Functional Coupling of Rab3-interacting Molecule 1 (RIM1) and L-type Ca2+ Channels in Insulin Release*

Insulin release by pancreatic β-cells is regulated by diverse intracellular signals, including changes in Ca2+ concentration resulting from Ca2+ entry through voltage-gated (CaV) channels. It has been reported that the Rab3 effector RIM1 acts as a functional link between neuronal CaV channels and the machinery for exocytosis. Here, we investigated whether RIM1 regulates recombinant and native L-type CaV channels (that play a key role in hormone secretion) and whether this regulation affects insulin release. Whole-cell patch clamp currents were recorded from HEK-293 and insulinoma RIN-m5F cells. RIM1 and CaV channel expression was identified by RT-PCR and Western blot. RIM1-CaV channel interaction was determined by co-immunoprecipitation. Knockdown of RIM1 and CaV channel subunit expression were performed using small interference RNAs. Insulin release was assessed by ELISA. Co-expression of CaV1.2 and CaV1.3 L-type channels with RIM1 in HEK-293 cells revealed that RIM1 may not determine the availability of L-type CaV channels but decreases the rate of inactivation of the whole cell currents. Co-immunoprecipitation experiments showed association of the CaVβ auxiliary subunit with RIM1. The lack of CaVβ expression suppressed channel regulation by RIM1. Similar to the heterologous system, an increase of current inactivation was observed upon knockdown of endogenous RIM1. Co-immunoprecipitation showed association of CaVβ and RIM1 in insulin-secreting RIN-m5F cells. Knockdown of RIM1 notably impaired high K+-stimulated insulin secretion in the RIN-m5F cells. These data unveil a novel functional coupling between RIM1 and the L-type CaV channels via the CaVβ auxiliary subunit that contribute to determine insulin secretion.

Insulin release by pancreatic ␤-cells is regulated by diverse intracellular signals, including changes in Ca 2؉ concentration resulting from Ca 2؉ entry through voltage-gated (Ca V ) channels. It has been reported that the Rab3 effector RIM1 acts as a functional link between neuronal Ca V channels and the machinery for exocytosis. Here, we investigated whether RIM1 regulates recombinant and native L-type Ca V channels (that play a key role in hormone secretion) and whether this regulation affects insulin release. Whole-cell patch clamp currents were recorded from HEK-293 and insulinoma RIN-m5F cells. RIM1 and Ca V channel expression was identified by RT-PCR and Western blot. RIM1-Ca V channel interaction was determined by co-immunoprecipitation. Knockdown of RIM1 and Ca V channel subunit expression were performed using small interference RNAs. Insulin release was assessed by ELISA. Co-expression of Ca V 1.2 and Ca V 1.3 L-type channels with RIM1 in HEK-293 cells revealed that RIM1 may not determine the availability of L-type Ca V channels but decreases the rate of inactivation of the whole cell currents. Coimmunoprecipitation experiments showed association of the Ca V ␤ auxiliary subunit with RIM1. The lack of Ca V ␤ expression suppressed channel regulation by RIM1. Similar to the heterologous system, an increase of current inactivation was observed upon knockdown of endogenous RIM1. Co-immunoprecipitation showed association of Ca V ␤ and RIM1 in insulin-secreting RIN-m5F cells. Knockdown of RIM1 notably impaired high K ؉ -stimulated insulin secretion in the RIN-m5F cells. These data unveil a novel functional coupling between RIM1 and the L-type Ca V channels via the Ca V ␤ auxiliary subunit that contribute to determine insulin secretion.
Release of insulin-containing vesicles by pancreatic ␤-cells is regulated by various intracellular signals, including Ca 2ϩ . Phys-iologically, glucose stimulation increases the [ATP]/[ADP] intracellular ratio that closes ATP-sensitive potassium (K ATP ) channels, thereby depolarizing ␤-cell plasma membrane. This process in turn activates plasma membrane voltage-gated (Ca V ) channels, allowing Ca 2ϩ to enter the cell and trigger insulin exocytosis (1,2). Ca V channels are classified according to their activation threshold as low voltage-activated or high voltage-activated. Based on pharmacological profiles, high voltage-activated channels can be divided into L-type and non-L-type channels, the latter including the N, P/Q, and R subtypes (3,4). Neurotransmitter release is attributed to Ca 2ϩ influx through P/Qtype (Ca V 2.1) and N-type (Ca V 2.2) channels, whereas L-type (Ca V 1.2 and Ca V 1.3) channels are considered to be responsible for hormone secretion (3). At the molecular level, Ca V channels are oligomeric complexes of at least three proteins or subunits, the pore-forming (Ca V ␣ 1 ) subunit and the auxiliary Ca V ␣ 2 ␦ and Ca V ␤ subunits (3,4).
Electrophysiological and molecular studies indicate that pancreatic ␤-cells express several subtypes of Ca V channels. In particular, dihydropyridine-sensitive, L-type Ca V channels are responsible for a significant portion of the high voltage-activated current (5,6), and given that dihydropyridines potently suppress insulin secretion, L-channels are considered crucial for ␤-cell function (7). Of the four genes that encode Ca V ␣ 1 subunits of L-channels, either Ca V 1.2 (formerly known as ␣ 1C ), Ca V 1.3 (␣ 1D ), or both have been identified in rodent and human islets as well as in various ␤-cell lines, including the rat insulinoma RIN-m5F cells (6,8). Although the relative expression levels of the two genes and their importance for insulin secretion remain uncertain, immunoprecipitation experiments suggests that Ca V 1.2 may represent ϳ50% of the L-type channels in this cell line (8).
In vivo and in vitro studies have shown that pancreatic islets respond to increases in extracellular glucose with a biphasic pattern of insulin release. The first phase lasts a few minutes and reflects the release of a pool of granules in close proximity to L-type channels (9,10). Two mechanisms possibly contribute to the second phase of insulin secretion: the replenishment of the immediately releasable pool from the reserve pool and exocytosis of granules located far from Ca V channels due to widespread increases in cytosolic Ca 2ϩ during depolarization.
The latter mechanism also involves non-L-type channels. Last, studies in mice lacking Ca V 1.2 and Ca V 1.3 channels have corroborated that L-type channels are crucial for ␤-cell physiology (11,12).
Interestingly, it has been found that different members of the RIM family (13)(14)(15)(16), putative effectors of Rab3, and some associated proteins (17) may functionally link Ca V channels to the machinery for exocytosis. Moreover, it has been reported that RIM1 modulates neuronal Ca V 2.1 channels through its interaction with the Ca V ␤ subunit, modifying the inactivation rate for a sustained Ca 2ϩ influx and anchoring neurotransmittercontaining vesicles in the vicinity of the channels (16). In contrast to these findings, no evidence has been reported for an N-type (Ca V 2.2) channel/RIM interaction at the presynaptic terminals using a chick calyx synapse preparation as well as in the heterologously expressed proteins in HEK293T cells (18,19). These results argue against the hypothesis that RIM proteins may be critical for neuronal channel localization at the active zone. On the other hand, recent studies have shown also that RIM1 or RIM2 and RIM3 could indeed interact with native and recombinant mammalian N-type channels (20). Although the reason for this discrepancy is presently unknown, a model has emerged that could reconcile the conflicting results regarding the N-and P/Q-type channel/RIM interaction. In this model, RIM is part of a complex that tethers the synaptic vesicle to the channel, acting as a switch for a link between the channel and the synaptic vesicles that changes from high to low affinity states (19,21).
In the present report, by using a strategy that combines patch clamp recordings with biochemical and molecular biology techniques, we provide evidence that RIM1 regulates recombinant L-type Ca V channels (of the Ca V 1.2 and Ca V 1.3 class) heterologously expressed in HEK-293 cells as well as native L-channels expressed in rat insulinoma RIN-m5F cells and also show that this regulation results in a facilitation of insulin secretion. These data stress the importance of RIM1 as a regulatory constituent of the insulin secretory machinery.
Recombinant Ca V Channel Expression and Electrophysiology-After splitting HEK-293 cells on the previous day and seeding at 60% confluence, cells were transfected using the Lipofectamine Plus reagent (Invitrogen) with 1.6 g of each plasmid cDNA encoding L-type channel pore-forming subunit Ca V 1.2 (GenBank TM accession number X15539) or Ca V 1.3 (AF370009) with Ca V ␤ 2 (M80545) or Ca V ␤ 3 (M88751), and Ca V ␣ 2 ␦-1 (M86621) in the presence or absence of RIM1 (NM_053270). For electrophysiology, 0.6 g of a plasmid cDNA encoding the green fluorescent protein (Green-Lantern; Invitrogen) was added to the transfection mixture to identify and select transfected cells.
Electrophysiological recordings were performed according to the whole cell configuration of the patch clamp technique (22) at room temperature (22-24°C) in a bathing solution containing 10 and 5 mM BaCl 2 (for Ca V 1.2 and Ca V 1.3, respectively), 125 mM TEA-Cl, 10 mM HEPES, and 10 mM glucose (pH 7.3). Patch pipettes were filled with a solution containing 120 mM CsCl, 10 mM HEPES, 10 mM EGTA, 5 mM MgCl 2 , 4 mM ATP, and 0.1 mM GTP (pH 7.3). Ba 2ϩ was used as the charge carrier instead of Ca 2ϩ for the following reasons: (i) conductance for Ba 2ϩ versus Ca 2ϩ ions through high voltage-activated Ca V channels is larger, thereby increasing the signal/noise ratio; (ii) it reinforces blockade of K ϩ currents; and (iii) rundown of the current is sometimes prominent, and the use of Ba 2ϩ attenuates this problem. More importantly, when experiments were performed with external solutions containing Ca 2ϩ , cells exhibited a prominent Ca 2ϩ -activated component, which difficult a clear evaluation of the action of RIM1 on L-type Ca V currents. This component was suppressed in external Ba 2ϩ conditions. It is worth noting, however, that the effect of RIM1 on L-type currents is qualitatively similar in external Ba 2ϩ and Ca 2ϩ conditions (supplemental Fig. 1).
Recordings were made using an Axopatch 200B amplifier (Molecular Devices). Data acquisition and analysis were performed using pClamp10 software (Axon CNS) and Sigma Plot 11.0 (Systat Software Inc.) as described elsewhere (23,24). Linear leak and parasitic capacitance components were subtracted on-line using a P/4 protocol. Membrane capacitance (C m ) was determined as described previously (25) and used to normalize currents.
RT-PCR-Total RNA was extracted from RIN-m5F cells by TRIzol reagent (Invitrogen). Reverse transcription was done with 5 g of total RNA using the SuperScript III first strand system for RT-PCR (Invitrogen). The sequences of forward and reverse primers used for RIM1 amplification were 5Ј-GTTCA-GTGATTTCCTTGATGGG-3Ј and 5Ј-TTACTATGACCGG-ATGCAGGG-3Ј (sense and antisense, respectively) (28). As a PCR control, ␤-actin was amplified using as a sense primer 5Ј-AAGATGACCCAGATCATGTT-3Ј and antisense primer 5Ј-GAGTACTTGCGCTCAGGAGG-3Ј. The PCR was carried out in a total volume of 50 l containing 5 l of cDNA solution, 1ϫ PCR buffer, 0.2 mM each deoxynucleotide triphosphate, 1.5 mM MgCl 2 , 0.5 M each primer, and 2.5 units of Taq DNA polymerase on a Thermal Cycler (Thermo Scientific) for 25 cycles. Denaturation was carried out at 94°C for 45 s, annealing at 55°C for 30 s, and elongation at 75°C for 1 min. PCRs were performed using Taq DNA polymerase (Invitrogen) with a 0.5 M concentration of each primer.
RNA Interference in RIN-m5F Cells-The siRNA sequences 5Ј-CUCAGAUUAUGAGGUUGAU (dT) and 5Ј-AUCAAC-CUCAUAAUCUGAG (dT) for RIM1 were transfected in RIN-m5F cells using the N-TER nanoparticle transfection system (Sigma-Aldrich). A scramble sequence was used as a control. Cells transfected with a 40 or 50 nM concentration of each siRNA were subjected to electrophysiological recording and insulin secretion assays 48 h after transfection. Protein extracts of transfected cells were obtained to confirm RIM1 silencing by Western blotting.
Data Analyses-Statistical analyses were carried out using the SigmaPlot 11 software (Systat Software Inc.). The significance of observed differences was evaluated by Student's unpaired t test. A probability less that 5% was considered to be significant. All experimental values are given as means Ϯ S.E. Curve fitting was performed as reported previously (29).

RESULTS
First, to investigate the coupling of the Rab3-interacting molecule 1 (RIM1) to L-type Ca V channels and the functional consequences of this interaction, we examined the effects of RIM1 on whole cell currents through recombinant L-channels using the HEK-293 cell line, a heterologous expression system that does not express endogenous Ca V channels (25, 30) (supplemental Fig. 3). Hence, Ca V 1.2␣ 1 or Ca V 1.3␣ 1 channels together with the Ca V ␤ (␤ 2 or ␤ 3 ) and the Ca V ␣ 2 ␦-1 auxiliary subunits were co-transfected in HEK-293 cells in the absence or presence of RIM1 48 h before electrophysiological recordings. Fig. 1 shows the average current density-voltage relationships (peak current amplitude normalized by C m ) in response to 2-s membrane depolarizations from a holding potential (V h ) of Ϫ80 mV and with 10-mV incremental steps from Ϫ50 to ϩ60 mV. As observed, no apparent differences in current densities were detected in the absence or presence of RIM1 (Table 1). It is worth noting that RIM1 expression in HEK-293 cells was confirmed after cDNA transfection by Western blot analyses ( Fig.  1, C and F). Although RIM1 had no effect on the density and voltage dependence of the expressed currents, its co-expression profoundly affected current inactivation kinetics, as we shall discuss below. Fig. 2A compares representative whole cell current traces obtained in control conditions and in the presence of RIM1. As shown, HEK-293 cells co-transfected with Ca V 1.2␣ 1 , Ca V ␣ 2 ␦-1, and Ca V ␤ 2 produced robust macroscopic Ba 2ϩ current (I Ba ) through recombinant Ca V channels. Likewise, as expected from previous results, peak current amplitude was increased, and inactivation kinetics fastened by expression of the Ca V ␤ 3 auxiliary subunit ( Fig. 2A, bottom). Notably, I Ba through Ca V 1.2 channels decayed with a significantly slower time course in RIM1-expressing cells than in the controls. The L-channel inactivation rate was quantitatively compared between RIM1-expressing cells and control cells by fitting L-current traces with single exponential functions. The time constant for Ca V 1.2 currents was ϳ1.5-2-fold slower in the presence of RIM1 (Fig. 2B, top). The parameters from the best fits are given in Table 1. As a consequence, the percentage of current inactivated at the end of the pulse with respect to the peak current amplitude (I remaining) was significantly larger in the RIM1-transfected cells when compared with cells not expressing RIM1 (Fig. 2B, bottom). These effects of RIM1 were also observed in cells expressing Ca V 1.3 channels (Fig. 2, C and D, and Table 1).

RIM1 Alters L-type Ca V Channel Inactivation and Increases Charge Transfer into Cells-
To investigate the functional significance of the L-type Ca V channels-RIM1 coupling in more detail, we next calculated the amount of charge mobilized (i.e. the number of ions that passed through the channels) during depolarization (Fig. 3). By integrating the whole cell current transients elicited by depolarizing commands to 0 from a V h of Ϫ80 mV, a net entry of ϳ354 Ϯ 42 picocoulombs of charge was estimated in control cells expressing Ca V 1.2 channels. In the RIM1-expressing cells, digital integration of the currents through Ca V 1.2␣ 1 /Ca V ␣ 2 ␦-1/ Ca V ␤ 2 channels during the imposed depolarization yielded a value of ϳ603 Ϯ 101 picocoulombs, corresponding to a 1.7-fold increase in charge entry (Fig. 3A). In a similar manner, the average charge transfer was significantly increased by RIM1 from a value of ϳ458 Ϯ 107 to 791 Ϯ 95 picocoulombs in cells expressing Ca V 1.3␣ 1 /Ca V ␣ 2 ␦-1/Ca V ␤ 2 channels (Fig. 3B). When Ca V ␤ 3 was co-transfected, qualitatively similar results were obtained for both Ca V 1.2-and Ca V 1.3-containing channels after RIM1 expression (Fig. 3, A and B, and Table 1).
To study whether RIM1 had an effect on L-type channel availability, the voltage dependence of inactivation was evaluated using 10-s prepulse depolarizations from Ϫ90 to ϩ40 mV, preceding a 140-ms test potential to 0 mV or Ϫ30 mV (for the Ca V 1.2␣ 1 -and Ca V 1.3␣ 1 -containing channels, respectively).
Normalized current amplitudes were compiled for 4 -7 cells and plotted against the prepulse voltage, and mean data points were well described by a sigmoid equation. Fits to the mean inactivation data points and the parameters from the best fits are given in Fig. 4 and Table 1. Interestingly, co-expression with RIM1 shifted V1 ⁄ 2 to the right about 10 mV for Ca V 1.3/Ca V ␣ 2 ␦-1/Ca V ␤ 2 channels. In contrast, the V1 ⁄ 2 value was not significantly altered by RIM1 expression in Ca V 1.3/Ca V ␣ 2 ␦-1/Ca V ␤ 3 and Ca V 1.2␣ 1 /Ca V ␣ 2 ␦-1/Ca V ␤ channels (Table 1). These results suggest that RIM1 affects mainly the rate of channel inactivation and has a minor impact on the inactivation of the channels at steady state. Therefore, we speculate that RIM1 has an important role in the transition rates between inactivation states while having less impact in the availability of the chan-

TABLE 1 Effects of RIM1 on Ca v 1.2 and Ca v 1.3-mediated macroscopic currents
The indicated Ca v channel subunits were co-expressed with RIM1 in HEK-293 cells, and the biophysical properties were determined using Ba 2ϩ (5 or 10 mM) as charge carrier. inact and V1 ⁄ 2 were obtained by fitting the data as described previously (29). Q was calculated by integration of the whole cell Ca v channel-mediated currents. pF, picofarads; pC, picocoulombs.  (16) established that RIM1 associates with different neuronal voltage-gated Ca 2ϩ channels (Ca V 2.1 and Ca V 2.2) via interactions with the Ca V ␤ subunit. In order to determine whether this mechanism is also valid for recombinant L-type channels (Ca V 1.2 and Ca V 1.3), a series of experiments using Ca V ␤ 2 and Ca V ␤ 3 antibodies were performed to study whether RIM1 could be immunoprecipitated in samples from transfected HEK-293 cells (Fig. 5). In these experiments, negative controls were obtained with anti-Sp1 antibodies. As shown in Fig. 5, A and B, immunoprecipitation with Ca V ␤ 2 antibodies results in a band between the size markers of 150 and 250 kDa (expected size 178 kDa) in both Ca V 1.2 and Ca V 1.3 channels. Similarly, using Ca V ␤ 3 antibodies, RIM1 could be immunoprecipitated in HEK-293 cells expressing Ca V 1.2 and Ca V 1.3 channels (Fig. 5, C  and D). In all cases, the negative control (IgG 0 ) did not co-precipitate with RIM1. These data provide direct evidence that expression of Ca V 1.2␣1 or Ca V 1.3␣1 does not hinder the formation of a complex between the Ca V ␤ subunits and RIM1.

Channel composition
In order to confirm that the functional effects of RIM1 on L-type channels occur through its interaction with the Ca V ␤ subunit, we characterized its effects on whole cell Ba 2ϩ currents through recombinant Ca V 1.2 and Ca V 1.3 channels expressed in HEK-293 cells in the absence of Ca V ␤. Fig. 6 shows that, as expected, RIM1 expression did not result in appreciable changes in current density recorded by applying depolarizing pulses to 0 and Ϫ30 mV for Ca V 1.2␣ 1 -and Ca V 1.3␣ 1 -containing channels, respectively ( Fig. 6A and Table 1). More importantly, co-expression of RIM1 did not affect inactivation kinetics of the L-type currents arising from recombinant Ca V 1.2 and Ca V 1.3 channels in the absence of the Ca V ␤ subunit (Fig. 6, B and C, and Table 1). In these experiments, RIM1 expression was confirmed after cDNA transfection by Western blot analysis (Fig. 6D). Taken together, these results confirm the role of the Ca V ␤ auxiliary subunit in structurally and functionally coupling RIM1 to L-type Ca V channel complexes.
RIM1 Regulates Native L-type Ca V Channel Activity-Once we established the regulation of recombinant L-type Ca V channels by RIM1, we checked whether native channels were also regulated by RIM1 using the rat insulinoma RIN-m5F cell line as a model. To this end, RT-PCR and immunoblotting were first used as methods to analyze the expression of RIM1 and different subunits that compose the L-type channel complex. By using antibodies, conspicuous signals were consistently  observed in the RIN-m5F cells for Ca V 1.2␣ 1 , Ca V 1.3␣ 1 , Ca V ␤ 2 , and Ca V ␤ 3 subunits in Western blot experiments (Fig. 7A). Likewise, using specific primers, an expected cDNA fragment of 512 bp for RIM1 was amplified from RIN-m5F cells and from mouse brain used as control tissue (Fig. 7B). The expression of RIM1 was confirmed by Western blot analyses (Fig. 7C). The possible interaction between RIM1 and the L-type channels was then studied by both co-immunoprecipitation and knockdown of RIM1 using specific siRNAs.
First, to evaluate the siRNA silencing efficiency, RIM1 expression was determined by semiquantitative analysis of Western blots. Fig. 7D shows that the RIM1 expression level in RIN-m5F cells transfected with the RIM1 siRNA was significantly lower than those with scrambled siRNA control, whereas the actin expression levels remained essentially unchanged. Semiquantitative analysis indicates that siRNA decreased the levels of RIM1 to ϳ20% of the levels observed in cells transfected with the control siRNA (Fig. 7E). Altogether, these data indicate that RIN-m5F cells express RIM1 and that RIM1 levels can be successfully down-regulated by RNA silencing.
In agreement with the results obtained with the heterologous expression system, we found that silencing endogenous RIM1 expression with siRNAs increases the extent of inactivation of whole cell native currents. Examples of normalized I Ba traces elicited in RIN-m5F cells by 2-s depolarizing pulses from a V h of Ϫ80 to 0 mV in the control condition and after knocking down RIM1 are shown in Fig. 7F. As noted, comparison of normalized superimposed records shows that currents from RIM1 knockdown cells inactivate more rapidly than those from control cells. The extent of inactivation defined by the I remaining decreased ϳ25% in the RIM1 knockdown cells (Fig. 7G).
RIM1 Associates with L-type Channels in RIN-m5F Cells and Regulates Insulin Secretion-Using antibodies against Ca V ␤ 2 and Ca V ␤ 3 , RIM1 could be co-immunoprecipitated in samples from RIN-m5F cells. Negative controls were obtained with Sp1 antibodies. Hence, probing of RIM1 immunoprecipitated with anti-Ca V ␤ 2 (Fig. 8A) and Ca V ␤ 3 (Fig. 8B) antibodies revealed a band of ϳ180 kDa in the co-immunoprecipitated sample lane but not in the IgG 0 control lane, indicative of the specificity of the immunoprecipitation.
Last, we investigated the physiological relevance of the RIM1 coupling to the L-type Ca V channels by assessing insulin release from RIN-m5F cells in control conditions and after transfection with RIM1 siRNA. Basal insulin release to the culture medium remained unchanged in non-stimulated cells (Fig. 8C). In contrast, insulin release triggered by Ca 2ϩ influx in response to high K ϩ -induced membrane depolarization (extracellular K ϩ concentration was increased from 5 to 40 mM for 30 min) was significantly decreased (ϳ25%) in RIM1 knockdown cultures (Fig. 8C). Likewise, insulin release triggered by depolarization with high K ϩ was also significantly decreased by siRNAs specific to the Ca V ␤ 2 and Ca V ␤ 3 subunits (Fig. 8, D and E). Altogether, these data demonstrate the importance of L-type channel regulation by RIM1 for fine tuning insulin release.  Table 1).

DISCUSSION
The present study reveals that the coupling between RIM1 and the Ca V ␤ auxiliary subunits is also operational in L-type Ca V channels. This interaction decelerates L-type current inactivation, producing a sustained depolarization-induced Ca 2ϩ influx in insulin-secreting cells that favors hormone release. RIM1 is a putative effector of Rab3 that associates selectively with the active form of the GTPase (31). RIM1 contains an N-terminal domain that interacts with Rab3 and two C2 domains located at the C terminus. Although mainly expressed in the brain, RIM1 is also expressed in pancreatic ␤-cells, where it is involved in insulin release (28). In line with this, we found that RIM1 expression significantly increased charge transfer into HEK-293 cells by slowing down the inactivation kinetics of L-type Ca V 1.2 and Ca V 1.3 channels. Likewise, our results show that siRNA-mediated RIM1 knockdown in RIN-m5F cells significantly affected L-type current inactivation and reduced insulin release triggered by depolarization with high K ϩ . Together, these results suggest that RIM1 might play a role in docking the Rab3-bound vesicles near Ca V channels, functionally coupling channel activity to the exocytotic machinery in insulin-secreting cells.
Likewise, by searching for possible binding partners of the RIM proteins, initial studies found that the C2A domain mediated the interaction of RIM1 with some synaptic proteins as well as with the pore-forming subunit of neuronal Ca V 1.2 channel (13). It has also been reported that the mouse RIM1 argin-ine-to-histidine substitution (R655H), which corresponds to the human autosomal dominant cone-rod dystrophy mutation, modifies RIM1 function in regulating L-type Ca V 1.4 channels of the retina (32) and that the II-III loop of the Ca V 1.2␣1 subunit binds directly to the C2A domain of RIM2 in the INS-1 cells (14) for lipid raft targeting of the channels (15). Last, RIM proteins expressed in cochlear inner hair cells seem to be capable of modulating L-type Ca V 1.3 channel function (33). Although this identifies RIM proteins as scaffolding proteins with a role in maintaining a high Ca V channel density at active zones, they have not yet attained general acceptance as critical tethering molecules. Wong and Stanley (19) found that co-immunostaining with RIM and anti-Ca V 2.2 antibodies neither colocalized nor co-varied at the transmitter release face and that the two proteins did not co-immunoprecipitate.
It should be noted, however, that parallel studies by Han et al. (34) and Kaeser et al. (35) have reported more recently an important role for RIM proteins in localizing Ca V channels to active zones. Based on protein/protein interaction studies, generation of conditional KO mice, electrophysiological recordings, Ca 2ϩ imaging, and quantitative immunofluorescence, these authors propose that the PDZ domains of RIM proteins stoichiometrically interact with Ca V 2 channels in vitro and that RIM proteins, by interacting directly through their PDZ domains with the Ca V ␣ 1 subunits, are essential for tethering Ca V channels to presynaptic terminals in vivo. This interaction   MAY 6, 2011 • VOLUME 286 • NUMBER 18 speeds the rate of transmitter release by increasing the intrinsic Ca 2ϩ sensitivity of release as well as by contributing to the tight co-localization of readily releasable vesicles with neuronal Ca V channels (34,35).

RIM1 and L-type Ca 2؉ Channel Interaction
Similarly, recent studies by Mori and colleagues (16,20) have documented a functional coupling of RIM1 with neuronal Ca V channels mediated by its physical association with the Ca V ␤ auxiliary subunit via the C2B domain at the C terminus region. This interaction significantly suppressed voltage-dependent channel inactivation (16,20,36), enhancing membrane docking of vesicles and potentiating neurotransmitter release (16,20). Likewise, different RIMs have been shown to physically associ-  . RIM1 interacts with native L-type channels through the Ca V ␤ subunit and contributes to determine insulin secretion. A and B, proteins from RIN-m5F cells were immunoprecipitated with anti-Ca V ␤ 2 , anti-Ca V ␤ 3 , or control (IgG 0 ) antibodies and subjected to Western blot analysis with anti-RIM antibody. The ϳ180 kDa RIM1 band is visualized in the immunoprecipitation (IP) lane. In both cases, control experiments with the irrelevant antibody as a substitute for the anti-Ca V ␤ antibodies failed to co-immunoprecipitate RIM1. Immunoprecipitation data were collected from the same experiment, and the images are shown separately because they were acquired with different time exposures. C-E, basal and high K ϩ -induced insulin secretion from RIN-m5F cells after transfection with RIM1-and Ca V ␤-targeted siRNA compared with control (non-transfected) cells. RIN-m5F cells were transfected with RIM1 siRNA (open bars) and 48 h later incubated with KRB buffer containing 5 mM (low K ϩ ) or 40 mM (high K ϩ ) KCl. Insulin content in the supernatants was measured by ELISA as described under "Experimental Procedures." The mean Ϯ S.E. of three independent experiments is displayed. ate with Ca V ␤ decelerating current inactivation, increasing depolarization-induced Ca 2ϩ entry and enhancing neurotransmitter release (20). Interestingly, the results of our co-immunoprecipitation experiments have identified a RIM1-Ca V channel complex formed by direct interaction of the Ca V ␤ 2 and Ca V ␤ 3 subunits with RIM1 heterologously expressed in HEK-293 cells. The identification of native RIM1-Ca V channel complexes in RIN-m5F cells and the effect of the RIM1 knockdown on insulin release support a physiological role for the RIM1-Ca V ␤ subunit interaction. In this context, it is well known that the Ca V ␤ subunit interacts with the pore-forming Ca V ␣ 1 subunit from the cytoplasmic side to enhance functional channel trafficking to the plasma membrane and to modify multiple kinetic properties (34,37). In particular, functional studies have shown that the Ca V ␤ subunit is a key determinant in Ca V channel inactivation (38). For many types of high voltage-activated Ca V channels, co-expression with Ca V ␤ tends to increase the rate of inactivation (30,39,40). Therefore, the possibility exists that RIM1 may act on the Ca V ␤ subunits to suppress the regulatory function of this auxiliary subunit on L-type Ca V inactivation. As a consequence, association with Ca V ␤ may enable RIM1 to play an important physiological role in hormone release. Decreased L-channel inactivation by RIM1 interaction implies that a substantially larger Ca 2ϩ current would be maintained during depolarization facilitating insulin release.