PubMed]
  • Regehr WG, Mintz IM. Participation of multiple calcium channel types in transmission at single climbing fiber to Purkinje cell synapses. Neuron. 1994;12:605–613. [PubMed]
  • Schneggenburger R, Meyer AC, Neher E. Released fraction and total size of a pool of immediately available transmitter quanta at a calyx synapse. Neuron. 1999;23:399–409. [PubMed]
  • Schoch S, Castillo PE, Jo T, Mukherjee K, Geppert M, Wang Y, Schmitz F, Malenka RC, Sudhof TC. RIM1alpha forms a protein scaffold for regulating neurotransmitter release at the active zone. Nature. 2002;415:321–326. [PubMed]
  • Schoch S, Mittelstaedt T, Kaeser PS, Padgett D, Feldmann N, Chevaleyre V, Castillo PE, Hammer RE, Han W, Schmitz F, et al. Redundant functions of RIM1alpha and RIM2alpha in Ca(2+)-triggered neurotransmitter release. Embo J. 2006;25:5852–5863. [PMC free article] [PubMed]
  • Shin OH, Xu J, Rizo J, Sudhof TC. Differential but convergent functions of Ca2+ binding to synaptotagmin-1 C2 domains mediate neurotransmitter release. Proc Natl Acad Sci U S A. 2009;106:16469–16474. [PMC free article] [PubMed]
  • Takahashi T, Momiyama A. Different types of calcium channels mediate central synaptic transmission. Nature. 1993;366:156–158. [PubMed]
  • Wang Y, Liu X, Biederer T, Sudhof TC. A family of RIM-binding proteins regulated by alternative splicing: Implications for the genesis of synaptic active zones. Proc Natl Acad Sci U S A. 2002;99:14464–14469. [PMC free article] [PubMed]
  • Wang Y, Okamoto M, Schmitz F, Hofmann K, Sudhof TC. Rim is a putative Rab3 effector in regulating synaptic-vesicle fusion. Nature. 1997;388:593–598. [PubMed]
  • Wang Y, Sugita S, Sudhof TC. The RIM/NIM family of neuronal C2 domain proteins. Interactions with Rab3 and a new class of Src homology 3 domain proteins. J Biol Chem. 2000;275:20033–20044. [PubMed]
  • Wiedemann U, Boisguerin P, Leben R, Leitner D, Krause G, Moelling K, Volkmer-Engert R, Oschkinat H. Quantification of PDZ domain specificity, prediction of ligand affinity and rational design of super-binding peptides. Journal of molecular biology. 2004;343:703–718. [PubMed]
  • Wojcik SM, Brose N. Regulation of membrane fusion in synaptic excitation-secretion coupling: speed and accuracy matter. Neuron. 2007;55:11–24. [PubMed]
  • Wu LG, Westenbroek RE, Borst JG, Catterall WA, Sakmann B. Calcium channel types with distinct presynaptic localization couple differentially to transmitter release in single calyx-type synapses. J Neurosci. 1999;19:726–736. [PubMed]
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...