Structure-Function Relationships of omega -Conotoxin GVIA
SYNTHESIS, STRUCTURE, CALCIUM CHANNEL BINDING, AND FUNCTIONAL ASSAY OF ALANINE-SUBSTITUTED ANALOGUES*

(Received for publication, November 15, 1996, and in revised form, February 6, 1997)

Michael J. Lew Dagger §, James P. Flinn Dagger par , Paul K. Pallaghy , Roger Murphy Dagger , Sarah L. Whorlow Dagger , Christine E. Wright Dagger , Raymond S. Norton ** and James A. Angus Dagger

From the Dagger  Department of Pharmacology, University of Melbourne and the  Biomolecular Research Institute, 343 Royal Parade, Parkville 3052, Victoria, Australia

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The structure-function relationships of the N-type calcium channel blocker, omega -conotoxin GVIA (GVIA), have been elucidated by structural, binding and in vitro and in vivo functional studies of alanine-substituted analogues of the native molecule. Alanine was substituted at all non-bridging positions in the sequence. In most cases the structure of the analogues in aqueous solution was shown to be native-like by 1H NMR spectroscopy. Minor conformational changes observed in some cases were characterized by two-dimensional NMR. Replacement of Lys2 and Tyr13 with Ala caused reductions in potency of more than 2 orders of magnitude in three functional assays (sympathetic nerve stimulation of rat isolated vas deferens, right atrium and mesenteric artery) and a rat brain membrane binding assay. Replacement of several other residues with Ala (particularly Arg17, Tyr22 and Lys24) resulted in significant reductions in potency (<100-fold) in the functional assays, but not the binding assay. The potencies of the analogues were strongly correlated between the different functional assays but not between the functional assays and the binding assay. Thus, the physiologically relevant assays employed in this study have shown that the high affinity of GVIA for the N-type calcium channel is the result of interactions between the channel binding site and the toxin at more sites than the previously identified Lys2 and Tyr13.


INTRODUCTION

The fish-hunting marine cone snails produce a range of polypeptide toxins that rapidly immobilize their prey (1, 2). A number of such toxins targeted at ion channels have been isolated from the venoms of these cone shells, including several that selectively block N- and P-type calcium channels (3). The toxin studied here, omega -conotoxin GVIA (GVIA),1 is a 27-residue polypeptide from Conus geographus (4) that selectively blocks N-type voltage-gated calcium channels (5, 6), an activity that may confer a number of useful therapeutic properties on GVIA, including antihypertensive, analgesic, and neuroprotective activities, as demonstrated for the closely related omega -conotoxin MVIIA (MVIIA) from Conus magus (7) .

The amino acid sequence of GVIA (with the cystine bridges indicated by lines) is as show below.
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O indicates hydroxyproline. The three-dimensional structure of GVIA in solution has been solved by a number of groups (8-11). The molecule is a member of the inhibitor cystine knot structural family (12, 13), the members of which share a triple-stranded beta -sheet and a three-disulfide bridge cystine knot. The structure of the homologous N-type calcium channel blocker MVIIA is essentially identical to that of GVIA (14, 15), and the structure of the closely related P-type calcium channel blocker from C. magus, omega -conotoxin MVIIC (MVIIC), is very similar (16, 17) .

A number of studies of GVIA have been carried out in an attempt to identify those residues that are essential for its high affinity interaction with the N-type calcium channel. The role of the ammonium groups was studied by selective acetylation, with the N terminus being found to be important for binding to rat hippocampal membranes and chick brain synaptosomes (18). Studies of GVIA biotinylated at Lys2 and/or Lys24 demonstrated that Lys2 was more important than Lys24 for binding to rat brain synaptic membranes (19). Studies of alanine substitutions at the basic residues (20) indicated that Lys2 was the only basic residue of importance for binding to chick brain synaptic membranes.

The roles of several structural residues in GVIA, including Gly5, the half-cystine residues, and the N- and C-terminal beta -strands, have also been investigated. An active analogue was produced with the substitution of Gly5 with D-Ala, implying that this residue was probably in a type II beta -turn (21), as subsequently confirmed in the three-dimensional structures. Full-length analogues with single cystine bridges deleted (21) or fragments with single disulfide bonds joining discontinuous segments of the polypeptide chain (22) were found to be inactive in a rat brain membrane binding assay. We reported recently that major alterations to the topology of the molecule through deletion of specific disulfide bridges or deletion of N-terminal or C-terminal residues resulted in analogues that were inactive on the N-type calcium channel in the rat isolated vas deferens (23).

To gain a more detailed understanding of the roles of individual amino acid residues in the biological activity of GVIA, we have undertaken an "alanine scan" of the molecule, in which each residue except the bridging half-cystine residues has been replaced individually by alanine. While this work was in progress, a chick brain binding study on a series of alanine-substituted analogues of GVIA was published by Kim et al. (24), in which only residues Lys2 and Tyr13 were found to be important for binding. That study also demonstrated that the post-translational modification from Pro to Hyp (hydroxyproline), for all three Hyp residues simultaneously, was unnecessary for activity. A similar investigation of MVIIA (25) yielded the result that Arg10, Leu11, and Arg21 were important for rat brain synaptosomal membrane binding, in addition to the conserved Lys2 and Tyr13 residues. The present study of GVIA differs from that of Kim et al. in several respects. First, the alanine-substituted analogues included separate substitutions of Hyp4, Hyp10 and Hyp21 with Ala. Second, the structures of the analogues were analyzed rigorously by one and two-dimensional 1H NMR spectroscopy to determine the extent of isomer purity and to assess conformational integrity. Third, in addition to binding studies, activity studies were performed with three functional organ bath assays (rat isolated vas deferens, right atrium, and small mesenteric artery), as well as in intact conscious rabbits. Our data provide a much more complete picture of the calcium channel binding surface of GVIA and point to the possibility of differences in subtype populations of the N-type channels in different tissues, and to differences between the epitopes important in binding and function.


EXPERIMENTAL PROCEDURES

Synthesis of GVIA and Analogues

N-alpha -Protected amino acids were obtained from Auspep Pty. Ltd. (Melbourne, Australia), Bachem, or Millipore (Sydney, Australia) and were of the L-configuration except for Gly. N-(9-Fluorenyl)methoxycarbonyl (Fmoc) amino acids included: Cys (trityl), Arg (2,2,5,7,8-pentamethylchroman-6-sulfonyl), Ser (t-butyl), Lys (t-butyloxycarbonyl), Tyr (t-butyl), Thr (t-butyl), Hyp (t-butyl), and Asn (trityl). All reagents and solvents (peptide synthesis grade) were purchased from Auspep Pty. Ltd. The polyethylene glycol-polystyrene resin was purchased from Millipore.

Peptide synthesis was performed using a semi-automatic NovaBiochem Gem peptide synthesizer at 0.13-0.2 mmol scale of polyethylene glycol-polystyrene resin. Fmoc amino acids (3 eq) were activated using 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (3 eq) with diisopropylethylamine (6 eq) used to initiate the in situ reaction. Peptide resins were cleaved, and all protecting groups were removed, typically by treatment with a solution containing trifluoroacetic acid (10 ml), ethane-1,2-dithiol (0.25 ml), thioanisole (0.5 ml), H2O (0.5 ml), and phenol (0.5 g) (26). After 4-6 h, resins were removed by filtration and the crude peptide was precipitated with diethylether, recovered by centrifugation, washed three times with diethyl ether, dried in vacuo, dissolved in 20% MeCN (aq), 0.1% trifluoroacetic acid, and lyophilized.

Crude peptides were oxidized by dissolution in 10% dimethyl sulfoxide in ammonium bicarbonate (50 mM, pH 8) at 1 mg/ml (27). These oxidations were monitored by analytical reversed-phase HPLC.

HPLC was performed on a Hewlett Packard 1050 chromatograph with detection by UV absorption at 220 nm. Preparative HPLC was conducted on an Alltech Econosil ODS column (250 mm × 22 mm, 10-µm particle size), semi-preparative chromatography was performed on a Phenomenex Spherisorb ODS column (250 mm × 10 mm, 10-µm particle size), and analytical chromatography was conducted on a Hypersil ODS column (250 mm × 2.1 mm, 5-µm particle size) or on an Adsorbosphere ODS column (150 mm × 2.1 mm, 5-µm particle size). Peptides were eluted using either 0.1% trifluoroacetic acid in H2O (buffer A) and 0.1% trifluoroacetic acid in acetonitrile (MeCN) (buffer B) or 0.6% triethylammonium phosphate at pH 2.25 (TEAP 2.25) (buffer A) and TEAP in MeCN (buffer B). Flow rates were 0.3 ml/min for analytical HPLC, 4 ml/min for semi-preparative HPLC, and 10 ml/min for preparative HPLC. Hereafter, these mobile phase systems will be abbreviated to 0.1% trifluoroacetic acid H2O/MeCN and TEAP 2.25/MeCN.

The crude (oxidized) peptides were loaded batchwise on to the preparative column, and eluted with a linear gradient of TEAP 2.25/MeCN. The gradient applied to the preparative column was 3-16% over 46 min. The generated fractions were analyzed by analytical HPLC using gradient elution with 0.1% trifluoroacetic acid H2O/MeCN. Appropriate fractions (>95% pure) were pooled, diluted and loaded onto a 250 mm × 10-mm semi-preparative column for desalting. The peptide was eluted with a linear gradient of 0.1% trifluoroacetic acid H2O/MeCN. Fractions containing the purified peptide were pooled and lyophilized.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used for confirmation of product identity and was performed on a Finnigan Lasermat. The absorbing matrix used was alpha -cyano-4-hydroxy-cinnamic acid. Capillary zone electrophoresis was used for confirmation of product homogeneity and was conducted on an Applied Biosystems model 270A instrument, using the following conditions: applied potential, 30 kV; buffer, sodium citrate (20 mM, pH 2.5); capillary length, 72 cm; capillary diameter, 50 µm; temperature, 30 °C.

NMR Analysis

All polypeptide analogues were analyzed by 1H NMR spectroscopy on a Bruker AMX-600 spectrometer. Two-dimensional spectra were acquired on 2-10-mg samples of polypeptide in 90% H2O, 10% 2H2O at pH 3.4 and 298 K (400 t1 increments, 4096 data points, and 64-96 scans/t1 increment were employed). The TOCSY spin-lock and NOESY mixing times were 70 and 400 ms, respectively. The methodology was otherwise as described by Pallaghy et al. (28).

NOESY spectra of analogues were analyzed in terms of which non-trivial (medium and long range) NOEs of the native molecule were lost in the analogues and which, if any, new NOEs were observed that were not seen in the native spectrum. The intensities of NOEs that were present in both spectra were not monitored. A custom-written computer program, NMRanalogue, was written to produce macros (suitable for input to the spectral analysis programs Felix 2.3 and Felix 95) that allowed this procedure to be partially automated for the series of NOESY spectra. The lost NOEs were categorized as very weak, weak, medium, strong, or very strong according to their intensities in NOESY spectra of the native molecule. Only peaks of intensity "weak" or greater for analogues of estimated sample quantity >= 7 mg were considered significant. The native sample quantity was approximately 10 mg, and only well shaped peaks were used in the native NOE list.2 The designation of a native NOE as "lost" for any given analogue was performed conservatively, so that even a very distorted peak near the required position was considered to be maintained. Insight II (version 95.0) was used for molecular graphics.

Binding Assay

Crude rat brain membranes were prepared by the method of Cruz and Olivera (29). Individual brains were homogenized using an Ultra-Turrax homogenizer (Janke and Kunkel) in 10 volumes of buffer (0.32 M sucrose, 5 mM HEPES-Tris, pH 7.4, 0.1 mM phenylmethylsulfonyl fluoride) and the homogenate spun at 1000 × g for 10 min at 4 °C. The pellet was resuspended in another 10 volumes of buffer and spun again. The combined supernatants were then spun at 17,000 × g for 60 min at 4 °C. The pellet was taken up in 100 volumes of buffer as above but also containing 50 mg/liter lysozyme. This gives membrane derived from about 200 µg of tissue/20 µl of homogenate. The suspension was divided into 3-ml aliquots and stored at -70 °C.

Binding assays were conducted in 96-well microtiter plates with 0.65-µm filters in the bottom (Millipore multiscreen system), using a modification of the method of Cruz and Olivera (29) and Haack et al. (30), as also used by Kim et al. (24).3 The binding buffer contained 0.32 M sucrose, 5 mM HEPES-NaOH (pH 7.4), 0.1 mM phenylmethylsulfonyl fluoride, 0.3% bovine serum albumin, and 50 mg/liter lysozyme. Wash buffer consisted of 150 mM NaCl, 5 mM HEPES-NaOH, pH 7.4, 1.5 mM CaCl2, and 0.1% bovine serum albumin. Tracer solution was prepared as follows; 50 µCi of 125I-[Tyr22]GVIA (DuPont NEN) was dissolved in 1 ml of water and divided into 50-µl aliquots, which were stored at -15 °C. Tracer for each assay was prepared by diluting one aliquot (2.5 µCi) into 3.5 ml of binding buffer for each 96-well microtiter plate to be used in the assay. Incubation mixtures (total volume 100 µl/well) consisted of 50 µl of binding buffer, containing displacing ligand, 20 µl of membrane preparation, and 30 µl of diluted 125I-[Tyr22]GVIA tracer solution, final concentration 0.13 nM. Nonspecific binding was determined in the presence of 1 µM unlabeled GVIA. Assays were incubated at 4 °C for 90 min and were terminated by filtration under vacuum. Each well was washed three times with 200 µl of wash buffer and then left under vacuum for 10 min to dry the filters. Filters were punched out and counted in a gamma  counter. Each measurement was determined in quadruplicate within an experiment, and each experiment was replicated at least three times. Data were analyzed using the computer program GraphPad Prism.

In Vitro Biological Assays

Male Harlan Sprague Dawley rats were killed by CO2 anesthesia followed by decapitation, and the vasa deferentia, heart, and a portion of intestine with attached mesentery removed and placed in a dish of cool Krebs solution (composition in mM: Na+ 144, K+ 5.9, Mg2+ 1.2, Ca2+ 2.5, HPO4- 1.2, Cl- 129, SO4- 1.2, HCO3- 25, glucose 11, EDTA 0.026, bubbled with 5% CO2 in oxygen) for further dissection.

Vas Deferens

Vasa deferentia were mounted in 5-ml organ baths, with the top of each tissue attached to an isometric force transducer (Grass FT03) and the bottom attached to a movable support and straddled with platinum stimulating electrodes. The vasa were stretched by a passive force of about 10 millinewtons and stimulated with single electrical field pulses (100 V, 0.2-ms duration) every 20 s. The resulting twitch responses were mediated by sympathetic nerves, being sensitive to inhibition by guanethidine (10 µM) or tetrodotoxin (0.1 µM), and were recorded on a chart recorder. GVIA (0.3-10 nM) caused a gradual concentration-related reduction in the size of the twitch response to electrical nerve stimulation in the rat vas deferens. Submaximally effective concentrations of GVIA elicited an initially rapid fall in the size of the twitch, and a continued very gradual decrease thereafter for over 90 min. This apparently slow equilibration meant that concentration-response curves with full equilibration of the toxin could not be constructed without interference from spontaneous fade of the twitch responses. To offset this problem, a protocol with fixed 20-min intervals between successive concentration increments was used. Cumulative concentration-response curves for GVIA or the analogues were constructed by addition of the peptides to the solution bathing the vas deferens in 10-fold concentration increments from 1 nM. A single concentration-response curve was constructed in each tissue.

Mesenteric Artery

Under a dissecting microscope, an artery (three branch orders proximal to the arteries that enter the intestine) was carefully dissected free of the fat and connective tissue around it, and a 2-mm-long segment was mounted on 40-µm wires in a Mulvany-Halpern style isometric myograph and warmed to 37 °C. The artery was incrementally stretched radially with about four steps, and the force measured and the arterial circumference calculated at each step, producing a diameter-force curve where the diameter is that of a circle with the same circumference as the vessel at each level of stretch. The diameter of the artery was then set to be 90% of the diameter predicted for distending pressure of 100 mm Hg using standard calculations (31). The rat mesenteric artery set up under these conditions does not develop any spontaneous active contractile force. Potassium depolarizing solution was applied for about 2 min to maximally activate the artery, and washed out. The prejunctional alpha 2-adrenoreceptors were blocked with the covalent antagonist benextramine (3 µM for 5 min) in the presence of prazosin (0.1 µM) to protect the postjunctional alpha 1-adrenoreceptors. The antagonists were then washed out, and noradrenaline (10 µM) was applied to confirm the washout of the antagonists. This procedure greatly increases the size of the responses to sympathetic nerve stimulation. Each jaw of the myograph was fitted with a platinum electrode about 1 mm away from the artery. Sympathetic nerves in the wall of the artery were stimulated with monopolar electrical field pulses of 0.25-ms duration at 30 V, stimulation parameters that give responses that are blocked >95% by tetrodotoxin. We have previously demonstrated that the responses are abolished by guanethidine and are thus mediated by sympathetic nerves. Stimulation of the sympathetic nerves produced contractile responses that had a short (<1 s) latency and decayed rapidly after the end of the train of stimuli. All responses were measured as the peak change in force. Nerve stimulation was applied as sets of three trains of 75 pulses at 25 Hz with a 60-s interval between trains, and 30 min between successive sets of stimuli. GVIA or analogues were applied in a cumulative fashion with 30 min of contact before each test stimulation.

Right Atrium

The right atrium was dissected free of the heart and placed in a 5-ml organ bath at 37 °C on a support having two fine platinum electrodes in contact with the atrium to collect the surface electrogram and another pair of electrodes for electrical stimulation. The spontaneous rate of contraction was continuously measured using the surface electrogram to trigger a period meter. Tachycardia responses mediated by sympathetic nerves were measured in response to sets of 4 electrical field pulses at 2 Hz, in the presence of atropine (1 µM) to abolish the effect of parasympathetic nerve stimulation. Increasing concentrations of GVIA or analogue were applied immediately following the second of two control stimulations, with the drug in contact with the tissue for 30 min before each stimulation.

Conscious Rabbit Experiments

The central ear artery and marginal ear vein of New Zealand White rabbits of either sex (weight 2.5 ± 0.1 kg) were cannulated under local anesthesia (1% lignocaine hydrochloride) for measurement of blood pressure (MAP) and for drug injections, respectively. The phasic blood pressure signal triggered a rate meter for the measurement of heart rate (HR). Phasic and mean arterial pressure and HR were recorded on a Grass polygraph. Following the minor procedures as outlined above, rabbits rested quietly for about 40 min in polycarbonate restrainers. The effects of intravenous administration of selected GVIA analogues were assessed on MAP, HR, and the baroreflex. The baroreflex was measured by eliciting alternate graded steady-state increases and decreases in MAP (± 5-35 mm Hg from base line) with phenylephrine and sodium nitroprusside, respectively (32). GVIA analogue potencies were assessed by comparing their effects to the effect of 10 µg/kg GVIA. The GVIA analogues were administered at an initial dose of 3 or 10 µg/kg. MAP and HR were monitored for 60 min and the dose of analogue increased (cumulative half-log10 dose increments) if its effect was less than that of 10 µg/kg intravenous GVIA (33, 34). The baroreflex curve was then reassessed 60 min after the highest dose of peptide was administered and the reflex parameters (gain, location, and plateaus) obtained by fitting the baroreceptor-heart rate reflex curve to a logistic equation (32).


RESULTS

Peptide Synthesis and Analysis

Synthesis of all linear peptides proceeded smoothly, as indicated by monitoring of the Fmoc deprotection peak at each cycle. Oxidative refolding of linear peptide analogues gave a complex mixture of products, but the structures corresponding to the native fold were readily separated from non-native structures by the TEAP 2.25/MeCN chromatography system (35) (Fig. 1A). Refolding of most of the linear peptide analogues gave purified yields of cyclized peptides of approximately 5% of the crude material.


Fig. 1. Reversed-phase HPLC analysis of oxidized peptides. A, native GVIA; B, G5A; C, N20A; D, T23A. All HPLC analyses were conducted using an Adsorbosphere ODS column (150 × 2.1 mm, 5-µm particle size) eluted with a linear gradient of A (TEAP 2.25) and B (MeCN), from 3 to 20% B over 34 min at a flow rate of 0.3 ml/min. Detection was by UV absorbance at 220 nm. Arrow in panel A indicates the correctly folded product.
[View Larger Version of this Image (20K GIF file)]

On the basis of HPLC analysis, only three of the oxidized analogues (G5A, N20A, and T23A) failed to show a major product with native fold that could be purified using the TEAP 2.25/MeCN chromatography system (Fig. 1, B-D). The amount of material with native-like structure in the oxidized mixtures of G5A, N20A, and T23A was therefore estimated by 1H NMR spectroscopy. The NMR spectrum of native GVIA contains five backbone amide proton resonances in the region 9.2 and 9.5 ppm (Fig. 2A), which were also present in correctly folded Ala-containing analogues (e.g. Fig. 2B) and thus served as a convenient marker of the native fold. Disulfide isomers or other incorrectly folded forms of the molecule could be distinguished from the native-like fold on the basis of these resonances (9). Thus, in cases where the purification scheme described above did not yield a significant quantity of the desired isomer, one-dimensional 1H NMR spectra were employed to estimate the proportion of "native-like" fold in the oxidized mixture, as illustrated for T23A in Fig. 2C. The estimate was based on the average height of the five downfield amide resonances indicative of native-like structure, with the spectrometer calibrated using a sample of native GVIA. By this means the contents of correctly-folded product in G5A, N20A, and T23A were calculated to be, respectively, 0.1, 1, and 3% of the oxidized peptide mixture (i.e. <0.15% of the crude material). Substitution of D-Ala at position 5, instead of L-Ala, resulted in a normal refolding pattern to give a readily-isolated component corresponding to the native fold of the peptide.


Fig. 2. One-dimensional 1H NMR spectra of synthetic polypeptides. Figure shows native GVIA (A), Y13A (B), and T23A (C) (oxidized mixture), each in 90% H2O, 10% 2H2O at pH 3.4 and 298 K.
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Conformational Analysis

Sequential assignments were derived from two-dimensional TOCSY and NOESY NMR spectra for all of the analogues except G5A, N20A, and T23A. The chemical shift deviations from native were small enough to suggest that the native fold was maintained for all of these analogues. The average of the magnitude of the chemical shift deviations from native, |Delta delta |, provides a convenient single-parameter characterization of the structure and is tabulated in Table I for the NH and Calpha H resonances. The deviations of the Calpha H resonances were typically half the size of the NH deviations and so were less sensitive to conformational changes. The largest deviations occurred for the K2A, O4A, G5dA, O10A, S12A, Y13A, R17A, O21A, and Y27A analogues.

Table I. Summary of NMR chemical shift and NOE data for the 22 Ala scan analogues


Analogue Approximate NMR quantitya |Delta delta |av from native (NH) |Delta delta |av from native (Calpha H) 39 very weak peaks lost No. of weak and medium NOEs lost No. of NOEs gainedb

mg ppm ppm %
K2A 7.0 0.094 0.045 0 1 2
S3A 7.0 0.013 0.016 5 1 0
O4A 2.0 0.084 0.054 41
G5dA 3.0 0.064 0.049 56
S6A 7.0 0.035 0.023 8 0 1
S7A 5.0 0.015 0.011 10
S9A 1.8 0.034 0.016 26
O10A 4.0 0.067 0.056 26
T11A 7.0 0.044 0.016 8 0 0
S12A 7.0 0.087 0.039 3 2 1
Y13A 7.0 0.058 0.040 5 1 1
N14A 7.0 0.043 0.026 21 2 0
R17A 7.0 0.073 0.045 15 2 1
S18A 6.0 0.020 0.024 15
O21A 3.5 0.085 0.039 44
Y22A 7.0 0.057 0.027 10 0 0
K24A 4.5 0.047 0.023 26
R25A 1.9 0.042 0.014 56
Y27A 5.0 0.079 0.040 23

a Approximately proportional to the yield of oxidized polypeptide.
b No gained NOEs were observed for analogues with an NMR quantity of less than 7 mg.

Most Ala substitutions caused chemical shift changes primarily in groups spatially close to the substitution, as typified by S3A in Fig. 3A. More widespread chemical shift changes occurred in the K2A, S6A, S12A, Y13A, and R17A analogues and in all three Hyp right-arrow Ala analogues, O4A, O10A, and O21A. The changes in the Hyp right-arrow Ala analogues were spread across the whole molecule (Fig. 3B) and, in the absence of significant NOE changes (see below), can be ascribed to a small conformational rearrangement throughout the molecule due to a different preferred local conformation of the substituted non-cyclized amino acid.


Fig. 3. Deviations of NH and Calpha H chemical shifts from GVIA native values in Ala-substituted analogues. Figure shows S3A (A), O4A (B), and K2A (C), each in 90% H2O, 10% 2H2O at pH 3.4 and 298 K.
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In the K2A analogue, there appeared to be a more specific conformational change associated with Ala substitution, with chemical shift perturbations evident for resonances from Ser6 and Asn14 (normally in contact with Lys2), and more interestingly, Ser9, Ser12, and Tyr13 (Fig. 3C). The Ser12 and Tyr13 perturbations could reflect a local structural change transmitted via Asn14, but there were no NOEs in the native spectra between Ser9 and Lys2, Ser6, or Asn14 to account for the perturbation of the chemical shifts of Ser9.

NOE intensities, due to their r-6 distance dependence, are not as sensitive as chemical shifts to long distance effects, and this is borne out by the small number of non-trivial NOEs lost or gained in the various analogues, as summarized in Table I. A total of 14 very strong, 26 strong, 51 medium, 57 weak, and 39 very weak NOEs between non-adjacent residues in the amino acid sequence (i-j > 1) defines the structure of native GVIA.2 Of these, none of the 40 strong or very strong NOEs was lost in any of the folded analogues, confirming the conclusion based on chemical shift deviations that the tertiary fold was maintained in all cases.

In K2A the long range effect evident from the chemical shifts was clarified by the NOE data and was the clearest example of a small conformational change among all of the correctly folded analogues. New NOEs were observed between Ala2NH and Asn14Ndelta H and between Ala2Cbeta H and Asn14Ndelta H (without corresponding native Lys2/Asn14 NOEs) and the NOE between Cys8 Cbeta H and Ser12 Cbeta H was lost. Visual inspection of the native structure (Fig. 4) indicated that Asn14 had probably moved relative to Lys2, Cys8, and Ser12, pushing Cys8 and Ser12 further apart (and causing the loss of that NOE).


Fig. 4. Stereo view of the structure of omega -conotoxin GVIA. The backbone (bold), disulfide bonds (gray), and side chains are shown. This structure is the closest to the ensemble average of a refined family of structures determined using NMR data (see Footnote 2).
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Another long range effect occurred in the S6A analogue, in which the chemical shift of Cys16NH was perturbed, even though that of Lys2, which is located between these two residues, changed very little. This chemical shift change correlated with a new NOE between Hyp4Cdelta H and Cys16Cbeta H, and it is likely that a small conformational change in turn I (Ser3-Ser6) was responsible for this change. In S12A there was a long range chemical shift change of Cys19NH, transmitted across the gap between beta -turns II (Ser9-Ser12) and IV (Hyp21-Lys24), which have only two NOE interactions between them (both involving Hyp10 and Cys19). However, Ser12 and Cys19 are quite close in the structure (Fig. 4), even though there are no NOEs between them. NOEs lost in the S12A analogue (Ser12NH-Cys8Cbeta H and Ser12NH-Asn14Cbeta H) were confined to the site of substitution, but there was a new NOE (Tyr22NH-Arg17Calpha H) in turn IV near Cys19, suggesting that the long range chemical shift effect on Cys19NH may be due to a small conformational change.

In Y13A the chemical shift of Cys19 moved downfield by 0.24 ppm. This long distance effect might be due to loss of the ring-current effects in this analogue. The only NOE lost was near the substituted residue (Ser12NH-Asn14Cbeta H), but there was a new NOE (Ala13Hbeta -Cys19NH) between the new Ala residue and Cys19NH (which had a perturbed chemical shift). This NOE may have been boosted in intensity due to the substitution of the beta -methylene of tyrosine with beta -methyl of alanine, and may not necessarily be indicative of a genuine conformational change.

Functional and Binding Assays

Specific binding of 125I-Tyr22-GVIA to rat brain membrane constituted 65-75% of the total binding. Nonlinear regression analysis indicated a single binding site with an apparent pKD of 8.95 ± 0.11 and Bmax of 43.7 ± 2.6 fmol/mg tissue. Unlabeled GVIA displaced the labeled tracer with pIC50 of 9.7 ± 0.1. The binding affinities of the Ala-substituted analogues are shown in Fig. 5D.


Fig. 5. Binding affinities and biological activities of Ala-substituted analogues of GVIA. Plots show the change in potency (Delta  pEC50: GVIA pEC50-analogue pEC50) of the alanine-substituted analogues of GVIA in the vas deferens (panel A), the right atrium (panel B), the mesenteric artery (panel C), and the brain binding assay (panel D). * indicates analogues N20A and T23A where a correction has been made for the fraction of the tested samples that appeared by NMR to have the native fold, and dagger  indicates G5A where D-alanine was used instead of L-alanine.
[View Larger Version of this Image (22K GIF file)]

GVIA inhibited the twitch response to single-pulse stimulation of the sympathetic nerves of the vas deferens with a pEC50 of 8.9 ± 0.05, and inhibited the responses of the mesenteric artery and atria to trains of sympathetic nerve stimuli with pEC50 values of 8.8 ± 0.06 and 9.0 ± 0.09, respectively. In the vas deferens and atria, GVIA completely inhibited the responses to the stimulation conditions used. However, in the artery the maximal inhibition by GVIA was only about 90%, and the residual response was sensitive to tetrodotoxin (0.1 µM). Thus, there was a component of the response of the artery to sympathetic nerve stimulation that was resistant to GVIA in the artery but not the other assays. GVIA-resistant sympathetic neurotransmission mediated by P-type and Q-type calcium channels has been reported in vas deferens from the rat and mouse (36) and the guinea pig (37). This GVIA-resistant response was seen only with high frequencies of stimulation, either because the P and Q channels were recruited only at high frequencies or because their effect accumulated to functionally important levels at high frequencies (38). This GVIA-resistant component was too small to affect our estimates of N-channel blocking potencies in the artery experiments, and was not present in the rat vas deferens and atrium experiments in this study, where only single- or four-pulse stimuli were used.

In each assay the incorrectly folded isomers of G5A were inactive, but substitution of Gly5 with D-Ala did not result in any reduction in potency. Both N20A and T23A, tested only as impure mixtures of isomers, were weak in all assays and were applied at a sufficiently high concentration for a reliable potency estimate only in the binding and vas deferens assays. After correction for the fraction of properly folded product estimated from NMR, their potencies were similar to native GVIA.

K2A and Y13A showed markedly reduced potency compared with native GVIA in both the binding assay and the functional assays (Fig. 5), but both retained full activity (i.e. inhibited the sympathetic nerve responses completely) when applied at a high enough concentration (Fig. 6). Arg17 was also significantly less potent than GVIA in all assays (3-fold in the binding and the vas deferens, 10-fold in the artery and atrium), but for the other analogues there was poor agreement between the binding assay and the functional assays. An obvious discrepancy was that none of the analogues was substantially more potent than native GVIA in the functional assays, but several were in the binding assay (Fig. 5). There are some common features among the different assays, with Arg17 being important in all four and the regions of Hyp21-Tyr22 and Lys24-Arg25 being important in the three functional assays, but significant differences between the assays were also apparent. A correlation analysis was performed (excluding the results for K2A and Y13A to avoid the otherwise inevitable overestimation of the quality of the correlation), and it was found that the analogue potencies were significantly correlated in the three bioassays (p < 0.01), but there was no significant correlation between the potencies in the binding assay and the potencies in the functional assays (Fig. 7). Two analogues appeared to have some selectivity between the tissue assays. Y22A was about 3-fold less potent than GVIA in the vas deferens but 30- and 100-fold less potent than GVIA in the atrium and artery, and O10A was almost equipotent with GVIA in the vas deferens and 5- and 15-fold less potent in the artery and atrium, respectively.


Fig. 6. Concentration-response curves in the rat isolated vas deferens for GVIA (open circle ), K2A (bullet ), Y13A (square ), and K24A (black-square).
[View Larger Version of this Image (19K GIF file)]


Fig. 7. Scattergrams of analogue potencies in the four assays (excluding analogues K2A, Y13A, N20A, and T23A). Potencies were significantly correlated between the three functional assays (p < 0.001) but not between the binding assay and any of the functional assays (p > 0.06). Least product regression lines are shown in the panels where a significant correlation exists. Panel A, atrium versus artery, r = 0.81 and slope = 0.70. Panel B, atrium versus vas deferens, r = 0.80 and slope = 1.62. Panel C, artery versus vas deferens, r = 0.84 and slope = 2.31. Panel D: binding versus artery, r = 0.23. Panel E, binding versus vas deferens, r = 0.46. Panel F, binding versus atrium, r = 0.37.
[View Larger Version of this Image (27K GIF file)]

Conscious Rabbit Experiments

GVIA (10 µg/kg, intravenous) caused a fall in MAP of 15 mm Hg and a tachycardia of about 80 beats/min (33). The onset was slow, with a peak response after 35-40 min, and the effect was maintained for several hours. GVIA also decreased the baroreflex HR range from 175 to about 70 beats/min and increased the lower HR plateau of the baroreflex curve, which indicated an attenuation of both the sympathetic (upper) and vagal (lower) components of the curve. The effects of some of the analogues closely mirrored those of GVIA. G5dA and S6A each at a dose of 10 µg/kg (intravenous) caused similar changes in MAP and HR to those elicited by GVIA. The baroreflex curves after these two analogues were also similar to the curve after GVIA, with the HR range of the curves reduced to about 70 beats/min in each case. For all other analogues tested in this assay, a dose >10 µg/kg (intravenous) was required to cause similar changes in MAP and HR, as well as to the baroreflex, to those observed after GVIA. The approximate potency ratios compared with GVIA were: N14A, 3; K2A, 10; R17A, Y22A and K24A, 30; and Y13A, >100.


DISCUSSION

In this structure-activity study of GVIA, we have found that Lys2 and Tyr13 are the most important individual amino acids for affinity for the N-type calcium channel, a result that has been reported previously by others (24). In contrast to previous studies, however, the functional assays used in this study show that several other amino acid residues are also important for the high affinity of GVIA for the N-type calcium channel, and the detailed NMR investigation of the alanine-substituted analogue structures shows that the potency reductions produced by the Ala substitutions are primarily the result of the loss of side-chain interactions with the binding site rather than perturbations of the tertiary structure of the molecule.

Synthesis and Folding of GVIA Analogues

Only three of the Ala-substituted analogues of GVIA (G5A, N20A, and T23A) failed to fold into a native-like conformation in substantial yield. The inability of G5A to fold into the same structure as the native toxin is not surprising, as Gly5 is an essential component of the type II beta -turn, which extends over residues 3-6 in the molecule (8-11). Introduction of D-Ala at this position, in contrast to L-Ala, resulted in a product that folded to a native-like structure, presumably because D-Ala mimics the positive phi -angle of Gly5 and thus supports a beta -turn structure (39). Surprisingly, Kim et al. (24) did not report any difficulty in refolding G5A. N20A and T23A failed to fold into a single major product, and our NMR studies indicated that only about 1% and 3%, respectively, of native-like structure were present in the mixtures, giving overall yields of <0.15% of the crude materials. Kim et al. (40) did report low yields of correctly folded N20A and T23A. Residues 20 and 23 may be important for the initial folding of the linear polypeptide chain as a precursor to formation of the disulfide bridges, a possibility supported by a side chain to side chain interaction between these residues evident in the refined NMR structures.2

Solution Structures of Analogues

The NMR studies confirmed that most Ala-substituted analogues of GVIA had conformations very similar to native GVIA, confirming that the modulation of activity by alanine substitutions can be ascribed to the altered chemical moiety rather than conformational changes. One possible exception to this is K2A, where some of the modulation of its activity may be due to a small conformational change. Even in this case, however, there were only three NOE changes out of the total of 192, so the conformational change was very small. An interesting feature of the NMR analyses was that the NOE changes were localized almost exclusively in the poorest defined region of the molecule, namely Cys8 to Cys15. It has been proposed that this part of the molecule may be genuinely flexible (9), and in the refined structures2 two families of conformations for this region of the molecule are present. This region may be more sensitive to substitutions at various positions throughout the molecule because of a lack of stabilizing interactions. Interestingly, Kim et al. did not report any perturbation of the structure of GVIA by the substitution of Ala for Lys2. This presumably reflects the fact that the circular dichroism methods used in their study are not nearly as sensitive to local conformational changes as NMR analysis.

N-type channel Blocking Activities

The potencies of GVIA and the analogues measured in the functional assays are not direct measurements of their affinity for the N-type calcium channel because there is an unknown relationship between occupation of the N-type calcium channel by the blockers and the fractional inhibition of the responses we measured, and because the analogues were not allowed sufficient time for full equilibration within the tissues and at the channels. The onset of inhibition of N-type calcium channels by GVIA has a very slow time course, with over 90 min required for full expression of the effect of low concentrations in the rat isolated small mesenteric artery (41). Drug contact time in our functional in vitro assays was too short for full equilibration of each concentration of peptide. The under-equilibration was largest in the vas deferens assay because of a shorter contact time (20 min, compared with 30 min in the other assays) and because the vas deferens is substantially thicker than the other tissues and diffusion of the peptides into the tissue should be slower. The time for equilibration at a receptor is shorter for any drug when it is applied at a higher concentration, and thus any affinity reduction in the GVIA analogues would be underestimated more in the vas deferens assay than in the other assays. Additionally, the truncated contact time for GVIA and its analogues dictates that any decrease in affinity that is the result of a slowed association rate constant will result in an exaggerated decrease in potency, because it would also increase the time required for full equilibration. The corollary applies that any decrease in affinity that is the result of a faster dissociation rate constant will be underestimated. Without knowledge of the rate constants involved, it is not possible to determine the magnitude of these effects. These issues may not be relevant to the binding assays, which had 90-min incubation times (albeit at 4 °C).

There was a strong correlation between the potency of each analogue in the three bioassays, but, excluding positions 2 and 13 to avoid an artifactually inflated correlation coefficient, there was no correlation between the binding potencies and the functional potencies (Fig. 7). Furthermore, none of the Ala-substituted analogues showed a significantly increased potency compared with native GVIA in the functional assays, but several were 3-fold more potent than GVIA in the binding assays in this study and a previously published study by others (24). The in vivo experiments yielded data that were rather qualitative in nature because issues of drug distribution and metabolism can alter apparent potencies, but it was clear that the order of potency of the analogues tested in vivo resembled that obtained in the other functional assays. In particular, the activity of Y22A in vivo (about 30-fold less potent than GVIA) was within the range of its potency in the functional assays (between 3- and 100-fold less potent than GVIA) but not its potency in the binding assay (about 3-fold more potent than GVIA). The discrepancies between the functional and binding assays might be explained in several ways. First, the conditions of the binding assay may alter the conformation of the N-type calcium channel from its native (physiological) conformation, maintaining the strong interaction between the channel and the Lys2 and Tyr13 residues of GVIA but significantly altering the weaker interactions between the channel and the other residues. Our binding buffer contained isotonic sucrose and only the sodium ions from adjusting the pH with NaOH, and this near absence of small ions might well have altered the interaction between GVIA and the N-type calcium channel or the conformation of the channel. Indeed, the binding of GVIA to the channel has been reported to be modified by physiological concentrations of calcium, sodium, potassium and magnesium ions (42). The N-type calcium channel is voltage-gated, and thus by definition its conformation is sensitive to changes in membrane potential. In the binding assays the membrane potential is zero, with an identical ionic milieu on each side of the membrane (presuming that vesicles are not formed), so it might be expected that the interaction between GVIA and the channel could be different in the binding experiments from the functional experiments. It has been shown that the interaction of the scorpion toxin charybdotoxin with the voltage-gated potassium channel expressed in Xenopus oocytes was affected by interaction of the channel with the cytoskeleton (43), and similar interactions of the calcium channel could contribute to the differences observed here for GVIA. If the properties of the N-type calcium channel were significantly altered by the conditions chosen for binding experiments then any rational drug discovery program based solely on the binding data would be compromised.

Another possible explanation for differences among the assays is tissue-dependent subtypes of N-type calcium channels. The binding experiments were conducted with brain membranes, and the functional assays used peripheral tissues. Any subtle differences between the N-type calcium channels expressed in different tissues would show up as differences among the orders of potency of the Ala-substituted analogues in the different assays. This could also contribute to both the modest discrepancies in the analogue potencies among the three in vitro functional assays, and the more striking differences between the potencies in the functional assays and the rat brain binding assay in this study and the chick brain assay of Kim et al. (whose binding conditions were nominally the same as this study) (24, 44). The pairwise correlations between the results from the different functional assays were all similar in magnitude (Fig. 7). However, the correlation slopes suggest that the vas deferens was least sensitive to changes in analogue potencies and the artery assay the most sensitive. This could be the result of tissue differences in N-type calcium channels, but is more likely to be the result of a combination of the shorter than ideal toxin contact time and restricted diffusion of the analogues to their sites of action in the vas deferens, as discussed above. Tissue and species differences in the N-type calcium channel would be very important for potential therapeutic applications of N-type calcium channel blockers, but more detailed studies would be required to confirm such differences.

In all of the assays, the least potent of the correctly folded analogues were Y13A and K2A, confirming that Tyr13 and Lys2 are the most significant determinants of GVIA binding affinity. Although our NMR studies showed that K2A had a slightly different structure from native GVIA, the conformational change appears to be minor and we believe that the activity losses from Ala substitutions at Lys2 and Tyr13 indicate that these side chains are important in the interaction between native GVIA and its binding site on the alpha -subunit of the N-type calcium channel. Other studies of Ala-substituted analogues have also identified these residues as being important in GVIA (24, 44). Additionally, GVIA biotinylated at Lys2 is about 10-fold less potent than native GVIA in binding to rat brain membranes (30). A structure-activity study of omega -conotoxin MVIIA, a selective N-type calcium channel blocker with a similar structure to GVIA, also pointed to the importance of Lys2 and Tyr13 (25). As the solution structures of GVIA and MVIIA are very similar (with only small rearrangements due to a single insertion and deletion) (14, 15) and the same residues, Lys2 and Tyr13 (25), have been identified as being important, it may be assumed that GVIA and MVIIA bind to the N-type calcium channel in the same way.

A study of chimeras of the alpha -subunits of the N-type and a GVIA-insensitive calcium channel pointed to a putative external loop between the IIIS5-IIIH5 regions as being important to the activity of GVIA. Substitution of Asn and Lys for each of two Glu residues caused a substantial decrease in the association rate of GVIA (45). It is possible that one or both of these negatively-charged Glu residues interacts with the positively charged Lys2, either decreasing the dissociation rate constant by acting as an energetically favorable interaction between bound GVIA and the N-type calcium channel, or increasing the association rate constant by contributing to the favorable alignment of GVIA when it approaches the channel (46).

Although Lys2 and Tyr13 are the most important individual residues for affinity of GVIA, the functional assays investigated in our study point to additional interactions that were not evident from the binding assay. These additional residues are distributed over a larger region of the molecular surface than that encompassed by Lys2 and Tyr13 (Fig. 8). This means that the pharmacophore of GVIA is larger and more complex than has been suggested by others previously. The interactions with the N-type calcium channel of GVIA and the structurally related MVIIA are probably similar. In the case of MVIIA, Arg10, Leu11, and Arg21 are as important as Lys2 and Tyr13 for the affinity of MVIIA for the N-type calcium channel, although it should be noted that these conclusions were based on rat (25) and chick (44) brain binding data. Fig. 8 shows the structures of GVIA and MVIIA with key residues highlighted. It can be seen that, in addition to the conserved Lys2 and Tyr13, the locations of Tyr22, Hyp21, and Hyp10 and to a lesser extent Lys24 in GVIA are similar to those of Arg21, Arg10, and Leu11 in MVIIA. Thus, although these residues are not identical, it is likely that they interact with similar regions of the calcium channel. The distributed binding surfaces are consistent with a model in which the toxins block the channel by interacting with both the pore and the vestibule of the channel, as in the case of scorpion toxins and the voltage-gated potassium channel (47). However, channel blockade due to toxin binding remote from the pore cannot be ruled out from our data.


Fig. 8. CPK representation of the structures of GVIA and MVIIA. The refined structure of GVIA (see Footnote 2), which is closest to the geometric average, and the first entry in the Protein Data Bank file 1OMG for MVIIA (15) are shown. The structures were aligned over the backbone heavy atoms of the three beta -strands. Residues shown to be important for binding or activity in GVIA (this study) and for rat brain binding in MVIIA (25) are colored. Red, >100-fold potency reduction (for GVIA, in at least 2 assay types); orange, >10-fold potency reduction (for GVIA, in at least 2 assay types); yellow, >10-fold potency reduction (for GVIA, in one assay type).
[View Larger Version of this Image (107K GIF file)]

It is concluded that there are important differences between the structure-activity relationships for GVIA analogues when tested in binding and functional assays, which may result from either tissue-specific distribution of subtypes of the N-type calcium channel, or from the non-physiological nature of the conditions of the binding assay. The functional assay data show that there are several residues on GVIA that make important interactions with the channel in addition to the previously identified residues Tyr13 and Lys2.


FOOTNOTES

*   This work was supported in part by Glaxo-Wellcome Australia.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.
§   To whom correspondence should be addressed. Fax: 61-3-9347-1452; E-mail: m.lew{at}pharmacology.unimelb.edu.au.
par    Supported by a National Heart Foundation of Australia Postgraduate Scholarship.
**   To whom correspondence should be addressed. Fax: 61-3-9903-9655; E-mail: ray{at}mel.dbe.csiro.au.
1   The abbreviations used are: GVIA, omega -conotoxin GVIA; MVIIA, omega -conotoxin MVIIA; Fmoc, N-(9-fluorenyl)methoxycarbonyl; NOE, nuclear Overhauser enhancement; NOESY, two-dimensional NOE spectroscopy; TOCSY, two-dimensional total correlation spectroscopy; HPLC, high performance liquid chromatography; MeCN, acetonitrile; TEAP, triethylammonium phosphate; MAP, mean arterial pressure; HR, heart rate.
2   P. K. Pallaghy and R. S. Norton, manuscript in preparation.
3   K. Sato, personal communication.

ACKNOWLEDGEMENTS

We thank Peter Coles, Mark Ross-Smith, and Angela Mountain for expert technical assistance.


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