Deletion of the synaptic protein interaction site of the N-type (Ca_V2.2) calcium channel inhibits secretion in mouse pheochromocytoma cells

Abstract

Presynaptic N-type Ca²⁺ channels (Ca_v2.2, α_1B) are thought to bind to SNARE (SNAP-25 receptor) complex proteins through a synaptic protein interaction (synprint) site on the intracellular loop between domains II and III of the α_1B subunit. Whether binding of syntaxin to the N-type Ca²⁺ channels is required for coupling Ca²⁺ ion influx to rapid exocytosis has been the subject of considerable investigation. In this study, we deleted the synprint site from a recombinant α_1B Ca²⁺ channel subunit and transiently transfected either the wild-type α_1B or the synprint deletion mutant into mouse pheochromocytoma (MPC) cell line 9/3L, a cell line that has the machinery required for rapid stimulated exocytosis but lacks endogenous voltage-dependent Ca²⁺ channels. Secretion was elicited by activation of exogenously transfected Ca²⁺ channel subunits. The current-voltage relationship was similar for the wild-type and mutant α_1B-containing Ca²⁺ channels. Although total Ca²⁺ entry was slightly larger for the synprint deletion channel, compared with the wild-type channel, when Ca²⁺ entry was normalized to cell size and limited to cells with similar Ca²⁺ entry (≈150 × 10⁶ Ca²⁺ ions/pF cell size), total secretion and the rate of secretion, determined by capacitance measurements, were significantly reduced in cells expressing the synprint deletion mutant channels, compared with wild-type channels. Furthermore, the amount of endocytosis was significantly reduced in cells with the α_1B synprint deletion mutant, compared with the wild-type subunit. These results suggest that the synprint site is necessary for efficient coupling of Ca²⁺ influx through α_1B-containing Ca²⁺ channels to exocytosis.

Release of neurotransmitter from presynaptic vesicles is triggered by Ca²⁺ influx through voltage-dependent Ca²⁺ channels (1-3). Presynaptic vesicles are docked at release sites by binding of the vesicular protein synaptobrevin (also called VAMP) to two plasma membrane proteins, syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) (4-6). Collectively, these three proteins form the SNAP-25 receptor (SNARE) complex, which is an essential component of the exocytotic machinery (7). In addition to participating in SNARE complex formation, syntaxin and SNAP-25 bind to and modulate voltage-dependent Ca²⁺ channels (8-10).

The association of Ca²⁺ channels with proteins of the SNARE complex was first demonstrated by coimmunoprecipitation of the N-type Ca²⁺ channel subunits with syntaxin and synaptotagmin (11-13). Like other voltage-dependent Ca²⁺ channels, the N-type channel is composed of at least three subunits: α₁, the pore-forming subunit, which is distinctive for each Ca²⁺ channel subtype (α_1B for the N-type Ca²⁺ channel), and two auxiliary subunits, α₂δ and β, which alter the voltage-dependent properties of the channels as well as surface expression of the channel complex (14, 15). Subsequent in vitro-binding assays with recombinant fusion proteins identified the minimal site of interaction between the N-type Ca²⁺ channel and syntaxin as amino acids 773-859 of the intracellular loop between transmembrane domains II and III of the α_1B subunit (16); this region was named the synaptic protein interaction (synprint) site (17). Subsequently, a longer region of the intracellular loop between domains II and III (amino acids 718-963) of the Ca²⁺ channel α_1B subunit was shown to bind syntaxin with higher affinity than the originally defined synprint region (16, 17). A similar synprint site was identified in the α_1A subunit of the P/Q-type Ca²⁺ channel (17). Short and long synprint peptides bind to syntaxin and SNAP-25 in a biphasic, Ca²⁺-dependent manner with maximal binding at 20 μM free Ca²⁺ and reduced affinity at higher Ca²⁺ concentrations (18). Functional effects on neurotransmitter release also have been attributed to the synprint site. Injection of peptides corresponding to the synprint region into presynaptic neurons resulted in a 24-42% reduction in synaptic transmission in rat sympathetic neurons (19) or ≈25% reduction in Xenopus spinal neurons (20). From these combined data, it was hypothesized that the synprint site docks vesicles near Ca²⁺ channels such that Ca²⁺ influx is efficiently coupled to secretion (9, 10). Consistent with this hypothesis, two recent papers by Mochida et al. (21, 22) suggested that the synprint site is necessary for both the localization of Ca²⁺ channels at sites of neurotransmitter release and for functional synaptic transmission.

To further examine the importance of the synprint site for exocytosis, we deleted the synprint site from the α_1B subunit (Ca_v2.2) and expressed this mutant as well as wild-type α_1B in mouse pheochromocytoma (MPC) 9/3L cells. We have shown in a previous study (23) that MPC 9/3L cells contain vesicles and many of the proteins involved in vesicle fusion, including SNAP-25 and syntaxin 1A. MPC 9/3L cells express little or no endogenous Ca²⁺ current and so exhibit no exocytosis when stimulated by trains of depolarizations. In contrast, when MPC 9/3L cells are transfected with exogenous Ca²⁺ channel subunits, they rapidly release vesicles in a Ca²⁺-dependent manner similar to PC12 cells and adrenal chromaffin cells (23). To assess the functional consequences of disrupting the synaptic protein interaction site between the N-type Ca²⁺ channel and the SNARE complex, we transiently transfected MPC 9/3L cells with both wild-type and mutant α_1B Ca²⁺ channel subunits that had the short synprint site (amino acids 772-856) deleted. No currents were detected in cells transfected with the α_1B long synprint site deletion mutants (amino acids 718-963). An earlier study by Mochida et al. (21) showed that P/Q-type Ca²⁺ channels with a similar long synprint deletion also did not express functional Ca²⁺ channels. For both wild-type and mutant Ca²⁺ channel expression, cells were chosen that had a similar Ca²⁺-influx. Measurements of release showed that the cells transfected with the short synprint deletion mutant had significantly reduced secretion, compared with wild-type cells. Furthermore, both the maximal rate of release and the amount of endocytosis 40 s after stimulation were significantly reduced in cells expressing the short synprint deletion mutant, compared with wild-type cells. Our results suggest that the synprint site of the N-type Ca²⁺ channel is necessary for efficient coupling of the channel to the release machinery.

Methods

Ca²⁺ Channel cDNAs. Cloning of the bovine chromaffin cell α_1B (GenBank accession no. AF173882) and β_2a (GenBank accession no. AF174417) subunits of the N-type Ca²⁺ channel has been described (24). Regions of the α_1B cDNA coding for portions of the synprint site (see Fig. 1) were deleted by using the “splicing-by-overlap extension” method (25). The cDNA flanking each deletion was sequenced on both strands to assure that the desired deletions had been accomplished without introducing other unintended mutations. A rat α₂δ Ca²⁺ channel subunit cDNA (GenBank accession no. M86621) was a kind gift from T. Snutch (University of British Columbia, Vancouver). All of these Ca²⁺ channel subunits were subcloned into pcDNA3.1(+) (Invitrogen) for expression in the MPC 9/3L cells.

Fig. 1.

Domain structure of the α_1B subunit of the N-type (Ca_v2.2) Ca²⁺ channel. (a) The II-III loop between domains II and III contains the synprint site. (b) Two mutant α_1B Ca²⁺ channel subunits were constructed by deleting portions of the intracellular loop between domains II and III (schematized as the thicker, darkened areas). The short synprint deletion (α_1B Δ772-856) and long synprint deletion (α_1B Δ717-954) in the bovine sequence correspond to the originally described short and extended synprint sites, respectively, for the rat α_1B Ca²⁺ channel subunit (16, 17).

Cell Culture and Transfection. MPC cell line 9/3L was grown in culture medium that consisted of RPMI medium 1640, 10% heat-inactivated horse serum, 5% FBS (HyClone), 2 mM glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen) in a humidified 5% CO₂ incubator at 37°C. The day before transfection, the cells were replated to collagen-coated coverslips. Cells were transfected with Qiagen-purified plasmids in a 5:5:5:1 μg ratio of the α₁:β_2a:α₂δ:enhanced GFP plasmids by using Lipofectamine 2000 (Invitrogen). Recordings were done 48-72 h after transfection.

Electrophysiology. A coverslip of cells was set into a recording chamber and perfused with an external Na⁺ solution that contained 135 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 5 mM CaCl₂, 12 mM Hepes, and 10 mM glucose (pH 7.3; ≈295 mOsm). A single transfected cell was selected based on its fluorescence due to enhanced GFP expression and voltage-clamped in the whole-cell configuration (26) with an Axopatch-1C amplifier (Axon Instruments, Union City, CA) modified for capacitance measurements. Gigaohm seals were obtained with sylgard-coated electrodes filled with an internal solution that contained 120 mM CsAsp, 5 mM MgCl₂, 0.1 mM EGTA, 40 mM Hepes, 2 mM ATP, and 0.3 mM GTP (pH 7.3; ≈310 mOsm). Ca²⁺ currents were isolated with an Na⁺-free external solution that contained 140 mM tetraethylammonium-Cl, 0.0001 mM tetrodotoxin, 5 mM CaCl₂, 10 mM Hepes, and 10 mM glucose (pH 7.3; ≈300 mOsm).

Capacitance measurements were made with the phase-tracking technique in which a 60-mV peak-to-peak sine wave was superimposed on a holding potential of -80 mV (27, 28). Details of the technique have been described (29). The data were collected at a 500-μs sampling rate and filtered at 2 kHz. All current records were compensated for series resistance and whole-cell capacitance. Single-pulse current data were leak-subtracted by an average of 10 hyperpolarizing sweeps. Experiments were carried out at room temperature (22-24°C).

Stimulation Protocol. Five minutes after the whole-cell configuration was obtained, each cell was stimulated with a train of five-step depolarizations. The membrane was depolarized to 20 mV for 200 ms with a 50-ms interpulse duration. The current-voltage (I-V) relationships were obtained by applying 50-ms step depolarizations to various test potentials every 15 s. Each current was leak- and capacitance-subtracted.

Data Analysis. Statistical analysis of the data are expressed as mean ± SEM, and an independent Student t test (origin, Microcal, Amherst, MA) was performed to test statistical significance (P < 0.05). For I-V curve calculations, the peak current from each depolarization was recorded and normalized to the whole-cell capacitance, and cells were pooled to calculate the mean and SEM. The maximal rate of secretion was calculated as the maximal release that occurred for a single depolarization divided by the duration of the depolarization. Ca²⁺ current inactivation was fit with a single exponential function by using origin.

Results

The Synprint Deletion Mutant of the α_1B Ca²⁺ Channel Subunit Produces Ca²⁺ Currents That Are Similar to Wild-Type α_1B Ca²⁺ Currents. N-type Ca²⁺ channels contain at least three subunits (α_1B, β, and α₂δ). The pore-forming α_1B subunit is composed of four homologous domains, each of which contains six membrane-spanning domains (15, 30) (Fig. 1a). Between domains II and III is a large cytoplasmic loop that is thought to interact with SNARE proteins (15). Previously, two overlapping synprint sites have been defined for the Ca²⁺ channel α_1B subunit, a short region and a long region of the II-III intracellular loop (16, 17). In our current study, each region was independently deleted from the bovine chromaffin cell α_1B subunit. One synprint deletion mutant was made that removed the short synprint site of bovine α_1B (Δ772-856) that correlated to the originally defined synprint site of rat α_1B (773-859; Fig. 1b) (16), whereas a second bovine α_1B mutant was constructed that deleted a longer synprint site (bovine α_1B Δ717-954) that correlated to the long synprint site of rat α_1B (718-963; Fig. 1b) (17). Each of these mutant α_1B subunits was transiently transfected into MPC 9/3L cells along with the auxiliary Ca²⁺ channel subunits, β_2a and α₂δ, and an enhanced GFP vector to allow visual selection of cells that had been successfully transfected. The external Na⁺ solution was exchanged with a Na⁺-free solution (5 mM Ca²⁺) designed to isolate the Ca²⁺ currents (I_Ca).

As we have reported (23), nontransfected MPC 9/3L cells express little if any endogenous I_Ca. In our earlier study, 94% of cells tested had an endogenous Na⁺ current (I_Na) that was blocked by bath application of tetrodotoxin, and 18% of cells exhibited a small endogenous I_Ca that averaged 70 ± 8 pA (n = 23). After transfection with either the wild-type α_1B Ca²⁺ channel subunit or the short synprint deletion mutant (and β_2A and α₂δ subunits), ≈75% of the GFP-positive cells expressed exogenous N-type Ca²⁺ currents (Fig. 2 a and b). No difference in current kinetics was observed for the two α_1B subunits. The time to peak was 6.42 ± 0.39 ms for wild-type channels and 5.03 ± 0.51 ms for synprint deletion channels, whereas the time constants for the decay rate were 184.4 ± 28.3 ms for wild-type channels and 199.0 ± 45.2 ms for synprint deletion channels. These currents were sensitive to the specific N-type Ca²⁺ channel blocker ω-CgTx GVIA (data not shown, but see ref. 23). Step depolarizations were applied to different test potentials from a holding potential of -80 mV. The currents elicited were normalized by cell size (whole-cell capacitance). Normalized current-voltage plots for wild-type and synprint deletion mutant channels are shown in Fig. 2 c and d.

Fig. 2.

Similar N-type Ca²⁺ currents were observed in cells expressing either wild-type or short synprint deletion α_1B subunits. Cells were transiently transfected with the α_1B (a) or α_1B short synprint deletion mutant (b) subunits (α_1B Δ772-856) in addition to β_2a and α₂δ subunits. I_Ca was recorded from individual cells in response to a voltage step stimulation to 20 mV and duration of 200 ms. The current vs. voltage plots show the peak current normalized by the whole-cell capacitance (pF) for α_1B (c) and α_1B short synprint deletion (d) Ca²⁺ channel subunits. Each plot shows data averaged from seven cells. The data are expressed as mean ± SEM. Please note that current decay was not completely consistent between cells. In some cells, two exponentials were required to fit the data, whereas in others a single exponential fit well.

Cells transfected with either the wild-type α_1B subunit or the short synprint deletion α_1B subunit (and accessory subunits) exhibited large Ca²⁺ currents that were similar in amplitude to each other. Average peak Ca²⁺ currents were measured from the first of five stimulations and averaged 1984 ± 319 pA (n = 44) for the wild-type α_1B subunit and 1812 ± 359 pA (n = 16; P = 0.76, compared with wild-type) for the short synprint deletion mutant subunit. These peak amplitudes were >25-fold greater than endogenous peak Ca²⁺ currents measured in untransfected cells. Cells transfected with the long synprint deletion α_1B subunit exhibited little if any Ca²⁺ current, which suggests that the long deletion mutant subunit did not form functional Ca²⁺ channels [see also Mochida et al. (21)].

Fig. 3 compares the average total Ca²⁺ influx in response to five-step depolarizations to +20 mV (200-ms duration) from cells transfected with either wild-type α_1B subunits or the short synprint deletion mutant. Fig. 3a plots representative current traces from two cells expressing either wild-type (Upper) or short synprint deletion mutant channels (Lower). Fig. 3b shows that, on average, the cells expressing wild-type α_1B subunits had a Ca²⁺ influx of 858 ± 100 × 10⁶ ions (n = 44), whereas cells expressing the short synprint deletion mutant subunit were not significantly different, with a Ca²⁺ influx of 1,067 ± 130 × 10⁶ ions (n = 16).

Fig. 3.

Similar Ca²⁺ influx was observed in cells expressing either wild-type or short synprint deletion α_1B subunits. (a) Current records from two cells expressing either wild-type (Upper) or short synprint deletion mutant channels (Lower). Five depolarizations were applied, separated by 50 ms. The numbers on top of the traces correspond to the order in which the currents were obtained. (b) The total number of Ca²⁺ ions to enter each cell in response to stimulation is shown for cells that were transfected with either wild-type α_1B or the short synprint deletion mutant subunit. (c) Ca²⁺ entry was normalized by the whole-cell capacitance (pF) for currents recorded in cells transfected with either the wild-type α_1B or the short synprint deletion mutant. A reduced pool of cells was selected from the total number of cells shown in b that had similar normalized Ca²⁺ influx to ensure that Ca²⁺ entry was the same between the two sets of cells (P = 0.56).

Neurotransmitter release is thought to be a nonlinear function of Ca²⁺-influx. Small differences in Ca²⁺-influx produce large changes in secretion. To compare secretion between MPC 9/3L cells expressing wild-type and short synprint deletion mutant α_1B subunits, the Ca²⁺ influx was normalized by cell size and limited to cells with similar Ca²⁺ influx per unit area (measured as pF; Fig. 3c). On average, normalized Ca²⁺ influx for the wild-type α_1B subunit was 148.6 ± 9.4 pA/pF (n = 19), and the short synprint deletion α_1B subunit was not significantly different (P = 0.56) at 158.9 ± 15.4 pA/pF (n = 9). This group of cells was subsequently used to determine the amount of secretion supported by each of the α_1B Ca²⁺ channel subunits.

Secretion Is Significantly Reduced in Cells That Express the Short Synprint Mutant α_1B Ca²⁺ Channel. To determine whether the synprint deletion mutant could support vesicle fusion, membrane capacitance was measured in response to stimulation with a train of five depolarizations to +20 mV (current was maximal at +20 mV, see Fig. 2 c and d) for 200 ms with a 50-ms interpulse duration. Representative capacitance traces for MPC 9/3L cells expressing either wild-type α_1B or the α_1B short synprint deletion mutant are shown in Fig. 4 a and b, respectively. The gaps in the capacitance trace represent the times the cell was stimulated with five-step depolarizations. In the capacitance traces, the dashed lines indicate the peak capacitance changes. It is interesting to note that exocytosis could be significant for all five depolarizations, even though the later Ca²⁺ currents were small (see Fig. 3a). This result may be due to residual Ca²⁺ summating with a relatively small Ca²⁺-influx, or, alternatively, intracellular Ca²⁺ stores may help support secretion. The bar graphs in Fig. 4 c and d plot averaged data for cells transfected with either the wild-type or the synprint deletion α_1B subunits. Peak membrane capacitance and the maximal rate of release for each cell was normalized by the cell size (pF), and the normalized values were then averaged. Secretion was significantly reduced in the synprint deletion mutant, compared with the wild-type α_1B subunit. The average peak change in membrane capacitance was 77 ± 10 fF/pF (n = 19) for the wild-type α_1B subunit and 43 ± 7 fF/pF (n = 9; P = 0.04, compared with wild type) for the α_1B synprint deletion mutant. Similarly, the rate of release was reduced in the α_1B synprint deletion mutant, with an average of 35 ± 6 fF/s per pF (n = 19), compared with wild-type α_1B, which averaged 76 ± 12 fF/s per pF (n = 9; P = 0.04). To ensure minimal interference by endocytosis in these measurements, Fig. 4e plots normalized capacitance changes produced by the first voltage depolarization. The average change in membrane capacitance was 42.5 ± 6.9 fF/pF (n = 19) for the wild-type α_1B subunit and 19.3 ± 3.4 fF/pF (n = 9; P = 0.04, compared with wild type) for the α_1B synprint deletion mutant. Therefore, deletion of the synprint site functionally alters release. Furthermore, this reduced secretion was not caused by a difference in Ca²⁺ influx during stimulation.

Fig. 4.

Cells transfected with the synprint deletion mutant exhibited reduced secretion, compared with cells expressing wild-type α_1B subunits. Representative capacitance traces are plotted as a function of time for individual cells transfected with either wild-type α_1B (a) or the short synprint deletion mutant (b). Each cell was stimulated with trains of five depolarizing voltage steps to +20 mV (duration 200 ms, interpulse 50 ms). The traces are shown on an expanded time scale (5 s). The bar graphs depict the averaged data for the cells transfected with either wild-type α_1B (n = 19) or the short synprint deletion mutant (n = 9). The graphs plot total amount of secretion (fF) normalized by cell size (pF) in response to stimulation (c), the maximal rate of secretion (fF/s) normalized by cell size (pF) (d), and secretion produced by the first depolarization (e) (indicated by the curly brackets in a and b).

Secretion of catecholamines from cells closely related to MPC cells (e.g., chromaffin cells and PC12 cells) can be measured by amperometry. To date, we have had no successful amperometric recordings from the MPC cells, possibly because these cells do not store sufficient quantities of oxidizable neurotransmitter. We attempted to load the cells with false neurotransmitters, a commonly used technique (31). However, even after loading, there were no amperometric events.

Membrane Retrieval Is Significantly Reduced in Cells That Express the Synprint Deletion Mutant α_1B. Fig. 5 a and b shows traces from the same cells as those shown in Fig. 4 a and b, except plotted as ≈80 s of capacitance data. Membrane retrieval was defined as the percentage recovery of the peak capacitance change (indicated by the dashed line in the figure). Forty seconds after stimulation, ≈40% of the membrane had been retrieved in the cell expressing the wild-type α_1B (Fig. 5a), but only ≈25% of the membrane had been retrieved in the cell expressing the α_1B synprint deletion mutant (Fig. 5b). The bar graph in Fig. 5c plots the average membrane retrieval values for both wild-type α_1B and the synprint deletion mutant. On average, the cells expressing wild-type α_1B retrieved 62 ± 6% (n = 12) of their membrane after 40 s compared with 23 ± 5% (n = 6; P = 0.001) for the synprint deletion mutant. These data indicate not only that the synprint deletion mutant channel is less effective at coupling the channel to neurotransmitter release, but also that membrane retrieval was less efficient in cells expressing the mutant channel.

Fig. 5.

Endocytosis was significantly reduced in cells expressing the short synprint deletion mutant. Capacitance traces are shown from cells transfected with wild-type α_1B (a) and short synprint deletion α_1B subunits (b). These are the same traces shown in Fig. 4 a and b, except on a compressed time scale. (c) The bar graph depicts the percentage of membrane recovered at 40 s post-stimulation for cells transfected with either the α_1B (n = 12) or α_1B short synprint deletion (n = 6) subunits. Forty seconds was chosen arbitrarily to include several cells for which only 40 s of data were available.

The capacitance technique used in this study measures the sum of exocytosis and endocytosis and does not distinguish between the two processes. It might be argued that an alternative explanation for the diminished exocytosis observed in cells expressing the short synprint deletion Ca²⁺ channel mutant is that the channels somehow augment endocytosis, thereby masking the true exocytosis. It also could be argued that the diminished endocytosis is due to delayed exocytosis masking endocytosis. Amperometry measures exocytosis exclusively. Haller et al. (32) made detailed comparisons between exocytosis measured with capacitance and amperometry and concluded that the data from both techniques were in agreement for measurements of exocytosis. Thus, our data, taken immediately after stimulation ends, should provide a reliable estimate of exocytosis. In addition, exocytosis in neuroendocrine cells is thought to end tens of milliseconds after Ca²⁺ influx has stopped (32). Therefore, our measurement of endocytosis tens of seconds after stimulation also should be free of overlap.

Discussion

Ca_v2 family members (N-, P/Q-, and R-type Ca²⁺ channels) are known to regulate neurotransmitter release (33-38). N- and P/Q-type Ca²⁺ channels are abundant in nerve terminals, where they colocalize with synaptic vesicles. The channels are thought to be in close physical proximity to neurotransmitter release proteins, including the SNARE proteins and the associated synaptic vesicles containing neurotransmitters (39-42). N- and P/Q-type Ca²⁺ channels bind to the SNARE proteins syntaxin 1A and SNAP-25 at their synprint sites (11, 16, 43). There has been considerable interest in understanding the functional consequences of this link between the synprint site on N- and P/Q-type Ca²⁺ channels and the SNARE complex.

We recently showed that a mouse pheochromocytoma cell line (MPC 9/3L), established by Powers et al. (44), is a useful model for studying Ca²⁺-dependent neurotransmitter release (23). MPC 9/3L cells express many of the proteins involved in Ca²⁺-dependent release but do not express endogenous voltage-dependent Ca²⁺ channels. They exhibit no exocytosis when stimulated by trains of depolarizations. MPC 9/3L cells therefore represent an excellent single-cell system in which to study the roles of specific Ca²⁺-channel subunits in secretion. Subtypes of known subunit composition can be expressed in isolation or in assorted combinations. Introducing exogenous N-type Ca²⁺ channels initiated vesicle fusion in these cells (23) in a manner similar to that found in neurons and neurosecretory cells (37, 38, 45). In an earlier study, we expressed L- and T-type Ca²⁺ channels (which do not possess synprint sites) in addition to N-type channels into MPC cells. All three types of Ca²⁺ channel were shown to elicit robust vesicle fusion (23), but no direct comparison of secretion elicited by each type of channel was attempted, in part due to the different gating and permeation properties of the channels. Furthermore, L-type Ca²⁺ channels seem to interact with SNARE proteins by means of a mechanism that does not involve the synprint site (46). In the present study, the importance of the synprint site was tested. N-type Ca²⁺ channels (α_1B, β_2A, and α₂δ), with and without the synprint site, were transfected into MPC cells to better understand the effect that this region of the α_1B subunit has on release. We found that the N-type channels are coupled less efficiently to secretion when the synprint site was deleted. Secretion was smaller and slower, even though Ca²⁺ current amplitude was not affected. In addition, endocytosis was slowed in cells expressing N-type channels that lacked the synprint site. The slowing of endocytosis may be a secondary consequence of the reduction in exocytosis because there would be less synaptic vesicle membrane available in the plasma membrane for retrieval.

After showing that N- and P/Q-type Ca²⁺ channels bound to SNARE proteins, different studies investigated the functional consequences of this interaction. The authors of these studies hypothesized that interaction was required for the efficient release of neurotransmitter (19, 20). If the release machinery and Ca²⁺ channels are tethered at the synprint site, then disrupting that interaction should interfere with release. Injection of fusion proteins containing the synprint site may dissociate N-type channels from the SNARE core. Mochida et al. (19) showed that introduction of the synprint peptides into presynaptic superior cervical ganglion neurons inhibited synaptic transmission. In this case, fast excitatory postsynaptic potentials due to synchronous transmitter release were partially inhibited, but late asynchronous release was increased. The injected peptides did not affect the Ca²⁺ current. Rettig and colleagues (20) showed that injection of the synprint peptide into embryonic Xenopus spinal neurons altered the Ca²⁺-dependence of release even though Ca²⁺ influx was not altered. In that study, the authors observed a 25% reduction in relative release when the cells were stimulated in an extracellular solution containing 1.8 mM Ca²⁺. The authors modeled their results and proposed that ≈70% of the previously linked synaptic vesicles became uncoupled by the addition of the synprint peptide (20). These earlier studies with the synprint peptide did not assay the peptide concentration at individual synapses. Thus, they could not assess the number of functional Ca²⁺ channel/SNARE interactions remaining in the presence of the synprint peptide. Our model system, MPC cells, allowed us to completely eliminate Ca²⁺ channel/SNARE interactions. Our data, which are consistent with these earlier studies, show that eliminating Ca²⁺ channel/SNARE interactions results in ≈50% reduction in release. This reduction in exocytotic amplitude may represent the fact that the Ca²⁺ channels are no longer optimally located for efficient release. Our results also indicate that the synprint site is not absolutely required for release.

Not only does the synprint site position Ca²⁺ channels appropriately for vesicle fusion, but it may be involved in targeting the N- and P/Q-type Ca²⁺ channels to synapses. In a recent study, Mochida et al. (21) deleted the synprint site in the α_1A subunit of P/Q-type channels and then expressed this construct in superior cervical ganglion neurons. They showed that these channels had a reduced effectiveness in synaptic transmission corresponding with a reduced localization to presynaptic terminals. Wild-type P/Q-type Ca²⁺ channels localized correctly. L-type channels are not typically localized at synapses, but, after Mochida et al. (21) spliced in a synprint site, the L-type channels localized correctly to the presynaptic nerve terminals and were able to establish synaptic transmission. Studies from other laboratories suggest that additional Ca²⁺ channel regions are used for targeting to synapses. Maximov and Bezprozvanny (47) showed that the C-terminal of the N-type Ca²⁺ channel contained a sequence that targeted the channel to synapses and that the modular adaptor proteins Mint1 and CASK may play a role in this targeting. More recently, Missler et al. (48) generated knockout mice in which one, two, or three of the α-neurexin genes were disrupted. In the triple-knockout animals, neurotransmitter release was greatly diminished due to loss of Ca²⁺ channel activity (48). Missler et al. (48) proposed that neurexins are required to couple the presynaptic N-type Ca²⁺ channels to the secretory machinery. If true, this finding would imply that there are at least two or more separate pathways for localizing Ca²⁺ channels to presynaptic nerve terminals and the secretory machinery. An alternate interpretation of the data presented in Missler et al. (48) is that the Ca²⁺ channels are correctly localized but that they are not functional in the absence of neurexin.

Not only do Ca²⁺ channels initiate neurotransmitter release, but also SNARE proteins, in turn, modify Ca²⁺ channel function, perhaps as a form of feedback modulation. For example, coexpressing syntaxin 1A with N-, P/Q-, and, in some cases, L-type Ca²⁺ channels results in decreased current amplitude, increased voltage-dependent inactivation, and/or enhanced G_βγ inhibition (8, 46, 49-55) presumably through interactions with the synprint site. The modulation of Ca²⁺ channel function by syntaxin 1A has also been demonstrated in nerve terminals. Stanley and Mirotznik (56) demonstrated that botulinum toxin, which cleaves syntaxin 1A, prevents G protein modulation of presynaptic Ca²⁺ channels. Although the mechanism(s) by which syntaxin 1A exerts its effects on Ca²⁺ channels are not well understood, it is clear that these types of interactions may have significant consequences for neurotransmitter release. Interestingly, other laboratories have shown that coexpression of other secretory proteins, like synaptotagmin, can relieve the syntaxin 1A-mediated inhibition of Ca²⁺ channels (46, 49, 57), suggesting that this kind of inhibition is dynamic. Our results showed that Ca²⁺ currents in MPC 9/3L cells were very similar regardless of whether or not the expressed Ca²⁺ channels lack a synprint site. Ca²⁺ channel availability was unchanged in the synprint deletion mutants. Our results therefore suggest that the N-type Ca²⁺ channels were in close proximity to mature SNARE complexes exclusively.

Although some aspects of the functional interactions between the synprint site and SNARE proteins remain to be elucidated, a wealth of information suggests that the site is important for both neurotransmitter release and Ca²⁺ channel function in both neurons and secretory cells. Further studies with MPC 9/3L cells and related cell lines will allow us to explore the functional links between a variety of different Ca²⁺ channels and secretion in greater detail.