Residue Gly1326 of the N-type Calcium Channel alpha 1B Subunit Controls Reversibility of omega -Conotoxin GVIA and MVIIA Block*

Zhong-Ping FengDagger §||, Jawed HamidDagger , Clinton DoeringDagger §, Gregory M. Bosey**, Terrance P. Snutch**DaggerDagger, and Gerald W. ZamponiDagger §§

From the Dagger  Departments of Physiology & Biophysics and Pharmacology & Therapeutics, Neuroscience Research Group, University of Calgary, Calgary, Alberta T2N 4N1, Canada, § NeuroMed Technologies Inc., Vancouver V6T 1Z4, Canada, and the ** Biotechnology Laboratory, University of British Columbia, Vancouver V6T 1Z3, Canada

Received for publication, January 16, 2001, and in revised form, February 2, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We recently reported that amino acid residues contained within a putative EF hand motif in the domain III S5-H5 region of the alpha 1B subunit affected the relative barium:calcium permeability of N-type calcium channels (Feng, Z. P., Hamid, J., Doering, C., Jarvis, S. E., Bosey, G. M., Bourinet, E., Snutch, T. P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 5726-5730). Since this region partially overlaps with residues previously implicated in block of the channel by omega -conotoxin GVIA, we assessed the effects of mutations in the putative EF hand domain on channel block by omega -conotoxin GVIA and the structurally related omega -conotoxin MVIIA. Both of the toxins irreversibly block the activity of wild type alpha 1B N-type channels. We find that in addition to previously identified amino acid residues, residues in positions 1326 and 1332 are important determinants of omega -conotoxin GVIA blockade. Substitution of residue Glu1332 to arginine slows the time course of development of block. Point mutations in position Gly1326 to either arginine, glutamic acid, or proline dramatically decrease the time constant for development of the block. Additionally, in the G1326P mutant channel activity was almost completely recovered following washout. A qualitatively similar result was obtained with omega -conotoxin MVIIA, suggesting that common molecular determinants underlie block by these two toxins. Taken together the data suggest that residue Gly1326 may form a barrier, which controls the access of peptide toxins to their blocking site within the outer vestibule of the channel pore and also stabilizes the toxin-channel interaction.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It is well established that peptide toxins isolated from marine snails have the propensity to potently and highly selectively inhibit the activities of a number of different types of voltage- and ligand-gated ion channels (1-5). These toxins include blockers of skeletal muscle sodium channels (µ-conotoxin (6-8)), the nicotinic acetylcholine receptor (alpha -conotoxins (9, 10)), and blockers of voltage-dependent calcium channels (omega -conotoxins (2)). Among the family of omega -conotoxins, two prominent members are omega -conotoxin GVIA, a 27-amino acid peptide derived from the Conus geographus sea snail, and omega -conotoxin MVIIA, a 25-amino acid toxin isolated from the venom of the Conus magus marine snail (2). Both of these toxins potently block N-type calcium channels from a variety of species and nerve tissues (11-14). Block develops rapidly and is only incompletely reversible even after prolonged washout (12). Both toxins have been reported to display a high degree of selectivity for the N-type calcium channel alpha 1B subunit (15), and omega -conotoxin GVIA has become a commonly used tool for the identification of native N-type calcium currents.

While little is presently known concerning the toxin structural requirements, which underlie the high affinity and selectivity of omega -conotoxins GVIA and MVIIA for N-type calcium channels, some of the N-type calcium channel regions that participate in the blocking interaction have been elucidated. Utilizing a chimeric channel approach in combination with site-directed mutagenesis, Ellinor and co-workers (16) identified crucial components of the binding site of omega -conotoxin GIVA in the external vestibule of the alpha 1B channel in the domain III S5-S6 region, suggesting that this toxin likely acts via physical occlusion of the pore. While the binding site for omega -conotoxin MVIIA has not yet been identified via molecular biological techniques, both biochemical and electrophysiological data suggest that omega -conotoxin MVIIA competes with omega -conotoxin GVIA for a common, or at least partially overlapping, receptor site (2, 17).

We have recently reported that point mutations in an external EF hand homology motif located on the N-type calcium channel alpha 1B subunit immediately adjacent to the omega -conotoxin GVIA binding site identified by Ellinor et al. (16) affects the permeation characteristics of the channel (18). In view of its proximity to the putative omega -conotoxin GVIA receptor site, we investigated whether mutations in this region could affect the characteristics of omega -conotoxin GVIA and MVIIA block of the channel. We report here that certain point mutations within the putative EF hand motif increase the affinity of the channel for omega -conotoxin GVIA. Moreover, one of the mutations, a replacement of residue Gly1326 with proline, is sufficient to dramatically enhance the unblocking rate of the toxin such that block by both omega -conotoxin GVIA and MVIIA is fully reversible over a time course of <10 min. Hence, residues immediately outside the region identified previously by Ellinor et al. (16) contribute to block of N-type calcium channels by omega -conotoxins, and a single amino acid residue in this region is sufficient to control the reversibility of toxin block.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Site-directed Mutagenesis-- The G1326P, E1332R, D1323R, and E1321R point mutation constructs have been described by Feng et al. (18). The R1325D, G1326K, G1326E, and G1326Y additional constructs were generated as described by Feng et al. (18). In brief, a SplI-AflII fragment (~4.5 kilobases) was excised from the full-length cytomegalovirus-alpha 1B (CMV) construct and ligated into pSL1180. Site-directed mutagenesis was carried out on using the QuikChange mutagenesis kit (Stratagene) and successful mutagenesis, and absence of sequence errors was confirmed via DNA sequencing. Subsequently, the mutated SplI-AflII fragment was introduced into the full-length clone in the cytomegalovirus (CMV) expression vector via restriction enzyme digestion an ligation and its presence confirmed by restriction endonuclease digestion and DNA sequencing.

Tissue Culture and Transient Transfection-- Human embryonic kidney tsa-201 cells were maintained in a 37 °C CO2 incubator in standard Dulbecco's modified Eagle's medium (supplemented with 10% fetal bovine serum, 200 units/ml penicillin, and 0.2 mg/ml streptomycin). The cells were split at 85% confluence using trypsin EDTA and plated on glass coverslips at 5-10% confluence. The cells were then allowed to recover for ~12 h at 37 °C before transfection. Immediately before transfection, the medium was replaced, and the cells were transiently transfected with cDNAs encoding for calcium channel alpha 1B, beta 1b, and alpha 2-delta subunits and EGFP (at a molar ratio of 1:1:1:0.3) via the calcium phosphate method. After 12 h, the cells were washed with fresh medium, allowed to recover for additional 12 h, and subsequently stored at 28 °C in 5% CO2 for 1-3 days before whole cell recording. Recordings were performed using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) linked to a personal computer equipped with pClamp version 7.0. Patch pipettes (Sutter borosilicate glass, BF 150-86-15) were pulled using a Sutter P-87 microelectrode puller and subsequently fire-polished using a Narashige microforge.

Whole Cell Patch Clamp Recordings and Data Analysis-- The whole cell patch clamp recording (membrane ruptured) procedures used here were similar to those described by Beedle and Zamponi (19). Briefly, cells were transferred to a 3-cm culture dish containing recording solution comprised of 20 mM BaCl2 (or CaCl2), 1 mM MgCl2, 10 mM HEPES, 40 mM TEA1-Cl, 10 mM glucose, 87.5 mM CsCl (pH 7.2 with TEA-OH). Pipettes with resistances of 2-4 MOmega were filled with internal recording solution containing 108 mM cesium methanesulfonate, 4 mM MgCl2, 9 mM EGTA, 9 mM HEPES (pH 7.2 with TEA-OH). After patch rupture, cell capacitance was neutralized, and series resistance was compensated. Currents were elicited by stepping from a holding potential of -100 mV to various test potentials using Clampex 7.0 software and an Axopatch 200 B amplifier (Axon Instruments, Foster City, CA). omega -Conotoxin GVIA (RBI Chemicals) and MVIIA (Sigma) were dissolved first, respectively, in water and acetic acid and then diluted in the external recording solution. Solutions containing various concentrations of toxin were delivered directly to the cells by means of a gravity-driven microperfusion system, which allows complete solution exchanges in less than 1 s. Currents underwent little if any rundown during the time course of a typical experiment. Data were filtered at 1 kHz and digitized at a sampling frequency of 2 kHz. Data were analyzed using Clampfit (Axon Instruments). All curve fittings were carried out using Sigmaplot 4.0 (Jandel Scientific). The time constant, tau , for onset of block was determined from the equation I(t)/I(t = 0) = exp(-t/tau ), where I(t) is the current amplitude at time t after application of the toxin, and I(t = 0) is the control current amplitude immediately prior to toxin application. Statistical analysis was carried out using SigmaStat 2.0 (Jandel Scientific). Differences between mean values from each experimental group were tested using a Student's t test for two groups and one way analysis of variance for multiple comparisons. Differences were considered significant if p < 0.05. All error bars given reflect S.E values, numbers in parentheses indicate numbers of cells.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

omega -Conotoxins GVIA and MVIIA Irreversibly Block N-type Calcium Channels-- Fig. 1 illustrates the effects of omega -conotoxin GVIA and MVIIA on N-type (alpha 1B+beta 1b+alpha 2-delta ) calcium channels transiently expressed in HEK tsa-201 cells. The application of 1 µM of either toxin completely removes current activity, which cannot be recovered following washout over the time course of 10 min. Fig. 1B compares the time courses of N-type channel block for various concentrations of both toxin. For both toxins the time course of development of block is accelerated with increasing toxin concentration, although on average development of omega -conotoxin MVIIA block occurs about three times more rapidly than that of omega -conotoxin GVIA. Similar to what has been previously reported for both native channels (14) and alpha 1B channels transiently expressed in Xenopus oocytes (16), block of both toxins was only poorly reversible such that even prolonged washout (7-15 min) resulted in the recovery of only a minor fraction of the N-type current. Fig. 1C illustrates the dependence of the inverse of the time constant for development of block on the toxin concentration. Consistent with a bimolecular interaction between the toxin molecule and the channel, the relation was well described by a linear fit in the case of each of the two toxins. The blocking rate constants (kon), obtained from the regression lines, were, respectively, 1.06 and 2.93 µM-1 min-1 for omega -conotoxins GVIA/MVIIA, consistent with the data shown in Fig. 1B. However, it is not possible to accurately determine the unblocking rate constants (koff) from either the regression lines or from the time course of recovery from block because of the poor reversibility of the blocking action.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 1.   Block of transiently expressed N-type (alpha 1B+beta 1b+alpha 2-delta ) calcium channels by omega -conotoxins (CTX) GVIA and MVIIA. A, current records illustrating the effects of the two toxins. Block is complete and virtually irreversible in response to washout for 10 min. Currents were elicited by stepping from a holding potential of -100 mV to a test potential of +20 mV. B, representative time courses of development of block and of recovery from block by omega -conotoxin GVIA and omega -conotoxin MVIIA. In both cases, the block develops more rapidly at higher toxin concentrations. Note that block by omega -conotoxin MVIIA develops more rapidly than that of omega -conotoxin GVIA. C, dependence of the inverse of the time constant for development of block, tau , on the toxin concentration, [CTX]. The solid lines are linear regressions (fitted with the equation: 1/tau  = kon*[conotoxin] + koff), whose slopes reflect the blocking rate constants (kon) and whose intercepts on the y axis are equivalent to the unblocking rate constants (koff). The rate constants obtained from the fits are as follows: omega -conotoxin GVIA, kon = 1.06 µM-1 min-1, koff = 0.011 min-1; omega -conotoxin MVIIA, kon = 2.93 µM-1 min-1, koff = 0.07 min-1). Error bars denote S.E. values.

Mutations in the Domain III S5-H5 Region Affect omega -Conotoxin GVIA Block-- We have previously reported that high voltage-activated calcium channels contain a highly conserved putative EF hand motif within the domain III S5-H5 region (18). As shown in Fig. 2 for the alpha 1B subunit, this region is localized immediately adjacent to a series of amino acid residues, which have been implicated in omega -conotoxin GVIA block of the channel (16). In a previous study we showed that replacement of the three negative charges in the putative EF hand motif with positively charged residues, or substitution of the central glycine (Gly1326), resulted in an ablation of the differential selectivity of the channel between barium and calcium ions without affecting other major biophysical properties of the channel such as activation, inactivation, or block by cadmium ions (18). To test whether this region of the channel might modulate the blocking action of omega -conotoxin GVIA, we first expressed the triple (E1321K,D1323R,E1332R) alpha 1B mutant in HEK tsa-201 cells (together with the ancillary beta 1b and alpha 2-delta subunits) and examined the dose dependence of omega -conotoxin GVIA blocking kinetics of the mutant channel. As shown in Fig. 3, the time course of development of omega -conotoxin GVIA block in the triple mutant channel was significantly slowed compared with that observed with the wild type channel, indicating that one or a combination of the three substituted residues contribute to omega -conotoxin GVIA block of the channel. Nonetheless, the application of 1 µM concentrations of the toxin was sufficient to eliminate all current activity, and as with the wild type channel, block of the mutant channel was irreversible (Fig. 3, A and B). To determine which of the three residues was responsible for mediating the slowing of the blocking kinetics, we examined omega -conotoxin GVIA block of both a single E1332R and a double (E1321K,D1323R) mutant. As seen in Fig. 3B, a single substitution of residue 1332 with arginine was sufficient to account for the slowing of the blocking kinetics, whereas the double mutant behaved similar to the wild type channel. This is shown quantitatively in form of a comparison of the blocking rate constants of omega -conotoxin GVIA block of the wild type and the mutant channels (Fig. 3C). Mutation in position Glu1332 to arginine resulted in a 2-fold reduction in kon and suggests that the glutamate contributes in a significant manner to omega -conotoxin GVIA block of N-type calcium channels.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2.   A, amino acid sequence of the alpha 1B domain III S5-H5 region. The putative EF hand calcium binding domain () spans residues Glu1321 to Glu1332. Amino acid residues implicated previously in omega -conotoxin (CTX) GVIA block (16) (open circle ) are located between Gln1327 and Gln1339. B, proposed membrane topology of the N-type calcium channel alpha 1 subunit (20) indicating the location of the putative EF hand motif. The inset illustrates the possible locations of the key residues relative to the pore forming p-loop structure of the channel as proposed by Ellinor et al. (16).


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of mutations in positions 1321, 1323, and 1332 on toxin block. A, current records obtained with mutant (E1321K,D1323R, E1332R), (E1321K,D1323R), or E1332R channels (coexpressed with beta 1b and alpha 2-delta ) in the absence and presence of 1 µM omega -conotoxin GVIA. Note that all three mutants are irreversibly and completely blocked at this toxin concentration. B, representative time course of development of block of wild type and mutant N-type calcium channels by 1 µM omega -conotoxin GVIA. Note that the triple mutant and the single E1332R mutant channel display slowed blocking kinetics compared with the wild type or the (E1321K,D1323R) mutant construct. C, blocking rate constants obtained with the wild type and mutant channels. The kon values were obtained by fitting linear regressions as shown in Fig. 1C for each individual experiment. Error bars denote S.E. values, numbers in parentheses reflect numbers of experiments, and the asterisks indicate statistical significance relative to the wild type channels (p < 0.05).

In our previous study examining permeation, we replaced the central glycine residue (Gly1326) within the putative external EF hand domain with proline (18). To determine whether this substitution affects block of the channel by omega -conotoxin GVIA, the G1326P mutant was expressed with the appropriate ancillary subunits in tsa-210 cells, and omega -conotoxin GVIA blocking kinetics were examined. Fig. 4 shows that development of block was accelerated 30-fold compared with the wild type channel (Fig. 4B). More strikingly, upon washout of the toxin current activity could be almost completely recovered over the time course of about 10 min (Fig. 4, A and B). Since the time course for development of block is inversely proportional to the sum of blocking and unblocking rates, such an increase in the rate of recovery from block is predicted to contribute to the observed 30-fold decrease in the time constant for development of block. To isolate any direct effects of the mutation on the true blocking rate constant, the time course for development of block was determined at several toxin concentrations, and the inverse of the time constant of development of block was plotted as a function of toxin concentration. As shown in Fig. 4C, the relation was nicely described by a linear fit. Furthermore, from the slope and the y intercept of the regression line, both the blocking (kon = 17.51 µM-1 min-1) and the unblocking (koff = 0.72 min-1) rate constants were found to be significantly increased in the G1326P mutant compared with the wild type channel, indicating that the access of the toxin to its binding site is enhanced in the mutant, but concomitantly the stability of the binding interaction is dramatically reduced.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 4.   omega -Conotoxin (CTX) GVIA block of the G1326P mutant. A, current records illustrating block of the G1326P (+beta 1b+alpha 2-delta ) construct by 100 nM omega -conotoxin GVIA. Note that the effects of the toxin are almost completely reversed following washout. B, representative time course of development of, and recovery from, block of the G1326P mutant by different concentrations of omega -conotoxin GVIA. Note the reversibility of toxin block. C, kinetic analysis of block of wild type and G1326P mutant channels by omega -conotoxin GVIA. The data for the wild type channel are the same as in Fig. 1. Note the dramatic increase in the slope of the regression line and the larger y intercept obtained with the mutant channel. The blocking and unblocking rate constants for the G1326P mutant obtained from the fit were, respectively, 17.5 µM-1 min-1 and 0.72 min-1. Error bars denote S.E. values.

To determine whether this effect is also observed with other substitutions at the G1326 position, we generated three additional mutations (G1326K, G1326E, and G1326Y). Interestingly, the G1326Y mutant resulted in channels that could no longer be functionally expressed. However, the G1326K and G1326E mutants expressed well, and as shown in Fig. 5A and B, the time course of development of block was accelerated, albeit not as dramatically as with the G1326P mutant. In both cases, block remained irreversible over the time course of a typical experiment. This is also evident from the kinetic analysis presented in Fig. 5, C and E, where the slope of the regression line (and hence, the blocking rate constant) is clearly increased, whereas the intercept on the ordinate is similar to that obtained with the wild type channels. To test whether the presence of a proline residue per se was sufficient to render the channels reversible from omega -conotoxin GVIA block, we generated an E1332P mutant. As shown in Fig. 5D, omega -conotoxin GVIA block of this mutant was not reversible, hence the presence of a proline per se near in the putative EF hand motif is insufficient to render the channels reversible from toxin block. Interestingly, the effect of the E1332P mutation on the blocking rate constant was opposite to that obtained with the E1332R construct, which tended to slow the blocking kinetics.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 5.   A and B, effects of different substitutions for Gly1326 on omega -conotoxin (CTX) GVIA block in the form of representative time courses. Note that unlike in case of G1326P, block of G1326K and G1326E channels remains irreversible. C, kinetic analysis of toxin block as described in Fig. 1C. Note that the blocking rate constant is increased in both mutants compared with the wild type channels. The blocking and unblocking rate constants obtained from the fits were as follows: G1326K, kon = 2.18 µM-1 min-1, koff = 0.5 min-1; G1326E, kon = 3.06 µM-1 min-1, koff = 0.21 min-1). D, effect of a substitution of Glu1332 to proline. Unlike the G1326P mutant, the E1332P mutant cannot be recovered from toxin block. E, comparison of the blocking rate constants obtained with wild type and mutant channels. The blocking rate constants were obtained as described in the legend to Fig. 3C. Error bars denote S.E. values, numbers in parentheses are numbers of experiments, and asterisks denote statistical significance relative to control. All mutants shown in the figure were coexpressed with beta 1b and alpha 2-delta subunits.

Overall, our data indicate that at least two additional residues besides those identified previously by Ellinor et al. (16) are important determinants of omega -conotoxin GVIA block and that residue 1326 is an important factor in controlling both access of the toxin and the strength of interaction with the channel.

The G1326P Mutant Results in Reversible omega -Conotoxin MVIIA Binding-- The striking effects observed with the G1326P mutant raise an obvious question: is omega -conotoxin MVIIA block affected in a similar manner? As shown in Fig. 6, both the development and the reversibility of block of the channel by omega -conotoxin MVIIA are dramatically enhanced in the Gly1326 mutant. However, there were subtle differences in the manner by which the mutation affected block by the two toxins. Whereas the effects of the mutation on recovery from omega -conotoxin MVIIA block was even more pronounced than those observed with the GVIA isoform (i.e. complete recovery from block after 5 min), the effect of the mutation on the blocking rate constant was greater for omega -conotoxin GVIA (16-fold increase in kon) than for omega -conotoxin MVIIA (7-fold increase). Nonetheless, these data indicate that there is significant overlap in the channel structural determinants of the access of omega -conotoxins GVIA and MVIIA to their binding sites, consistent with biochemical data showing that both toxins compete for a common binding site (11).


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of mutations in positions 1326 and 1332 on omega -conotoxin (CTX) MVIIA block. A, representative time course of development of block and recovery from block for the G1326P mutant. Note the rapid reversibility of blocking action. B, kinetic analysis of omega -conotoxin MVIIA block of wild type alpha 1B or mutant (E1321K,D1323R,E1332R), and G1326P mutant channels (coexpressed with beta 1b and alpha 2-delta ). The blocking and unblocking rate constants obtained from the fits were as follows: G1326P, kon = 19.87 µM-1 min-1, koff = 2.11 min-1; triple mutant, kon = 0.68 µM-1 min-1, koff = 0.23 min-1). C and D, blocking and unblocking rate constants obtained with wild type, G1326P and (E1321K,D1323R,E1332R) channels. Data were obtained via linear regressions as shown in Fig. 1C for each individual experiment. The error bars denote S.E. valuess, numbers in parentheses reflect the numbers of individual experiments, and asterisks denote significance relative to wild type channels.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Peptide toxins isolated from venomous animal species are among the most potent inhibitors of voltage-gated calcium channels (2, 21). Calcium channel blocking peptides appear to fall into two major classes: gating blockers, which allosterically inhibit channel opening (e.g. omega -agatoxin IVA (22, 23) and omega -grammotoxin-S1A (24-26)), and pore blockers, which physically prevent ion permeation (e.g. omega -conotoxins GVIA (16) and MVIIC (11, 17) and omega -agatoxin III (27, 28)). To date, the exact sites of action of the gating inhibitors on the calcium channel alpha 1 subunit remain enigmatic. Furthermore, unlike in the case of potassium channels (5, 29) and sodium channels (7, 30-32), the detailed molecular mechanisms by which calcium channels are inhibited by pore-blocking toxins remain poorly understood. To date, there has only been one comprehensive study that has examined the channel structural determinants of omega -conotoxin GVIA block. Using a series of chimeras between alpha 1A P/Q-type and alpha 1B N-type calcium channels, Ellinor et al. (16) were able to show that the domain III S5-S6 region was critically important for block of N-type calcium channels by omega -conotoxin GVIA. In particular, residues Gln1327, Glu1334, Glu1337, and Gln1339 were found to contribute to omega -conotoxin GVIA block, with Glu1337 mediating the largest effect. In the present study, we have identified two additional residues that appear to be important for the interaction of the channel with pore-blocking toxins, residues Gly1326 and Glu1332. Since these residues are conserved in alpha 1A and alpha 1B channels (33), their contributions to toxin block could not have been detected with a chimeric approach. It is interesting to note that amino acid substitutions adjacent to the region identified by Ellinor et al. (16) (i.e. Glu1332 and Gly1326) mediated a more pronounced effect on omega -conotoxin GVIA block, compared with mutations that occur further upstream (i.e. Glu1321 and Asp1332). This could perhaps suggest that residue 1326 might form an outer boundary for the omega -conotoxin GVIA receptor site. This is further supported by data obtained with an R1325E construct, which did not display altered toxin block (not shown).

We have previously reported that the expression of the G1326P, E1332R, and a double (E1321K,D1323R) mutant resulted in currents that no longer supported a differential permeability to calcium and barium ions, but otherwise displayed biophysical properties that did not differ significantly from those of the wild type channels, including activation, inactivation, and block by divalent cations (18). We attributed the effects on ion selectivity to a highly localized specific disruption of the function of a putative calcium binding EF hand spanned by residues 1321-1332, rather than a global structural rearrangement of the alpha 1B subunit. The observation that substitutions in positions 1321 and 1323 did not affect omega -conotoxin GVIA block is consistent with a highly localized and specific effect of amino acid substitutions in these positions on ion selectivity. Moreover, that substitutions in positions Gly1326 and Glu1332 did not affect channel gating, and cadmium block (18) is also consistent with a localized effect of these mutations on the toxin-channel interaction.

The most pronounced effect on toxin action was observed with the G1326P construct in that the blocking rate constant was increased 16-fold, and block became entirely reversible. This suggests that the mutation increases both the accessibility of the binding site and at the same time results in a less stable interaction between the toxin and the channel. Interestingly, other substitutions in this position, while increasing the rate of development of block did not affect the reversibility of block. It is well established that proline residues act as helix breakers and thus may mediate a more pronounced effect on the local structure of the channel. However, the presence of a proline in this region of the channel per se is not sufficient to render toxin block reversible, since the E1332P construct did not affect recovery from toxin block. The dramatic effects on the reversibility of block seen with the G1326P mutant cannot be explained by a simple change in toxin affinity, since this mutation resulted in only a ~4-fold increase in the equilibrium dissociation constants of the two toxins as determined from the ratios of unblocking and blocking time constants. Instead, we envision a scenario in which residue Gly1326 restricts the access of the toxin to its docking site (Fig. 7). Once the toxin has docked to its receptor site, the barrier formed by Gly1326 would aid in the stabilization of the channel toxin interaction. Upon mutation of Gly1326 to proline, this barrier may be removed, thereby providing greater access to the blocking site, but concomitantly resulting in a less stable interaction. Substitutions of Gly1326 with other types of amino acid residues may not be as effective in removing the putative access barrier and, hence, affect the time constant for development of block and unblock to a smaller degree.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 7.   Possible model accounting for the effects of the G1326P mutation on the time course of development and recovery from omega -conotoxin (CTX) block. In the wild type channel, residue Gly1326 may form a barrier that restricts access of the toxin to its blocking site but also prevents the dissociation of the toxin once docked to the receptor. The introduction of the proline residue may remove this barrier, thus providing more ready access and concomitantly, a less stable binding interaction.

It is more difficult to speculate about the basis for the effects of substitutions in position Glu1332. It is possible that this residue forms a low affinity contact point between the toxin and the channel. Alternatively, changes in this residue may allosterically affect the interaction of the toxin with residue Glu1334, which was previously identified as an important determinant of omega -conotoxin GVIA block (16). The change in the time constant of development of block observed with the E1332R and E1332P constructs are unlikely due to a change in net charge of the channel, since neither the double (E1321K,D1323R) nor the R1325E substitution significantly affected toxin block.

A major finding is that mutations that affect block of the channel by omega -conotoxin GVIA also affect the action of omega -conotoxin MVIIA. To date, the only evidence suggesting an overlap between the blocking sites for the two toxins had come from competitive binding studies (2). Here, we have demonstrated for the first time at the amino acid level that the two toxins share some of the same channel molecular determinants. The two toxins share a similar disulfide bond arrangement comprised of six conserved cysteine residues, but only about 27% in sequence identity among their non-cysteine residues (2, 34). This suggests that one or more of the conserved toxin residues may perhaps interact with residue 1326 on the calcium channels or that the three-dimensional folding of the toxin backbone is a key determinant for blocking action. The latter scenario would fit with the model shown in Fig. 7, which predicts that toxins with an overall similarity in their backbone structure would be similarly affected by the presence/absence of an access barrier. Regardless of the underlying mechanism, our data show that a single amino acid residue in the domain III S5-H5 region is sufficient to control access of two structurally distinct peptides from two different cone snail species, suggesting that the basic mechanism by which these two toxins block channel activity is highly conserved.

In summary, our data identify two residues (Gly1326 and Glu1332) in the alpha 1B subunit, which are important determinants of omega -conotoxin block. The G1326P mutant may serve as a convenient tool for further investigation into the detailed mechanisms underlying omega -conotoxin block of N-type calcium channels by allowing direct access to the effects of any additional mutations on the channel or the toxin molecule on both blocking and unblocking kinetics. In view of the clinical use of omega -conotoxin MVIIA as an analgesic (35, 36), such information may aid in the design of more effective calcium channel therapeutics.

    FOOTNOTES

* This work was supported by operating grants (to G. W. Z.) from the Canadian Institutes of Health Research (CIHR) and the Heart and Stroke Foundation of Alberta and the Northwest Territories and through a Scholarship Award (to G. W. Z.) from the EJLB Foundation.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.

These authors contributed equally to this study.

|| Recipient of a postdoctoral fellowship from the Natural Science and Engineering Research Council of Canada.

Dagger Dagger Holds CIHR operating funds and a Senior Scientist award from the CIHR.

§§ Holds Faculty Scholarships from the Alberta Heritage Foundation for Medical Research (AHFMR) and the CIHR and is the Novartis Investigator in Schizophrenia Research. To whom correspondence should be addressed: Dept. of Physiology and Biophysics, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada. Tel.: 403-220-8687; Fax: 403-210-8106; E-mail: zamponi@ucalgary.ca.

Published, JBC Papers in Press, February 2, 2001, DOI 10.1074/jbc.M100406200

    ABBREVIATIONS

The abbreviation used is: TEA, tetraethylammonium.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Quik, M. (1992) Mol. Neurobiol. 6, 19-40[Medline] [Order article via Infotrieve]
2. Olivera, B. M., Miljanich, G. P., Ramachandran, J., and Adams, M. E. (1994) Annu. Rev. Biochem. 63, 823-867[CrossRef][Medline] [Order article via Infotrieve]
3. Favreau, P., Le Gall, F., Benoit, E., and Molgo, J. (1999) Acta Physiol. Pharmacol. Ther. Latinoam. 49, 257-267[Medline] [Order article via Infotrieve]
4. Possani, L. D., Becerril, B., Delepierre, M., and Tytgat, J. (1999) Eur. J. Biochem. 264, 287-300[Abstract/Free Full Text]
5. Tenenholz, T. C., Klenk, K. C., Matteson, D. R., Blaustein, M. P., and Weber, D. J. (2000) Rev. Physiol. Biochem. Pharmacol. 140, 135-185[Medline] [Order article via Infotrieve]
6. Moczydlowski, E., Olivera, B. M., Gray, W. R., and Strichartz, G. R. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5321-5325[Abstract]
7. Becker, S., Prusak-Sochaczewski, E., Zamponi, G., Beck-Sickinger, A. G., Gordon, R. D., and French, R. J. (1992) Biochemistry 31, 8229-8238[Medline] [Order article via Infotrieve]
8. French, R. J., Prusak-Sochaczewski, E., Zamponi, G. W., Becker, S., Kularatna, A. S., and Horn, R. (1996) Neuron 16, 407-413[Medline] [Order article via Infotrieve]
9. Kulak, J. M., Nguyen, T. A., Olivera, B. M., and McIntosh, J. M. (1997) J. Neurosci. 17, 5263-5270[Abstract/Free Full Text]
10. Luo, S., Kulak, J. M., Cartier, G. E., Jacobsen, R. B., Yoshikami, D., Olivera, B. M., and McIntosh, J. M. (1998) J. Neurosci. 18, 8571-8579[Abstract/Free Full Text]
11. Woppmann, A., Ramachandran, J., and Miljanich, G. P. (1994) Mol. Cell. Neurosci. 5, 350-357[CrossRef][Medline] [Order article via Infotrieve]
12. Fox, J. A. (1995) Pfleugers Arch. 429, 873-875[Medline] [Order article via Infotrieve]
13. Boland, L. M., and Bean, B. P. (1993) J. Neurosci. 13, 516-533[Abstract]
14. Boland, L. M., Morrill, J. A., and Bean, B. P. (1994) J. Neurosci. 14, 5011-5027[Abstract]
15. Williams, M. E., Brust, P. F., Feldman, D. H., Patthi, S., Simerson, S., Maroufi, A., McCue, A. F., Velicelebi, G., Ellis, S. B., and Harpold, M. M. (1992) Science 257, 389-395[Medline] [Order article via Infotrieve]
16. Ellinor, P. T., Zhang, J. F., Horne, W. A., and Tsien, R. W. (1994) Nature 372, 272-275[CrossRef][Medline] [Order article via Infotrieve]
17. McDonough, S. I., Swartz, K. J., Mintz, I. M., Boland, L. M., and Bean, B. P. (1996) J. Neurosci. 15, 2612-2623
18. Feng, Z. P., Hamid, J., Doering, C., Jarvis, S. E., Bosey, G. M., Bourinet, E., Snutch, T. P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 5726-5730[Abstract/Free Full Text]
19. Beedle, A. M., and Zamponi, G. W. (2000) Biophys. J. 79, 260-270[Abstract/Free Full Text]
20. Catterall, W. A. (2000) Annu. Rev. Cell Dev. Biol. 16, 521-555[CrossRef][Medline] [Order article via Infotrieve]
21. Adams, M. E., Myers, R. A., Imperial, J. S., and Olivera, B. M. (1993) Biochemistry 30, 12566-12570
22. Mintz, I. M., Venema, V. J., Swiderek, K. M., Lee, T. D., Bean, B. P., and Adams, M. E. (1992) Nature 27, 827-829
23. McDonough, S. I., Mintz, I. M., and Bean, B. P. (1997) Biophys. J. 72, 2117-2128[Abstract]
24. Lampe, R. A., Defeo, P. A., Davison, M. D., Young, J., Herman, J. L., Spreen, R. C., Horn, M. B., Mangano, T. J., and Keith, R. A. (1993) Mol. Pharmacol. 44, 451-460[Abstract]
25. Piser, T. M., Lampe, R. A., Keith, R. A., and Thayer, S. A. (1995) Mol. Pharmacol. 48, 131-139[Abstract]
26. McDonough, S. I., Lampe, R. A., Keith, R. A., and Bean, B. P. (1997) Mol. Pharmacol. 52, 1095-1104[Abstract/Free Full Text]
27. Ertel, E. A., Warren, V. A., Adams, M. E., Griffin, P. R., Cohen, C. J., and Smith, M. M. (1994) Biochemistry 33, 5098-5108[Medline] [Order article via Infotrieve]
28. Mintz, I. M. (1994) J. Neurosci. 14, 2844-2853[Abstract]
29. Miller, C. (1995) Neuron 15, 5-10[Medline] [Order article via Infotrieve]
30. Chahine, M., Sirois, J., Marcotte, P., Chen, L., and Kallen, R. G. (1998) Biophys. J. 75, 236-246[Abstract/Free Full Text]
31. Li, R. A., Tsushima, R. G., Kallen, R. G., and Backx, P. H. (1997) Biophys. J. 73, 1874-1884[Abstract]
32. Li, R. A., Ennis, I. L., Velez, P., Tomaselli, G. F., and Marban, E. (2000) J. Biol. Chem. 275, 27551-27558[Abstract/Free Full Text]
33. Stea, A., Soong, T. W., and Snutch, T. P. (1995) in Handbook of Receptors and Channels: Ligand- and Voltage-gated Ion Channels (North, R. A., ed) , pp. 113-141, CRC Press Inc., Boca Raton, FL
34. Olivera, B. M., Gray, W. R., Zeikus, R., McIntosh, J. M., Varga, J., Rivier, J., de Santos, V., and Cruz, L. J. (1985) Science 230, 1338-1343[Medline] [Order article via Infotrieve]
35. Ridgeway, B., Wallace, M., and Gerayli, A. (2000) Pain 85, 287-289[CrossRef][Medline] [Order article via Infotrieve]
36. Penn, R. D., and Paice, J. A. (2000) Pain 85, 291-296[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.