Phoneutria nigriventer (cid:1) -Phonetoxin IIA Blocks the Ca v 2 Family of Calcium Channels and Interacts with (cid:1) -Conotoxin-binding Sites*

-Phonetoxin IIA ( (cid:1) PtxIIA), a peptide from spider venom ( Phoneutria nigriventer ), inhibits high threshold voltage-dependent calcium currents in neurons. To de-fine its pharmacological specificity, we have used patch-clamp methods in cell lines expressing recombinant v 2.2 higher concentrations, 50%, did not a distinct plateau. Subtraction of the Ca v 2.2 component v 2.1 may at least 25% of sites, this may be an underestimation. These are comparable to data recently reported using 125 I- (cid:5) AgaIIIA, a ligand that labels all high threshold calcium channels in synaptosomes and that is also partially displaced by (cid:5) -conotoxins The fraction of I- AgaIIIA binding to synaptosomes displaced by (cid:5) -conotoxins is smaller (cid:1) 40% (cid:5) MVIIC), consistent with the idea that I- AgaIIIA wider spectrum I- PtxIIA.

Voltage-gated calcium channels play a crucial role in coupling the electrical activity of neurons to a variety of cellular processes, including gene expression, morphological differentiation, and the synaptic release of neurotransmitters. Neurons express multiple types of calcium channels that were initially classified according to the biophysical and pharmacological properties of transmembrane currents. Channels were subsequently characterized in molecular terms as hetero-oligomers composed of ␣ 1 , ␣ 2 /␦, ␤, and ␥ subunits and are generally defined by the nature of the pore-forming ␣ 1 subunit (1-3). The high threshold calcium channels of the Ca v 2 family carry P/Q-, N-, or R-type currents and have been designated as Ca v 2.1 (␣ 1 A subunits), Ca v 2.2 (␣ 1 B subunits), and Ca v 2.3 (␣ 1 E subunits), respectively (4).
Ca v 2 channels are insensitive to the 1,4-dihydropyridine (DHP) 1 drugs that block high threshold channels of the Ca v 1 (L-type) family. However, a panel of natural peptide antagonists has been identified in animal venoms, providing molecular probes to analyze Ca v 2 channel structure and function.
-Conotoxins GVIA (GVIA) and MVIIA purified from the venoms of the marine mollusks Conus geographus and Conus magus, respectively, are specific ligands for Ca v 2.2 channels (3). Another C. magus peptide, -conotoxin MVIIC (MVIIC), blocks both Ca v 2.1 and Ca v 2.2 channel currents at micromolar concentrations, whereas at subnanomolar concentrations 125 I-MVIIC constitutes a specific radioligand for Ca v 2.1 channels (5)(6)(7). -Agatoxins from the spider Agenelopsis aperta also inhibit currents generated by Ca v 2 channels, and blockade by -agatoxin (Aga) IVA is considered as diagnostic for Ca v 2.1 channel activity (8). Differential sensitivity to -AgaIVA initially defined the P-and Q-type currents (9), which were subsequently shown to be supported by Ca v 2.1 splice variants (10). A second A. aperta peptide, AgaIIIA, blocks all high threshold calcium channels and provides a broad spectrum antagonist (11,12). Finally, SNX-482 from the venom of the tarantula Hysterocrates gigas inhibits a subclass of Ca v 2.3 channels (13) initially designated as R-type channels because of their resistance to blockade by DHPs, GVIA, and AgaIVA.
Thus, peptide antagonists isolated from animal venoms have been instrumental in pharmacologically defining neuronal Ca v 2 channels and particularly in demonstrating their role in transmitter and neuropeptide release. Furthermore, radiolabeled derivatives of these molecules are used as ligands to evaluate calcium channel density, to monitor biochemical isolation of channel proteins, and to assay anti-calcium channel antibodies for the diagnosis of human autoimmune disease (14,15). In this context, we have examined the properties of -phonetoxin IIA (PtxIIA), a neurotoxic peptide from the South American spider Phoneutria nigriventer. Subnanomolar concentrations of PtxIIA have been recently reported to block native N-type currents (Ca v 2.2) in rat dorsal root ganglion neurons, but to be inactive on low voltage-activated T-type currents (Ca v 3) (16).
In this study, we describe the purification of PtxIIA from P. nigriventer venom and evaluation of its activity on calcium currents generated by recombinant Ca v 2.1, Ca v 2.2, and Ca v 2.3 channels expressed in stable baby hamster kidney (BHK) cell lines. Our electrophysiological data indicate that PtxIIA is a potent and practically irreversible antagonist of both Ca v 2.1 and Ca v 2.2, whereas it displays partial and rapidly reversible blockade of Ca v 2.3. Recombinant Ca v 2.1 and Ca v 2.2 channels each constitute a single class of high affinity binding sites for 125 I-PtxIIA. In contrast, nerve terminals contain multiple classes of binding sites, and competition with -conotoxins suggests that a fraction of the high affinity sites are associated with native Ca v 2.1 and Ca v 2.2 channels.

EXPERIMENTAL PROCEDURES
Purification and Sequencing of PtxIIA-P. nigriventer venom was obtained by electrical stimulation of anesthetized spiders. The collection and storage of venom and fraction P24C4 were performed as described by Rezende et al. (17). ⌻he pooled fractions of P24C4 (causing flaccid paralysis) were obtained by reverse chromatography on a Vydac C 4 column (0.46 ϫ 25 cm, 5 m, 300 Å), dissolved in 0.1% (v/v) trifluoroacetic acid, and subjected to reverse-phase HPLC on a Vydac C 18 (218TP54) analytic column (0.46 ϫ 25 cm) equilibrated in the same solvent. The column was eluted (1 ml/min) with a linear gradient of 0 -21% acetonitrile in 0.1% trifluoroacetic acid for 15 min, followed by a second linear gradient from 21 to 29.4% for 35 min. Separation was conducted on a Hewlett-Packard HP1100 system coupled to a UV detector while monitoring elution at 215 nm. A second purification step was carried out with a linear gradient of 0 -40% acetonitrile in 0.3% trifluoroacetic acid for 80 min on the same column. The toxin eluted in the second step was rechromatographed under the same conditions and sequenced with an Applied Biosystems 476A sequencer using a standard protocol.
Mass spectrometry of the native toxin dissolved in ␣-cyano-4-hydroxycinnamic acid matrix was carried out on a MALDI-TOF mass spectrometer (Voyager DE-RP, Perseptive Biosystems, Framingham, MA) with the linear mode and positive polarity using an internal calibration method with a mixture of bovine insulin (M ϩ H ϩ ϭ 5734.59 Da, average mass) and horse apomyoglobin (M ϩ 2H ϩ /2 ϭ 8476.6 Da, average mass). Data were analyzed using GRAMS386 software.
Cell Culture and Electrophysiology-Stable BHK cell lines expressing Ca v 2.1 splice variant BI-2 (18), Ca v 2.2, or Ca v 2.3 splice variant BII-2 (19) with the ␣ 2 a/␦ and ␤ 1 a auxiliary subunits were cultured as previously described (20). Whole cell currents were recorded from isolated cells using wide tipped patch pipettes (1.5-3 megaohms). pClamp6 and Sigmaplot software were used for experimental protocol and analysis. Whole cell current traces (with inward currents downward) are shown with leak subtraction, after digitization (4 kHz) and low pass filtering (1 kHz). Dashed lines near the traces indicate zero current level. The voltage stimulation protocol consisted of steps to ϩ10 mV (100 ms) applied every 30 s from a holding potential of Ϫ90 mV. In figures, the peak calcium (or sodium in Fig. 2H) current was plotted against episode starting time. The transient sodium current present in some cells (e.g. Fig. 2C) did not impair the measurement of peak calcium currents, performed in the absence of tetrodotoxin. The pipette solution was composed of 130 mM CsCl, 1 mM MgCl 2 , 10 mM HEPES, 10 mM BAPTA, and 3 mM ATP adjusted to pH 7.2 with CsOH. The extracellular solution contained 150 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 10 mM glucose, and 10 mM HEPES adjusted to pH 7.4 with NaOH; and the solutions superfused before or during PtxIIA application also contained bovine serum albumin (1 g/liter, fraction V, Roche Molecular Biochemicals). Except during application of LaCl 3 , a continuous solution flow was applied near the cell using a local multi-way superfusion system (70 -100 l/min). Solution flow was stopped for manual applications of LaCl 3 (50 l, 1 mM) near the cell.
Analysis of Electrophysiological Data-We analyzed calcium current inhibition data assuming the channel to be functional when free (R) and blocked when bound to a toxin molecule (RL), with rate constants k ϩ1 for association and k Ϫ1 for dissociation of the toxin (L)-to-channel reversible binding reaction (R ϩ L 7 RL). In this model, for a given stimulation protocol (tens of millisecond range), the ratio of peak calcium currents observed in the presence of a given toxin concentration (L) versus absence of toxin (peak I Ca , fraction remaining) (see Fig. 3, A and B) is assumed to be at any time (minute range) equal to the ratio of toxin-free channel concentration (R[t]) to total channel concentration (R T ).
Blockade at Equilibrium-The fraction of peak calcium current inhibition at the plateau of blockade or the fraction of channel occupancy at equilibrium (RL eq /R T ) is linked to the constant toxin concentration applied (L eq ϭ L) by the Michaelis equation, RL eq /R T ϭ 1/(1 ϩ K D /L), where K D ϭ k Ϫ1 /k ϩ1 . Therefore, a measurement of K D is derived from peak current inhibition data by K D ϭ L⅐(fraction remaining/fraction inhibited).
Off Kinetics-Following toxin washout at time 0, the toxin dissociation reaction in the absence of free toxin follows a simple law, . Therefore, the toxin dissociation rate constant (k Ϫ1 ) is measured directly as the reciprocal of the time constant of calcium current recovery: 1/ off ϭ k Ϫ1 .
On Kinetics-During superfusion of a constant toxin concentration (L), starting at time 0, the kinetic equation . Therefore, the reciprocal of the time constant of calcium current blockade or the apparent inhibition time constant (k app ) is k app ϭ 1/ on ϭ k ϩ1 ⅐L ϩ k Ϫ1 , and a plot of k app ϭ 1/ on against toxin concentration gives k ϩ1 (slope) and k Ϫ1 (extrapolation to null toxin concentration) (see Fig. 3C).
Iodination of PtxIIA-PtxIIA contains two tyrosine residues. 1 nmol of native PtxIIA was reacted for 2 min with 0.3 nmol of carrierfree Na 125 I (Amersham Biosciences) in the presence of lactoperoxidase and H 2 O 2 in 50 mM phosphate buffer at pH 7.2. Monoiodotoxins were separated by reverse-phase liquid chromatography on a C 18 column (0.46 ϫ 25 cm; 5-m particles). The column was eluted with a linear gradient of 20 -33% acetonitrile for 65 min in constant 0.1% trifluoroacetic acid at a flow rate of 1 ml/min. Native toxin was eluted after 29 min, and two radioactive peaks (I and II) corresponding to the two possible monoiodo-PtxIIA derivatives were detected at 27 and 40 min. Mono[ 125 I]iodo-PtxIIA from peak II was used in this study, calculating the concentration from the specific radioactivity of 2200 Ci (81.4 TBq)/mmol.
Binding Assays-The standard buffer contained 10 mM Tris, 140 mM NaCl, and 0.1% bovine serum albumin adjusted to pH 7.2 with HCl. Binding studies with synaptosomes or membranes from BHK cell lines were carried out in 0.2 or 0.3 ml of buffer. Bound ligand was separated by rapid filtration over glass-fiber filters (Whatman GF/C) pretreated with 0.3% polyethyleneimine in aqueous solution. After washing the filters three times with 2 ml of ice-cold binding buffer, bound ligand was measured by ␥-counting with 60% efficiency. In all experiments, the level of nonspecific binding (data not shown) was determined in the presence of 50 -100 nM unlabeled PtxIIA and subtracted from the total binding to yield the specific component. At 0.2 nM 125 I-PtxIIA, nonspecific binding accounted for ϳ10% (BHK cells) or 30% (synaptosomes) of the total binding. Membrane protein concentrations were chosen so that Ͻ8% of the radioligand was bound in the course of the experiment. Under these conditions, the total ligand concentration is considered to be an acceptable approximation of the free ligand concentration.

RESULTS
PtxIIA was purified from P. nigriventer venom using a published method with certain modifications (see "Experimental Procedures"). The molecular mass was determined by MALDI-TOF spectrometry using internal calibration. Data analysis yielded a molecular mass of 8364 Da for ion M ϩ H ϩ , which, together with Edman sequencing of the first 41 residues, was entirely consistent with published data, allowing identification of the purified peptide as PtxIIA (Fig. 1).
Voltage-dependent currents were measured in BHK cells expressing Ca v 2.1, Ca v 2.2, and Ca v 2.3 channels using the whole cell configuration of the patch clamp with 2 mM Ca 2ϩ as the charge carrier.
Step depolarization from a holding potential of Ϫ90 mV to a test potential of ϩ10 mV evoked inward calcium currents that were reduced by perfusion of PtxIIA (Fig. 2, A, C, and E). Inhibition of Ca v 2.1 and Ca v 2.2 developed over tens of minutes, reaching Ͼ70% blockade at nanomolar concentrations of PtxIIA (Fig. 2, A-D). The effect of PtxIIA on Ca v 2.1 and Ca v 2.2 channels was practically irreversible, displaying Ͻ15% unblockade after a 30-min washout period (Fig. 2, B and D). In contrast, 17 nM PtxIIA inhibited Ca v 2.3 channel current only by 20%, with significantly more rapid off rates (Fig. 2, E and F). Some BHK cells, including cells from the recipient cell line tk-ts13 (Fig. 2G), express an endogenous fast, tetrodotoxin-sensitive sodium current, which was unaffected by the application of PtxIIA (Fig. 2, C and H). Additional experiments with a Chinese hamster ovary cell line expressing the voltage-gated sodium channel Na v 1.2 indicated that sodium currents were totally insensitive to PtxIIA (data not shown). These results indicate that nanomolar concentrations of PtxIIA inhibit the three channels of the Ca v 2 family. PtxIIA displays a similar high apparent affinity for Ca v 2.1 and Ca v 2.2, but its action on Ca v 2.3 is less potent and more readily reversible.
We analyzed calcium current inhibition data assuming a simple mechanism, the channel being functional when free and blocked when bound to a toxin molecule, with rate constants k ϩ1 for association and k Ϫ1 for dissociation (see "Experimental Procedures"). For Ca v 2.3 channels, fitting on and off peak current inhibition data produced time constants off ϭ 85 s and on ϭ 68 s using 17 nM PtxIIA, corresponding to k Ϫ1 ϭ 1.2 ϫ 10 Ϫ2 s Ϫ1 , k ϩ1 ϭ 1.8 ϫ 10 5 M Ϫ1 s Ϫ1 , and k Ϫ1 /k ϩ1 ϭ K D ϭ 67 nM. For Ca v 2.1 and Ca v 2.2 channels, washout reactions were not accessible; therefore, we used a graphical method to derive both k Ϫ1 and k ϩ1 from the apparent on rate constants (k app ) at various toxin concentrations ([Tx]) according to ( on ) Ϫ1 ϭ k app ϭ k ϩ1 ⅐[Tx] ϩ k Ϫ1 (Fig. 3). Association rate constants, measured as the slopes of linear fits of k app versus toxin concentration (Fig.  3C), were as follows: for Ca v 2.1 channels, k ϩ1 ϭ 7.1 ϫ 10 4 M Ϫ1 s Ϫ1 ; and for Ca v 2.2 channels, k ϩ1 ϭ 2.6 ϫ 10 5 M Ϫ1 s Ϫ1 . Extrapolation to null toxin concentration should then give k Ϫ1 . But due to some spontaneous channel rundown during whole cell recording, it is here in fact the sum of k Ϫ1 and a rate constant of rundown. Therefore, this graphical analysis gives an upper limit for k Ϫ1 and K D values. For Ca v 2.1 channels, k Ϫ1 Ͻ 3 ϫ 10 Ϫ4 s Ϫ1 and K D Ͻ 4 nM. For Ca v 2.2 channels, k Ϫ1 Ͻ 10 Ϫ3 s Ϫ1 and K D Ͻ 4 nM. Another evaluation of K D and k Ϫ1 values was obtained considering the fraction of current remaining at equilibrium (see "Experimental Procedures") and k ϩ1 values derived from on kinetics. For Ca v 2.1 channels, Ͼ90% of blockade at 10 nM gives K D Ͻ 1 nM; and therefore, k Ϫ1 Ͻ 7 ϫ 10 Ϫ5 s Ϫ1 and off Ͼ 230 min. For Ca v 2.2 channels, Ͼ95% of blockade at 3.5 nM gives K D Ͻ 0.2 nM; and therefore, k Ϫ1 Ͻ 5 ϫ 10 Ϫ5 s Ϫ1 and off Ͼ 330 min.
To directly address the binding properties of PtxIIA, 125 I iodination was performed by the lactoperoxidase method, and a mono-125 I-iodinated derivative was purified by reverse-phase HPLC (Fig. 4). 125 I-PtxIIA was used to titrate binding sites associated with recombinant Ca v 2.1, Ca v 2.2, and Ca v 2.3 channels using membranes prepared from stable transfected BHK cell lines. 125 I-PtxIIA displayed saturable binding to both Ca v 2.1 and Ca v 2.2 as illustrated by plots of the specific binding component (Fig. 5A). Scatchard plots of these data indicate a single class of binding sites for both Ca v 2.1 (equilibrium dissociation constant, K D ϭ 160 pM; and binding site capacity, B max ϭ 0.4 pmol/mg of protein) and Ca v 2. M MVIIC and GVIA, respectively (data not shown). Thus, -conotoxin binding to recombinant channels can totally occlude the PtxIIA-binding site.
In contrast, in the same concentration range, binding of 125 I-PtxIIA to membranes expressing Ca v 2.3 channels was barely detectable. This is unlikely to be due to the fact that Ca v 2.3 channel expression is significantly lower, as whole cell currents in the 1-nA range were recorded (Fig. 2E), which were generally comparable or superior to those in Ca v 2.1 and Ca v 2.2 cell lines. A small specific binding component was detected at ligand concentrations Ͼ0.4 nM, indicative of low affinity binding sites. However, for practical reasons, it was impossible to quantify interactions with Ca v 2.3 channels using 125 I-PtxIIA. Binding assays with recombinant Ca v 2 channels were thus globally consistent with the electrophysiological analysis, indicating that Ca v 2.1 and Ca v 2.2 each constitute a single class of sites with similar high affinity for 125 I-PtxIIA, whereas Ca v 2.3 channels form low affinity sites.
The binding properties of 125 I-PtxIIA were also assessed using rat brain synaptosomes. The association and dissociation kinetics of 125 I--PtxIIA with rat brain synaptosomal membranes are shown in Fig. 6. Semilogarithmic plots (Fig. 6A, inset) of the association data indicated the presence of at least two components, suggesting heterogeneity of 125 I-PtxIIAbinding sites. Linearization of the initial time points gave k app ϭ 10.4 ϫ 10 Ϫ4 s Ϫ1 . Binding was reversible (Fig. 6B), and the semilogarithmic plot (Fig. 6B, inset) yielded the dissociation rate constant k Ϫ1 ϭ 2.2 ϫ 10 Ϫ4 s Ϫ1 . The equation k ϩ1 ϭ (k app Ϫ k Ϫ1 )/[ 125 I-PtxIIA] gave the association rate constant k ϩ1 ϭ 4.1 ϫ 10 6 M Ϫ1 s Ϫ1 . The equilibrium dissociation constant was therefore K D ϭ k Ϫ1 /k ϩ1 ϭ 55 pM.
Displacement assays using 0.2 nM 125 I-PtxIIA can be used to evaluate some of the properties of the high affinity binding sites in synaptosomes. Native PtxIIA competes with 125 I-PtxIIA for interaction with synaptosomal membranes from rat brain (Fig. 7A) (Fig. 7A), suggesting that at least half of the high affinity PtxIIA-binding sites are associated with Ca v 2.1 and Ca v 2.2 channels. In contrast, similar experiments with GVIA indicated a maximum of 25% displacement of 125 I-PtxIIA achieved with 1 nM GVIA (Fig. 7A). Thus, at least 25% of high affinity 125 I-PtxIIA-binding sites in nerve terminals are associated with the Ca v 2.2 channel; and by subtracting GVIA-sensitive sites (25%) from MVIIC-sensitive sites (50%), ϳ25% are associated with the Ca v 2.1 channel. Thus, a significant residual fraction of high affinity 125 I-PtxIIA-binding sites in synaptosomes (ϳ50%) appear to be associated with MVIIC-insensitive calcium channels.
Reciprocal experiments were performed to confirm the binding of PtxIIA to native Ca v 2.1 and Ca v 2.2 channels in synaptosomes using 125 I-MVIIC and 125 I-GVIA. Total displacement of 125 I-MVIIC and 125 I-GVIA was achieved with calculated K i values of 100 and 80 pM, respectively (Fig. 7, B   FIG. 6. Kinetics of 125 I-PtxIIA binding to rat brain synaptosomes. A, association kinetics. 125 I-PtxIIA (0.2 nM) was added to rat brain synaptosomes (10 g of protein), and the membrane-bound radioactivity was determined after the indicated times at 30°C. Nonspecific binding estimated in the presence of 0.1 M unlabeled PtxIIA was subtracted. B, dissociation kinetics. At equilibrium, 0.1 M unlabeled PtxIIA was added, and the remaining specifically bound radioactivity was measured at the indicated times. Insets, linear semilogarithmic plots of kinetic data. B ϭ specifically bound membrane radioactivity; Beq ϭ B at equilibrium. The data illustrated are the results of one of two independent experiments that yielded quasi-identical rate constants. 125 I-GVIA and 125 I-MVIIC binding is known to be inhibited by millimolar concentrations of Ca 2ϩ ions (21,22). The fact that a fraction of PtxIIA-binding sites in synaptosomes overlap with -conotoxin-binding sites led us to examine the effect of Ca 2ϩ ; however, the presence of 1.5 mM EGTA or 1.5 mM CaCl 2 did not modify 125 I-PtxIIA binding (data not shown).
Finally, equilibrium binding experiments were also performed in which increasing concentrations of 125 I--PtxIIA were added to rat brain synaptosomes in the absence or presence of unlabeled -PtxIIA. Although specific binding increased with increasing ligand concentration, saturation was not achieved at concentrations approximately six times that of the K D calculated from binding kinetics (data not shown). Thus, Scatchard analysis was not performed, and the binding site capacity in synaptosomes cannot be accurately determined. These results suggest that rat brain nerve terminals express multiple classes of 125 I-PtxIIA-binding sites. They include high affinity sites associated with Ca v 2.1 and Ca v 2.2 that are occupied at subnanomolar concentrations and additional lower affinity sites that saturate at nanomolar concentration ranges. A reasonable interpretation, consistent with binding assays performed on recombinant channels, would be that at least some of the low affinity sites correspond to native Ca v 2.3 channels. Table I summarizes the binding parameters of PtxIIA, indicating that results from radioligand binding assays were generally consistent with patch-clamp recording data. The only discrepancy concerned the fact that the dissociation of 125 I-PtxIIA from synaptosomes was significantly more rapid than relief from toxin blockade in BHK cells expressing Ca v 2.1 and Ca v 2.2. This may be due to differences in methodology (binding assays versus electrophysiology) or in the molecular composition of channels (native presynaptic channels versus heterologously expressed channels). DISCUSSION P. nigriventer, the South American "armed" spider, is an aggressive species frequently responsible for human envenomation in Brazil. In 1993, Cordeiro et al. (23) reported purification and partial sequencing of several neurotoxic peptides from P. nigriventer venom, including a 40-amino acid N-terminal sequence designated as Tx3-4. Tx3-4 was subsequently shown to inhibit 45 Ca 2ϩ influx into rat brain synaptosomes (24). Tx3-4 displays identity at 38 positions to the 40-amino acid N-terminal sequence of PtxIIA recently described by Cassola et al. (16). The peptide that we have characterized pharmacologically displays molecular mass and sequence identical to those of PtxIIA; and thus, we have maintained this nomenclature. PtxIIA has significant homology to AgaIIIA and AgaIIIB from A. aperta (11). The three sequences display 49% homology when aligned with two gaps, although two substitutions suggest that PtxIIA may be more similar to AgaIIIB (16).
An initial report on the activity of PtxIIA demonstrated that it blocks high threshold DHP-resistant calcium current in dorsal root ganglion neurons, but spares low voltage-activated T-type current (16). This suggests that PtxIIA inhibits channels of the Ca v 2 family that generate P/Q-, N-, and R-type currents, but does not allow a more precise definition of pharmacological specificity. We therefore evaluated the effects of PtxIIA on whole cell calcium currents in stable BHK cell lines expressing recombinant Ca v 2.1 (␣ 1 A subunits), Ca v 2.2 (␣ 1 B subunits), or Ca v 2.3 (␣ 1 E subunits) channels, associated with the same auxiliary subunits. PtxIIA in the 10 nM range produced total and practically irreversible blockade of Ca v 2.1 and Ca v 2.2, whereas Ca v 2.3 was only partially inhibited in a rapidly reversible manner. We have not examined whether PtxIIA inhibits recombinant channels of the DHP-sensitive Ca v 1 family. Relatively high concentrations of PtxIIA have been shown to block a component of high voltage-activated calcium current in pancreatic ␤ cells that express a major DHP-sensitive current component (16). PtxIIA may thus block some Ca v 1 channels with lower affinity than Ca v 2.1 and Ca v 2.2 channels, but additional studies will be required to resolve this issue. The pharmacological profile of PtxIIA that is emerging from electrophysiological studies suggests that it represents an intermediate between AgaIIIA, which inhibits all classes of high threshold calcium channels with equivalent potency, and peptides such as AgaIVA and GVIA that are diagnostic for single channel subtypes. In keeping with the structural homology discussed above, PtxIIA may resemble AgaIIIB, which inhibits N-type (Ca v 2.2) current more potently than L-type (Ca v 1.2) current (11). 125 I-PtxIIA displayed specific high affinity binding to membranes containing recombinant calcium channels and to rat brain synaptosomes. Results with BHK cell membranes heterologously expressing Ca v 2 channels were consistent with electrophysiological analysis of channel blockade. Ca v 2.1 and Ca v 2.2 channels each constitute a single class of high affinity sites with practically identical K D values, indicating that PtxIIA does not distinguish between these two channel subtypes. Although cells expressing Ca v 2.3 subunits displayed robust calcium currents in the 1-nA range, the specific binding of 125 I-PtxIIA to Ca v 2.3 was barely detectable, and the low affinity of this interaction precluded further binding studies. 125 I-PtxIIA displayed specific binding to rat brain synaptosomes, but complex kinetics, partial displacement by other antagonists, and lack of saturation revealed multiple classes of sites. This heterogeneity is unlikely to result from the nature of the ligand or other aspects of methodology. A purified monoiodinated radioligand was used, and saturable binding to a single class of sites was demonstrated when membranes expressed a homogeneous population of recombinant channels. Thus, binding site heterogeneity in synaptosomes is likely to result from the expression of multiple types of calcium channel.
Interactions between -conotoxins and PtxIIA indicated that Ca v 2.1 and Ca v 2.2 account for at least 50% of the high affinity sites in synaptosomes. PtxIIA totally occluded 125 I-GVIA and 125 I-MVIIC binding, in a manner consistent with competitive inhibition, indicating that it binds to native Ca v 2.1 and Ca v 2.2 channels. Furthermore, binding experiments with recombinant Ca v 2.1 and Ca v 2.2 channels indicated that submicromolar concentrations of -conotoxins totally displaced 125 I-PtxIIA. In synaptosomes, however, displacement of 125 I- 3) expressed in BHK cells or native calcium channels in rat brain synaptosomes were determined. The constants indicated in boldface were derived from radioligand binding assays. Other constants were calculated from the kinetics of blockade during toxin application and relief from blockade upon washout (see "Experimental Procedures"), determined by patch-clamp recording of calcium currents. Constants derived from electrophysiological data at equilibrium are not included. ND, not determined. PtxIIA by increasing concentrations of GVIA was only partial and reached a distinct plateau, suggesting that Ca v 2.2 accounts for ϳ25% of high affinity PtxIIA-binding sites. Displacement by MVIIC, which binds to Ca v 2.1 but also occludes Ca v 2.2 at higher concentrations, reached 50%, but did not attain a distinct plateau. Subtraction of the Ca v 2.2 component indicates that Ca v 2.1 may contribute at least 25% of sites, but this may be an underestimation. These results are comparable to data recently reported using 125 I-AgaIIIA, a ligand that labels all high threshold calcium channels in synaptosomes and that is also partially displaced by -conotoxins (12). The fraction of 125 I-AgaIIIA binding to synaptosomes displaced by -conotoxins is smaller (ϳ40% with MVIIC), consistent with the idea that 125 I-AgaIIIA labels a wider spectrum of calcium channels than 125 I-PtxIIA. Which high threshold calcium channels account for the significant fraction of 125 I-PtxIIA binding that is not inhibited by -conotoxins? It is unlikely that DHP-sensitive channels of the Ca v 1 family provide a major contribution, as channel blockade occurs only at relatively high concentrations of PtxIIA (16). A comparison of the binding capacities of synaptosomes for 125 I-GVIA and [ 3 H]DHP in our laboratory has indicated a ratio of about six Ca v 2.2/one Ca v 1 (21,25). If Ca v 2.2 constitutes ϳ25% of 125 I-PtxIIA-binding sites, then Ca v 1 can account for only Ͻ5%. It is equally improbable that Ca v 2.3 channels contribute to residual 125 I-PtxIIA binding. Experiments with recombinant Ca v 2.3 channels did not reveal significant specific binding at the ligand concentration (0.2 nM) used in displacement assays. However, Ca v 2.3 and possibly Ca v 1 may contribute to the low affinity sites that appear as "non-saturable" binding at higher concentrations of 125 I-PtxIIA. The most parsimonious explanation for the residual sites is that MVIIC receptor occupation is incomplete, as suggested by the lack of plateau in the displacement curve. Our experiments were performed at relatively high ionic strength, which decreases the affinity of MVIIC for its binding sites (5). Thus, part of the residual 125 I-PtxIIA binding may involve Ca v 2.1 channel variants with a low sensitivity to MVIIC. Further work will be necessary to unequivocally identify this channel population.
In conclusion, PtxIIA is a potent blocker of Ca v 2.1 and Ca v 2.2 channels, although it interacts with Ca v 2.3 and possibly certain channels of the Ca v 1 family at higher concentrations. At subnanomolar concentrations, 125 I-PtxIIA simultaneously labels Ca v 2.1 and Ca v 2.2 channels, the major channel subtypes implicated in neurotransmitter release. These channels are the target for autoantibodies in Lambert-Eaton myasthenic syndrome, a human autoimmune disease affecting neurotransmission. Current antibody assay protocols used for diagnosis involve immunoprecipitation of native calcium channels labeled with 125 I-MVIIC and/or 125 I-GVIA. The specificity and stability of 125 I-PtxIIA suggest that it may provide a useful alternative reagent.