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J. Biol. Chem., Vol. 275, Issue 28, 21309-21316, July 14, 2000

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The Spider Toxin -Aga IIIA Defines a High Affinity Site on Neuronal High Voltage-activated Calcium Channels^*

Lizhen Yan and Michael E. Adams^§

From the Environmental Toxicology Graduate Program and Departments of Entomology and Neuroscience, University of California, Riverside, California 92521

Received for publication, January 11, 2000, and in revised form, April 24, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The spider toxin -agatoxin IIIA (-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 -Aga-IIIA to a single population of high affinity sites, with K_d = ~9 pM and B_max = ~1.4 pmol/mg protein. Partial inhibition of -Aga-IIIA binding by -conotoxins GVIA, MVIIA, and MVIIC identifies N and P/Q channels as components of this population. -Aga-IIIA binds to recombinant _1B and _1E calcium channels with a similar high affinity (K_d = ~5-9 pM) in apparent one-to-one fashion. Results from recombinant _1B binding experiments demonstrate virtually identical B_max values for -Aga-IIIA and -conotoxin MVIIA, providing further evidence for a one-to-one stoichiometry of agatoxin binding to calcium channels. The combined evidence suggests that -Aga-IIIA defines a unique, high affinity binding site on N-, P/Q-, and R-type calcium channels.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Calcium channels play key roles in numerous physiological processes including neurotransmitter release, hormone secretion, neurite outgrowth, and gene expression (1-4). 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 (5-7). For instance, L-type HVA channels are uniquely sensitive to dihydropyridines, N-type channels to -conotoxin GVIA (-CTX-GVIA), and P-type 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).

HVA calcium channels are hetero-oligomeric complexes consisting of subunits designated as ₁, ₂/, , and  (11). In the rat central nervous system, five distinct genes encoding the pore-forming ₁ subunit are designated as _1A, _1B, _1C, _1D, and _1E (12, 13), and corresponding genes are found in the human brain (14-16). Functional expression and immunoprecipitation studies identified _1C and _1D as dihydropyridine-sensitive L-type calcium channels (15, 17, 18). The _1B gene encodes the N-type (14, 19, 20), and splice variants of _1A specify P- or Q-type channels (21, 22). The neuronal _1E transcript, widely distributed in the brain, corresponds to R-type channels (13, 16, 23, 24).

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

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 Na¹²⁵I 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 µM 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 × 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 × 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 × 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 × g) using a JA-20.1 rotor. The pellet was resuspended in sucrose solution, aliquoted, and stored at 80 °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% CO₂. The medium also contained penicillin (100 units/ml), streptomycin sulfate (100 units/ml), hydromycin B sulfate (400 µg/ml), and G418 (600 µg/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 (~20-30 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 Na₂HPO₄·7H₂O, 1.4 mM KH₂PO₄, 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% Me₂SO 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 × 10⁶ cells/200-600 µl, and homogenized with a 2-ml glass homogenizer (Pyrex, catalog no. 7727). After a brief centrifugation at 1000 × 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 µl of 0.2 M sodium phosphate buffer at pH 7.4 in a siliconized tube. Approximately 0.67 nmol (4 µl) of Na¹²⁵I and 2 nmol (10 µl 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 µl of 10 mM) of cysteine. Monoiodinated -Aga-IIIA (¹²⁵I--Aga-IIIA) was isolated by reversed-phase liquid chromatography on a Microsorb-MV C₁₈ column with a linear gradient of acetonitrile/water at 0.1%/min in constant 0.1% trifluoroacetic acid (Fig. 1). Purified ¹²⁵I--Aga-IIIA was stored in 40% methanol with 0.1% of BSA at 20 °C. The concentration of carrier-free ¹²⁵I--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 µl of "binding buffer" (50 mM HEPES and 0.1% BSA, adjusted to pH 7.4 with NaOH) followed by addition of 100 µl of ¹²⁵I--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-µm retention at size 102 × 256 mm (catalog no. 11734; Skatron Instruments, Ltd, Newmarket, Suffolk, United Kingdom). The nonspecific binding of -Aga-IIIA was determined as the amount of ¹²⁵I--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 µl of the binding buffer and ~20 pM ¹²⁵I--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 BaCl₂, 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 µl 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. ¹²⁵I--Aga-IIIA (100 µl) 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 ¹²⁵I--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 = A*(1  e^BX) + 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 ¹²⁵I--Aga-IIIA of different incubation duration. The saturation curves were fitted with a rectangular hyperbola function (Y = P₁*X/(P₂ + X), where P₁ = the maximal binding; P₂ = a measure of the affinity of the receptor for a ligand), X = the concentration of free ¹²⁵I--Aga-IIIA. Y = the amount of specifically bound ¹²⁵I--Aga-IIIA at different concentration.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 ¹²⁵I--Aga-IIIA was purified by reversed-phase liquid chromatography (Fig. 1). The procedure yielded approximately 320 µCi (=144 pmol) of carrier-free ¹²⁵I--Aga-IIIA from 1 nmol of starting material. We confirmed the identity of purified ¹²⁵I--Aga-IIIA using an equivalent nonradioactive -Aga-IIIA derivative (¹²⁷I--Aga-IIIA) prepared and purified by the same method. The ¹²⁷I derivative, which co-eluted with ¹²⁵I--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, ¹²⁷I--Aga-IIIA inhibited the specific binding of ¹²⁵I--Aga-IIIA (IC₅₀ ~20 pM) to rat brain synaptosomal membranes, suggesting that the iodinated toxin was suitable for radioligand binding assays (Fig. 2).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Iodination of -Aga-IIIA. [125]-Aga-IIIA was synthesized by direct iodination of tyrosine residues using chloramine T as described under "Experimental Procedures." The purification procedure employed a Microsorb MV C18 reversed-phase column (4.6 × 150 mm) with a linear gradient of acetonitrile/water (0.1%/min) in constant 0.1% trifluoroacetic acid at a flow rate of 1 ml/min. Elution of native -Aga-IIIA and 125I--Aga-IIIA are indicated in the figure. The identity of 125I--Aga-IIIA was confirmed by mass analysis as discussed under "Results."

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Comparison of native and iodinated -Aga-IIIA to rat synaptosomal membranes. Shown is inhibition of 125I--Aga-IIIA binding by native toxin (solid squares) versus the non-radioactive 127I derivative (open circles). Synaptosomal membranes (0.7 µg) were incubated with 2.5 pM 125I--Aga-IIIA in a reaction volume of 2 ml. Nonspecific binding of the radioligand was defined as the amount of binding remaining in the presence of 10 nM unlabeled toxin. Each point is the average of three experiments, with standard error of the mean. The curves were fitted with the logistic function.

Kinetics of -Aga-IIIA Binding-- The association kinetics of ¹²⁵I--Aga-IIIA to rat brain synaptosomal membranes were determined at four different ¹²⁵I--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 ~2 h at 2 pM. The apparent association rates (k_obs) are positively correlated with ¹²⁵I--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 = 5.9 × 10⁷ M¹ s¹, k_off = 5.7 × 10⁴ s¹. The equilibrium dissociation constant K_d = k_off/k_on derived from these studies is ~9.7 pM.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Kinetics of 125I--Aga-IIIA binding to synaptosomal membranes. A, association curves of 125I--Aga-IIIA. 0.4 µg of synaptosomal membrane proteins was incubated with different concentrations of 125I--Aga-IIIA for different duration. The concentrations of 125I--Aga-IIIA are indicated in the figure. 125I--Aga-IIIA concentrations are indicated on the left side of the figure. The data were fitted with a singular exponential function. B, plot of estimated kobs values from A versus the corresponding 125I--Aga-IIIA concentrations. The parameters kon, koff, and Kd were estimated as indicated. C, dissociation kinetics of 125I--Aga-IIIA. In the experiment, 20 pM 125I--Aga-IIIA was incubated with 0.4 µg of synaptosomal membrane protein for 1 h and the dissociation was monitored by addition of 10 nM unlabeled -Aga-IIIA. Bo, amount of bound 125I--Aga-IIIA prior to the addition of unlabeled -Aga-IIIA (or at time 0); B, amount of bound 125I--Aga-IIIA at various times after addition of unlabeled -Aga-IIIA. The data were fitted by linear regression, and the koff was estimated as indicated. The results of both the association and dissociation kinetic experiments were the average of three independent experiments. The error bars are standard error of the mean.

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 × 10⁴ s¹, 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 ¹²⁵I--Aga-IIIA to rat brain synaptosomal membranes using increasing concentrations of ¹²⁵I--Aga-IIIA and a fixed amount of membranes. The saturation curve showed that the total amount of binding increased in proportion to ¹²⁵I--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 ¹²⁵I--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 ¹²⁵I--Aga-IIIA increased linearly with increasing concentrations of synaptosomal membranes (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Equilibrium binding assay of 125I--Aga-IIIA binding to rat brain synaptosomal membranes. 0.8 µg of synaptosomal membrane proteins was incubated with increasing concentrations of 125I--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, Bmax, were indicated. C, Hill plot. Hill coefficient, nH, is the slope of the curve as indicated.

Scatchard analysis yielded a linear relationship, with an equilibrium constant (K_d) of 8.5 pM and B_max of ~1.4 pmol/mg of protein (Fig. 4B). Consistent with Scatchard analysis, a unitary Hill coefficient (n_H = 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 (N-type channel-specific blocker), -CTX-MVIIA (N-type channel-specific blocker), and -CTX-MVIIC (N/P/Q-type channel-specific blocker) on the equilibrium binding of ¹²⁵I--Aga-IIIA to rat brain membranes. As expected, unlabeled -Aga-IIIA inhibited the binding of ¹²⁵I--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 ~18% of total -Aga-IIIA binding. In contrast, a significantly higher proportion (~38%) 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.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   The inhibitory effects of -neurotoxins on the binding of 125I--Aga-IIIA to its receptors in synaptosomal membranes. Different concentrations of the unlabeled toxins (-Aga-IIIA, -CTX-GVIA, -CTX-MVIIA, or -CTX-MVIIC) were incubated with 0.7 µg of rat brain synaptosomal membrane proteins suspended in 1.89 ml of HEPES buffer for 2 h. 100 µl of 50 pM 125I--Aga-IIIA was added to each of the reaction tubes (final concentration of 125I--Aga-IIIA was 2.5 pM). After another 2-h incubation, the reactions were terminated by rapid filtration. The nonspecific binding of 125I--Aga-IIIA was defined as the amount of binding in the presence of 10 nM unlabeled -Aga-IIIA, while 100% of total 125I--Aga-IIIA binding was defined as the amount of 125I--Aga-IIIA binding in the absence of toxins. The curves were fitted with logistic function for the average of three experiments performed in duplicate. The error bars were standard error of the mean.

-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 N- and 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, ¹²⁵I--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 ¹²⁵I--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 ¹²⁵I--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 ¹²⁵I--Aga-IIIA indeed binds specifically to recombinant _1B and _1E calcium channels.

View this table: [in this window] [in a new window]   Table 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 125I--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 125I--Aga-IIIA in the presence of unlabeled excess -Aga-IIIA. S, specific binding or the total amount of bound 125I--Aga-IIIA in the absence of unlabeled -Aga-IIIA minus nonspecific binding.

We determined the affinity of ¹²⁵I--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 = 2.43, p = 0.19 at the 0.05 level) (Figs. 6B and 7B). We note that these values agree well with the K_d value of ~9 pM obtained with rat brain membranes (Figs. 3 and 4).

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Equilibrium binding assay of [125]-Aga-IIIA binding to 1B subunits of calcium channels. 0.8 µg of S3 cell membrane proteins was incubated in the presence of increasing concentrations of [125]-Aga-IIIA for 2 h. Shown is the average of three experiments. Error bars are standard error of the mean. A, direct plot of specific binding data of equilibrium binding assay. Specific binding was obtained by subtracting the nonspecific binding from the total. B, Scatchard analysis of the specific binding data shown in A.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Equilibrium binding assay of 125I--Aga-IIIA binding to 1E channels. Experiments were carried out as described in Fig. 5 legend. 1.5 µg of 192C cell membrane proteins was incubated in the presence of increasing concentrations of 125I--Aga-IIIA for 2 h. A, direct plot of 125I--Aga-IIIA specific binding data. B, Scatchard analysis. The results were the average of three independent experiments. The error bars were standard error of the mean.

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 ¹²⁵I--Aga-IIIA and ¹²⁵I--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. 8B). This provides independent evidence confirming one-to-one binding of -Aga-IIIA to N-type calcium channels.

View larger version (17K): [in this window] [in a new window]   Fig. 8.   Comparing the number of 125I--Aga-IIIA and 125I--CTX-MVIIA binding sites on the 1B subunits of calcium channels. 1.5 µg of S3 cell membrane proteins was incubated in the presence of increasing concentrations of a radiolabeled toxin (125I--CTX-MVIIA or 125I--Aga-IIIA) for 2 h. A, direct plot of saturation data. B, Scatchard analysis. Squares and triangles represent the specific binding of 125I--CTX-MVIIA or 125I--Aga-IIIA, respectively. Shown are results from one of the representative experiments.

Sensitivity to Divalent Ions-- The affinity of -Aga-IIIA to calcium channels as determined by the above binding experiments (K_d = 5.1-9.7 pM) is about 3 orders of magnitude higher than that determined by previous electrophysiological studies (K_d = 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 BaCl₂), 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 ¹²⁵I--Aga-IIIA to synaptosomal membranes in binding buffer as compared with that in the electrophysiological recording buffer. The results showed that the binding of ¹²⁵I--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 = 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.

View larger version (22K): [in this window] [in a new window]   Fig. 9.   The effect of different buffers on 125I--Aga-IIIA binding to synaptosomal membranes. Equilibrium binding assays were performed as described under "Experimental Procedures" in either binding buffer or electrophysiological recording buffer (see "Experimental Procedures" for details). A, saturation curves. B, Scatchard analysis. The results were the average of three independent experiments. The error bars are standard error of the mean.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 ~5-9 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. 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 ¹²⁵I--Aga-IIIA to native and recombinant channels is saturable, specific, and high affinity (K_d = 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 = ~0.3-1 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 IC₅₀ 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 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 ¹²⁵I--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 (~18%) of total -Aga-IIIA binding. A somewhat larger component (~40%) 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 N- and 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 ~0.3-1 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.

	ACKNOWLEDGEMENTS

We thank Drs. James Offord and David Rock (Parke-Davis Pharmaceuticals) for supplying the _1B and _1E calcium channel clones and helpful information, Dr. George Miljanich (Elan Pharmaceuticals) for supplying -conotoxin MVIIC, and Drs. David Johnson, Baldomero Olivera and Guoqiang Jiang for helpful advice and comments during the course of this study.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Membrane Biochemistry and Biophysics, Merck Research Laboratories, Rahway, NJ 07065.

^§ To whom correspondence should be addressed: Dept. of Neuroscience, 5419 Boyce Hall, University of California, Riverside, CA 92521. Tel.: 909-787-4746; Fax: 909-787-3087; E-mail: adams@mail.ucr.edu.

Published, JBC Papers in Press, May 2, 2000, DOI 10.1074/jbc.M000212200

	ABBREVIATIONS

The abbreviations used are: HVA, high voltage-activated; LVA, low voltage-activated; CTX, conotoxin; Aga, agatoxin; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES