The spider toxin omega-Aga IIIA defines a high affinity site on neuronal high voltage-activated calcium channels.

The spider toxin omega-agatoxin IIIA (omega-Aga-IIIA) is a potent inhibitor of high voltage-activated calcium currents in the mammalian brain. To establish the biochemical parameters governing its action, we radiolabeled the toxin and examined its binding to native and recombinant calcium channels. In experiments with purified rat synaptosomal membranes, both kinetic and equilibrium data demonstrate one-to-one binding of omega-Aga-IIIA to a single population of high affinity sites, with K(d) = approximately 9 pm and B(max) = approximately 1.4 pmol/mg protein. Partial inhibition of omega-Aga-IIIA binding by omega-conotoxins GVIA, MVIIA, and MVIIC identifies N and P/Q channels as components of this population. omega-Aga-IIIA binds to recombinant alpha(1B) and alpha(1E) calcium channels with a similar high affinity (K(d) = approximately 5-9 pm) in apparent one-to-one fashion. Results from recombinant alpha(1B) binding experiments demonstrate virtually identical B(max) values for omega-Aga-IIIA and omega-conotoxin MVIIA, providing further evidence for a one-to-one stoichiometry of agatoxin binding to calcium channels. The combined evidence suggests that omega-Aga-IIIA defines a unique, high affinity binding site on N-, P/Q-, and R-type calcium channels.

Calcium channels play key roles in numerous physiological processes including neurotransmitter release, hormone secretion, neurite outgrowth, and gene expression (1)(2)(3)(4). Neurons express several distinct types of voltage-gated calcium channels, which fall into two main categories: high voltage-activated (HVA) 1 and low voltage-activated (LVA) channels. HVA channels are classified as L-, N-, P/Q-, and R-types, based primarily on pharmacological criteria (5)(6)(7). For instance, Ltype HVA channels are uniquely sensitive to dihydropyridines, N-type channels to -conotoxin GVIA (-CTX-GVIA), and Ptype channels to low concentrations of -Aga-IVA. More recently, Q-and R-type channels have been identified (8,9). Q-type channels are less sensitive to -Aga-IVA than P-type channels but retain high sensitivity to -conotoxin MVIIC (-CTX-MVIIC). R-type channels are resistant to all agents mentioned above, but a new toxin called SNX-482 from tarantula venom blocks a subset of these (10).
In addition to their usefulness in defining specific calcium channel subtypes and their physiological functions, drugs and toxins can be used as biochemical probes for estimating channel densities in cells and tissues. As yet, no estimates of overall HVA channel density are available for the brain or for strategic parts of nerve cells, such as terminals. A peptide toxin that may be useful for this purpose is -Aga-IIIA, a calcium channel antagonist identified from venom of the funnel web spider Agelenopsis aperta (25). In contrast to -Aga-IVA, which is a selective blocker of P-type calcium currents, -Aga-IIIA blocks all HVA currents, but spares those of LVA channels (26 -29). This unique specificity has made this toxin a useful tool for distinguishing HVA from LVA currents in various types of cells (30,31). -Aga-IIIA also has proved useful in the analysis of L-type channel gating currents.
In this paper, we present kinetic and saturation isotherm data showing -Aga-IIIA binding to native and recombinant HVA calcium channels. Our results indicate that this toxin identifies a single, high affinity binding site in mammalian synaptosomal membranes, and in membranes of cells expressing recombinant calcium channels. We estimate the total density of toxin binding sites in purified mammalian nerve terminals, and find that subsets of these sites are pharmacologically discernible as N-and P/Q-type channels based on competition with -conotoxins. Since a large component of -Aga-IIIA binding remains following competition with conotoxins, it appears that a considerable proportion of additional calcium channel subtypes co-exist with N-and P/Q-type channels in nerve terminals.

EXPERIMENTAL PROCEDURES
Source of Toxins and Iodine-125--Aga-IIIA was purified from whole venom of the funnel web spider A. aperta by three steps of reversed-phase liquid chromatography (25). -Conotoxin MVIIA (-CTX-MVIIA) and -CTX-MVIIC were kindly provided by Neurex Corp. (now Elan Pharmaceuticals, Menlo Park, CA). -CTX-MVIIC also was purchased from either Peptides International or Sigma, -CTX GVIA was from Bachem, and Na 125 I was from NEN Life Science Products.
Preparation of Rat Synaptosomal Membranes-Synaptosomal membranes were prepared from whole brains of 14 -20-day-old Harlan Sprague-Dawley rats. Rat brains were collected in 20 ml of 0.32 M sucrose plus 1 M pepstatin and leupeptin, then homogenized with a motor-driven Teflon-glass homogenizer (Wheaton) using 10 strokes at 3000 rpm. The homogenate was centrifuged at 6000 rpm (3000 ϫ g) for 10 min in a Beckman JA-20 rotor, and the resulting pellet was discarded. The supernatant was centrifuged at 15,500 rpm (18,800 ϫ g) for 20 min with the same rotor. The pellet was resuspended in 0.32 M sucrose. The suspension was subjected to Ficoll gradient (5-9%-12%) centrifugation for 50 min at 21,000 rpm (75,619 ϫ g) in a Beckman L8 -55 ultracentrifuge using SW41 Ti rotor. Synaptosomal membranes were collected from the interfaces between 5-9% and 9 -12% Ficoll layers, diluted in 50 mM HEPES at pH 7.4, and pelleted at 14, 500 rpm (25,000 ϫ g) using a JA-20.1 rotor. The pellet was resuspended in sucrose solution, aliquoted, and stored at Ϫ80°C for future use. All centrifugations were carried out at 4°C. Membrane protein was quantified using the Bradford reagent (Bio-Rad) with bovine serum albumin (BSA) as a standard.
Preparation of Membranes Containing Recombinant Calcium Channels-HEK293 cells providing stable expression of cloned calcium channels ␣ 1B and ␣ 1E were kindly provided by Parke-Davis Pharmaceutical Research. The S3 cell line expresses a human neuronal ␣ 1B clone prepared as described previously (32). The 192C cell line expresses a human neuronal ␣ 1E subunit. Both cell lines express a rabbit skeletal muscle calcium channel ␣ 2 /␦ subunit and a human neuronal ␤ 2 subunit. Details of transfection and selection are described by Rock et al. (31). Both cell lines were maintained as monolayer cultures in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum at 37°C with 10% CO 2 . The medium also contained penicillin (100 units/ml), streptomycin sulfate (100 units/ml), hydromycin B sulfate (400 g/ml), and G418 (600 g/ml). The cells were split 1:10 and fed medium every 2-3 days. All the chemicals except DMEM (Life Technologies, Inc.) used in cell culture were purchased from Sigma.
To obtain sufficient membrane protein for multiple binding studies, both cell lines were grown up to a large scale (ϳ20 -30 10-cm dishes). After the removal of medium, the cells were rinsed twice with phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 ⅐7H 2 O, 1.4 mM KH 2 PO 4 , pH 7.4), trypsinized, and collected by centrifugation. Aliquots of the cells were then kept frozen in DMEM plus 20% fetal bovine serum and 10% Me 2 SO in liquid nitrogen until use.
To prepare membranes, aliquots of the frozen cells were thawed, rinsed twice in phosphate-buffered saline buffer, resuspended in phosphate-buffered saline at 1 ϫ 10 6 cells/200 -600 l, and homogenized with a 2-ml glass homogenizer (Pyrex, catalog no. 7727). After a brief centrifugation at 1000 ϫ g to remove large debris, the supernatant containing membranes was saved and the protein concentration was determined using the Bradford reagent (Bio-Rad) with BSA as a standard.
Iodination of -Aga-IIIA-Purified -Aga-IIIA (1 nmol) was dissolved in 20 l of 0.2 M sodium phosphate buffer at pH 7.4 in a siliconized tube. Approximately 0.67 nmol (4 l) of Na 125 I and 2 nmol (10 l of 0.2 mM) of chloramine T were added to start the reaction. The reaction was terminated 5 min later by addition of 100 nmol (10 l of 10 mM) of cysteine. Monoiodinated -Aga-IIIA ( 125 I--Aga-IIIA) was isolated by reversed-phase liquid chromatography on a Microsorb-MV C 18 column with a linear gradient of acetonitrile/water at 0.1%/min in constant 0.1% trifluoroacetic acid (Fig. 1). Purified 125 I--Aga-IIIA was stored in 40% methanol with 0.1% of BSA at Ϫ20°C. The concentration of carrier-free 125 I--Aga-IIIA was calculated directly by utilizing a constant specific activity (2200 Ci (81.4 TBq)/mmol), due to the catastrophic decay of labeled ligand as described previously (33).
Kinetic Binding Assays-For measurement of ligand-receptor association, membranes were added to each of the reaction tubes containing 400 l of "binding buffer" (50 mM HEPES and 0.1% BSA, adjusted to pH 7.4 with NaOH) followed by addition of 100 l of 125 I--Aga-IIIA in binding buffer at specified time intervals. Reactions were terminated at the same time by dilution with two 4-ml volumes of ice-cold washing buffer (100 mM NaCl, 20 mM HEPES, 0.1% BSA, pH 7.4) using the Skatron cell harvester. The filtermat used in all binding assays was 1.0-m retention at size 102 ϫ 256 mm (catalog no. 11734; Skatron Instruments, Ltd, Newmarket, Suffolk, United Kingdom). The nonspecific binding of -Aga-IIIA was determined as the amount of 125 I--Aga-IIIA in the presence of unlabeled -Aga-IIIA at three time points. Radioactivity on filtermats was quantified using a 1272 Clinic ␥ counter (Amersham Pharmacia Biotech).
For dissociation experiments, rat brain synaptosomal membranes were added to each of the reaction tubes containing 400 l of the binding buffer and ϳ20 pM 125 I--Aga-IIIA. After incubation for 1 h to establish equilibrium, unlabeled -Aga-IIIA was added to the different reactions to achieve a final concentration of 10 nM at a specified time interval to initiate dissociation. Reactions were terminated by dilution and rapid filtration as described above.
Equilibrium Binding Assays-Equilibrium binding assays were performed at room temperature (22°C) in 4-ml polypropylene tubes. Two buffers were used, binding buffer or "recording buffer," the latter designed to mimic ionic conditions used for patch clamp recordings (29). Recording buffer contained 5 mM BaCl 2 , 160 mM tetraethylammonium chloride, and 10 mM HEPES, adjusted to pH 7.4 with tetraethylammonium-OH. Briefly, rat brain synaptosomal or HEK293 cell membranes were added to each of the reaction tubes containing 400 l of buffer with or without 10 nM unlabeled toxin. Reactions were incubated for 2 h to allow equilibrium binding of the unlabeled toxin to membranes. 125 I--Aga-IIIA (100 l) was added to achieve increasing concentrations (0.1-85 pM), and reactions were incubated for an additional 2 h prior to termination as described for kinetic binding assays. Binding of 125 I-neurotoxins in the presence of the 10 nM unlabeled toxin represented nonspecific binding. Specific binding was determined by subtracting the amount of nonspecific binding from the amount of total binding in the absence of unlabeled toxin.
Data Analysis-All the data generated from the binding experiments were analyzed using a nonlinear least square fitting program (Origin; MicroCal Software Inc., Northampton, MA). Association curves were fitted with a single exponential function (Y ϭ A*(1 Ϫ e ϪBX ) ϩ E, where A is the magnitude of the curve and B is the apparent rate constant (k obs ). E was starting value of the curve. Y was the amount of specifically bound 125 I--Aga-IIIA of different incubation duration. The saturation curves were fitted with a rectangular hyperbola function (Y ϭ P 1 *X/(P 2 ϩ X), where P 1 ϭ the maximal binding; P 2 ϭ a measure of the affinity of the receptor for a ligand), X ϭ the concentration of free 125 I--Aga-IIIA. Y ϭ the amount of specifically bound 125 I--Aga-IIIA at different concentration.

RESULTS
Iodination of -Aga-IIIA-Since -Aga-IIIA contains three tyrosine residues and lacks methionine (25), it was iodinated directly using chloramine T as described under "Experimental Procedures." The labeling reaction resulted in a mixture of unreacted -Aga-IIIA, mono-and di-iodinated species. Monoiodinated 125 I--Aga-IIIA was purified by reversed-phase liquid chromatography (Fig. 1). The procedure yielded approximately 320 Ci (ϭ144 pmol) of carrier-free 125 I--Aga-IIIA from 1 nmol of starting material. We confirmed the identity of purified 125 I--Aga-IIIA using an equivalent nonradioactive -Aga-IIIA derivative ( 127 I--Aga-IIIA) prepared and purified by the same method. The 127 I derivative, which co-eluted with 125 I--Aga-IIIA, was found to have an average molecular mass of 8633 daltons by laser desorption mass spectrometry (data not shown), indicating addition of a single iodine atom. Furthermore, 127 I--Aga-IIIA inhibited the specific binding of 125 I--Aga-IIIA (IC 50 ϳ20 pM) to rat brain synaptosomal membranes, suggesting that the iodinated toxin was suitable for radioligand binding assays (Fig. 2).
Kinetics of -Aga-IIIA Binding-The association kinetics of 125 I--Aga-IIIA to rat brain synaptosomal membranes were determined at four different 125 I--Aga-IIIA concentrations: 2, 6, 20, and 60 pM. The data show that the time to reach equilibrium is concentration dependent (Fig. 3A), ranging from 30 min at 60 pM to ϳ2 h at 2 pM. The apparent association rates (k obs ) are positively correlated with 125 I--Aga-IIIA concentration (Fig. 3B), indicating that the toxin-receptor interaction on synaptosomal membranes is a simple, reversible bimolecular interaction with a single class of binding sites. Association and dissociation rate constants estimated from the association experiment are: k on ϭ 5.9 ϫ 10 7 M Ϫ1 s Ϫ1 , k off ϭ 5.7 ϫ 10 Ϫ4 s Ϫ1 . The equilibrium dissociation constant K d ϭ k off /k on derived from these studies is ϳ9.7 pM.
The results of the dissociation experiment, conducted over a period of 2 h, are shown in Fig. 3C. The k off value estimated directly from this experiment was 2.2 ϫ 10 Ϫ4 s Ϫ1 , which is similar to that obtained indirectly from the association experiment. In summary, the results of kinetic studies indicate high affinity binding of -Aga-IIIA to a single class of receptors in rat synaptosomal membranes.
Equilibrium Binding Studies-We performed equilibrium binding of 125 I--Aga-IIIA to rat brain synaptosomal membranes using increasing concentrations of 125 I--Aga-IIIA and a fixed amount of membranes. The saturation curve showed that the total amount of binding increased in proportion to 125 I--Aga-IIIA concentration (Fig. 4A). Nonspecific binding of the radioligand was determined in parallel experiments in the presence of 10 nM unlabeled toxin. After subtracting nonspecific from total binding, the amount of bound 125 I--Aga-IIIA plateaued with increasing toxin concentrations, confirming that binding was saturable and therefore specific. Consistent with this, other experiments showed that the specific binding of a constant concentration of 125 I--Aga-IIIA increased linearly with increasing concentrations of synaptosomal membranes (data not shown).
Scatchard analysis yielded a linear relationship, with an equilibrium constant (K d ) of 8.5 pM and B max of ϳ1.4 pmol/mg of protein (Fig. 4B). Consistent with Scatchard analysis, a unitary Hill coefficient (n H ϭ 1) was obtained (Fig. 4C). Taken together, the results from both the kinetic and the equilibrium binding experiments suggest that the synaptosomal membranes contain a single class of high affinity binding sites for -Aga-IIIA.
-Aga-IIIA Binds to N and P/Q Calcium Channels in Rat Brain Synaptosomes-Previous electrophysiological studies (26,29,31) showed that -Aga-IIIA inhibits L-, N-, P/Q-, and R-type currents, but spares currents through low threshold channels (29,30). To assess the correspondence between block of currents in physiological experiments and receptor (channel) binding, we examined the abilities of several conotoxins to compete with -Aga-IIIA binding to purified synaptosomal membranes under equilibrium conditions. Specifically, we tested for the effects of unlabeled conotoxins -CTX-GVIA (Ntype channel-specific blocker), -CTX-MVIIA (N-type channelspecific blocker), and -CTX-MVIIC (N/P/Q-type channel-spe- cific blocker) on the equilibrium binding of 125 I--Aga-IIIA to rat brain membranes. As expected, unlabeled -Aga-IIIA inhibited the binding of 125 I--Aga-IIIA in a concentration-dependent fashion (Fig. 5). The N-type-specific conotoxins -CTX-GVIA and -CTX-MVIIA partially inhibited -Aga-IIIA binding, and it is noteworthy that they inhibit to a similar degree. Maximal inhibition with these toxins was ϳ18% of total -Aga-IIIA binding. In contrast, a significantly higher proportion (ϳ38%) of -Aga-IIIA binding was inhibited by -CTX-MVIIC, which provides compelling evidence that -Aga-IIIA interacts with both N-and P/Q-type calcium channels in rat brain synaptosomal membranes.
-Aga-IIIA Binds to Recombinant ␣ 1B and ␣ 1E Calcium Channels-The above experiments showed that -Aga-IIIA interacts with a single class of binding sites in rat brain synaptosomes and that this population of binding sites contains Nand P/Q-type calcium channels. These data support the hypothesis that -Aga-IIIA identifies a common binding site on multiple subtypes of calcium channels. To further test this hypothesis, we examined the binding of -Aga-IIIA to two recombinant ␣ 1B (N-type) and ␣ 1E (R-type) calcium channels expressed in the HEK cell lines S3 and 192C, respectively (31). Our purpose was twofold. First, we wanted to confirm the parameters of -Aga-IIIA binding to native N-type channels pharmacologically defined in synaptosomal membranes. Second, we sought to ascertain whether the toxin bound to R-type channels with similar high affinity.
As shown in Table I, 125 I--Aga-IIIA binding to membranes of both 192C and S3 cell membranes is high as compared with those of control HEK 293 cells. Binding of 125 I--Aga-IIIA to HEK 293 cells was nonspecific, since it could not be inhibited by adding excess unlabeled. On the other hand, whereas the binding of 125 I--Aga-IIIA to S3 and 192C cells exhibits a similar level of nonspecific binding, approximately half of total binding is specific. These results suggest that 125 I--Aga-IIIA indeed binds specifically to recombinant ␣ 1B and ␣ 1E calcium channels.
We determined the affinity of 125 I--Aga-IIIA binding to each recombinant calcium channel by equilibrium binding experiments. Toxin binding to membranes of both S3 and 192C cell lines was saturable (Figs. 6A and 7A). Scatchard analysis yielded similar K d values for -Aga-IIIA binding to ␣ 1B and ␣ 1E calcium channels of 5.1 and 8.6 pM, respectively (F ϭ 2.43, p ϭ 0.19 at the 0.05 level) (Figs. 6B and 7B). We note that these values agree well with the K d value of ϳ9 pM obtained with rat brain membranes (Figs. 3 and 4).
Binding of -Aga-IIIA to Recombinant Calcium Channels Is One to One-Our results suggest that -Aga-IIIA binds with similar high affinity to homogeneous populations of recombinant N-and R-type calcium channels. To obtain additional confirmation that toxin binding is one-to-one, we compared B max values for 125 I--Aga-IIIA and 125 I--CTX-MVIIA binding to recombinant N-type channels in S3 cells. It was reported previously that -CTX-MVIIA and -CTX-GVIA, both N-type calcium channel-specific blockers, exhibit 1:1 binding to N-type calcium channels (34 -37). We therefore reasoned that, if one -Aga-IIIA binding site occurs per calcium channel, B max values for -Aga-IIIA and -CTX-MVIIA should be identical. Equilibrium binding assays showed that binding of both toxins was saturable (Fig. 8A), and that B max values for each toxin were approximately 0.65 pmol/mg of membrane protein (Fig.   FIG. 4. Equilibrium binding assay of 125 I--Aga-IIIA binding to rat brain synaptosomal membranes. 0.8 g of synaptosomal membrane proteins was incubated with increasing concentrations of 125 I--Aga-IIIA for 2 h. Shown is the average of three experiments, and the error bars are standard error of the mean. A, direct plot of saturation data. The total (triangles) and nonspecific binding (squares) represent the amount of binding in the absence or presence of 10 nM -Aga-IIIA, respectively. Specific binding (circles) was obtained by subtracting the nonspecific binding from the total. B, Scatchard analysis of specific binding data in A. The maximal binding sites, B max , were indicated. C, Hill plot. Hill coefficient, n H , is the slope of the curve as indicated. -Aga-IIIA Binding to Calcium Channels 8B). This provides independent evidence confirming one-to-one binding of -Aga-IIIA to N-type calcium channels.
Sensitivity to Divalent Ions-The affinity of -Aga-IIIA to calcium channels as determined by the above binding experiments (K d ϭ 5.1-9.7 pM) is about 3 orders of magnitude higher than that determined by previous electrophysiological studies (K d ϭ 0.5-1.5 nM) (25,26,29). Since physiological experiments are conducted under conditions of relatively high ionic strength, we suspected that the discrepancy could be due to differences in the buffers used in the two types of experiments. The recording buffer for electrophysiological studies contains high concentrations of divalent cations (5 mM BaCl 2 ), which are absent in the binding buffer typically used in radioligand binding experiments. Indeed, divalent cations have been shown to affect the affinity of other toxins for calcium channels (35,36,38).
We therefore tested the binding of 125 I--Aga-IIIA to synaptosomal membranes in binding buffer as compared with that in the electrophysiological recording buffer. The results showed that the binding of 125 I--Aga-IIIA appeared to approach saturation at higher concentrations in the electrophysiological recording buffer than in the binding buffer (Fig. 9A), suggesting that buffer conditions do affect -Aga-IIIA binding. However, subsequent Scatchard analysis (Fig. 9B) showed that the difference in the affinity is merely 3-fold (K d ϭ 8.3 pM in the recording buffer versus 2.9 pM in binding buffer) and that the total levels of the binding (B max ) remained virtually unchanged. Similar results were obtained using the S3 and 192C cell membranes (data not shown). Taken together, these results indicate that buffer conditions (and potentially the divalent cations) indeed have an affect on binding but the effect is insufficient to explain the 3 orders of magnitude difference in the binding affinity observed in the current binding studies and the previous electrophysiological studies. DISCUSSION We have described the interactions of -Aga-IIIA with neuronal calcium channels using a biochemical approach, consisting of kinetic and equilibrium binding analyses. Our experiments encompassed toxin binding to: 1) a heterogeneous set of native calcium channels in rat brain synaptosomes, and 2) recombinant ␣ 1B (N-type) and ␣ 1E (R-type) channels expressed in cell lines. In each instance, -Aga-IIIA identifies a single, high affinity (K d ϳ5-9 pM) binding site. In synaptosomal membranes, this site occurs on multiple subtypes of calcium channels. Some of these are discernible as N-and P/Q-type channels, based on inhibition of -Aga-IIIA binding by conotoxins. I Specific binding of -Aga-IIIA to recombinant calcium channels Binding assays were performed essentially as described for equilibrium binding assays (see "Experimental Procedures" for details) by incubating 20 pM 125 I--Aga-IIIA with membranes prepared from the S3 cells, 192C cells, or the parental HEK 293, in the presence or absence of unlabeled excess 10 nM -Aga-IIIA for 2 hrs. NS, nonspecific binding or the amount of bound 125 I--Aga-IIIA in the presence of unlabeled excess -Aga-IIIA. S, specific binding or the total amount of bound 125 I--Aga-IIIA in the absence of unlabeled -Aga-IIIA minus nonspecific binding.  -Aga-IIIA Binding to Calcium Channels However, since a substantial component of -Aga-IIIA binding is conotoxin-resistant, we suggest the presence of additional HVA channels, including R-and L-type channels. This is based on the specificity of -Aga-IIIA for HVA channels, its high affinity binding to ␣ 1E (R-type) channels (this study), and previous evidence showing that -Aga-IIIA is a potent blocker of both R-and L-type channels (10, 26 -28, 30, 31).
-Aga-IIIA Binding to Native and Recombinant Calcium Channels Is Specific and High Affinity-Equilibrium assays showed that binding of 125 I--Aga-IIIA to native and recombinant channels is saturable, specific, and high affinity (K d ϭ 5 -9 pM). Interestingly, K d values measured for -Aga-IIIA in our radioligand binding assays are considerably lower than affinity estimates derived from electrophysiological and synaptosome flux studies (K d ϭ ϳ0.3-1 nM) (25,26,28). Given the difference between -Aga-IIIA binding affinity observed here and its efficacy in blocking calcium currents or calcium flux in functional assays, it is important to examine critically whether toxin binding sites in synaptosomal membranes are authentic calcium channels. Several lines of evidence argue in favor of this. First, we find that -conotoxins GVIA, MVIIA, and MVIIC inhibit binding of -Aga-IIIA to synaptosomal membranes, with IC 50 values in the low picomolar range. Second, -Aga-IIIA exhibits specific binding to membranes of S3 and 192C cells expressing N-and R-type calcium channels, respectively, but not to control HEK cells. Third, -conotoxin MVIIA inhibits -Aga-IIIA binding to ␣ 1B calcium channels. Fourth, B max values for -conotoxin MVIIA and -Aga-IIIA binding to ␣ 1B calcium channels are identical. These data provide compelling circumstantial evidence that the -Aga-IIIA binding sites we have described in rat synaptosomal membranes are indeed calcium channels.
Similar discrepancies between toxin binding affinity (35,36,39) and potency for calcium channel block (37,40) have been reported for -conotoxins GVIA, MVIIA, and MVIIC. Ionic strength and the presence of divalent ions are factors considered to be responsible for such differences. For instance, calcium ions cause a 400-fold reduction in the binding affinity of -CTX-GVIA to rat brain synaptosomal membranes (39). However, in other reports, calcium produced a mere 7-9-fold reduction in the binding affinities of conotoxins MVIIA and MVIIC (36), suggesting that the divalent ions alone cannot explain all such discrepancies. We also find that the presence of divalent ions and variations in ionic strength are insufficient to explain differences in binding affinity of -Aga-IIIA obtained from binding and electrophysiological experiments.
It seems more likely that such discrepancies stem from the fact that most electrophysiological experiments are not conducted under equilibrium conditions. Our data show that up to 1 h is required for -Aga-IIIA binding at low concentrations, and the time course of toxin unbinding involves many hours. Since electrophysiology experiments are rarely conducted over such an extended time course, accurate K d measurements from real time studies of -Aga-IIIA block and unblock are difficult. However, a study in which hours-long incubation times were used to block calcium-dependent serotonin release in intact neurons showed picomolar affinities for conotoxins similar to those observed in binding assays (41). This suggests that, under the appropriate conditions, intact cell assays can yield results similar to those obtained in binding assays.
One-to-one Binding of -Aga-IIIA to Calcium Channels-Our saturation binding and kinetic data are consistent with a single -Aga-IIIA binding site on each HVA calcium channel. We nevertheless conducted additional experiments to critically examine this issue. Using the S3 cell line, which expresses the ␣ 1B (N-type) channel, we found that B max values for -Aga-IIIA and -CTX-MVIIA are virtually identical (Fig. 7). Since -CTX-MVIIA binds to the N-type channel in a one-to-one fashion, this provides independent confirmation that the N-type calcium -Aga-IIIA Binding to Calcium Channels channel has a single -Aga-IIIA binding site.
The -Aga-IIIA Binding Site Occurs on Multiple HVA Calcium Channels-Our data from toxin association (linear plot of K obs versus 125 I--Aga-IIIA concentration, Fig. 3B), dissociation (linear plot of ln(B/B o ) versus time, Fig. 3C), and equilibrium binding experiments (linear Scatchard plot, Fig. 4B; unitary Hill coefficient, Fig. 4C) show that -Aga-IIIA interacts with a single high affinity binding site in rat brain synaptosomal membranes. Nevertheless, competition experiments with -conotoxins indicate that this site occurs on pharmacologically unique subtypes of calcium channels. One component of total -Aga-IIIA binding can be identified as N-type calcium channels, based on competition with -conotoxin GVIA and -conotoxin MVIIA, which inhibit the same percentage (ϳ18%) of total -Aga-IIIA binding. A somewhat larger component (ϳ40%) of total -Aga-IIIA binding is inhibited by the high affinity P/Q-type ligand -conotoxin MVIIC (36). This evidence for -Aga-IIIA binding to N-and P/Q-type channels is in accord with electrophysiological evidence showing that -Aga-IIIA not only exerts functional block of these channels, but occludes -conotoxin GVIA block of N-type channels (26) and -conotoxin MVIIC block of P-type channels (42,43). The substantial proportion of N-and P/Q-type channels in this overall population of binding sites is consistent with their demonstrated importance in transmitter release from nerve terminals (9, 44 -46). To summarize, our results from synaptosomal binding assays show that -Aga-IIIA defines a high affinity binding site, which occurs on both N-and P/Q-type channels.
A Diversity of Calcium Channels in Mammalian CNS Nerve Terminals-Since a large residual component of synaptosomal -Aga-IIIA binding remains after competition with conotoxins, the presence of calcium channel subtypes extending beyond Nand P/Q-type channels is indicated. Given that -Aga-IIIA blocks HVA but not LVA currents (27, 29 -31), this residual component probably includes R-and/or L-type channels, which comprise the remaining HVA subtypes currently recognized. Our data show that -Aga-IIIA binds with high affinity to the recombinant ␣1E clone, which corresponds to the R-type channel (8,30), and recent studies demonstrate involvement of R-type channels in exocytotic transmitter release at synapses and neurosecretory endings (48,49). Therefore, it is possible if not likely that R-type channels contribute a component of -Aga-IIIA binding in synaptosomal membranes. With respect to L-type channels, direct measurement of -Aga-IIIA binding has not been accomplished as yet, but electrophysiological studies show the toxin is a potent blocker (K d ϳ0.3-1 nM) of L-type channels in both cardiac myocytes and neurons (26 -29). Additionally, although L-type channels generally are considered to be only minimally involved in exocytotic release in neurons, their presence in nerve terminals is clear (47).
Our evidence suggests that the high affinity -Aga-IIIA binding site occurs on multiple HVA calcium channels. From previous studies on this toxin, it is possible to make several inferences regarding the location of this site on these channels. First, -Aga-IIIA inhibits the binding of -conotoxin GVIA to the ␣1 pore-forming subunit of the N-type calcium channel (50) and this inhibition is competitive (51). Since the -conotoxin GVIA binding site has been localized to the extracellular-facing vestibule of the N-type channel pore (32), these data are consistent with -Aga-IIIA binding to extracellular face of the channel pore. Further evidence in support of this comes from electrophysiological evidence demonstrating that -Aga-IIIA blocks ionic conductance but not gating current in L-type calcium channels (27), consistent with pore occlusion in a manner analogous to sodium channel block by tetrodotoxin. It is interesting to note that, while -Aga-IIIA exerts complete block of L-type channels, it occludes N-, P/Q-, and R-type channel currents partially. This may be due to the fact that the toxin acts like a leaky plug, reducing but not eliminating the flow of ions through the pore. Considering these data along with the conservation in structure of HVA calcium channel ␣1 subunits, it seems quite likely that -Aga-IIIA binds to a common, high affinity site in the outer vestibule of L-, N-, P/Q-, and R-type channels to block ionic current. The fact that -Aga-IIIA does not block LVA calcium channels suggests that the toxin recognizes an evolutionary point of departure between HVA and LVA channels.