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Br J Pharmacol. Jul 1999; 127(6): 1375–1387.
PMCID: PMC1760671

Differential blockade of rat α3β4 and α7 neuronal nicotinic receptors by ω-conotoxin MVIIC, ω-conotoxin GVIA and diltiazem


  1. Rat α3β4 or α7 neuronal nicotinic acetylcholine receptors (AChRs) were expressed in Xenopus laevis oocytes, and the effects of various toxins and non-toxin Ca2+ channel blockers studied. Nicotinic AChR currents were elicited by 1 s pulses of dimethylphenylpiperazinium (DMPP, 100 μM) applied at regular intervals.
  2. The N/P/Q-type Ca2+ channel blocker ω-conotoxin MVIIC inhibited α3β4 currents with an IC50 of 1.3 μM; the blockade was non-competitive and reversible. The α7 currents were unaffected.
  3. At 1 μM, ω-conotoxin GVIA (N-type Ca2+ channel blocker) inhibited by 24 and 20% α3β4 and α7 currents, respectively. At 1 μM, ω-agatoxin IVA (a P/Q-type Ca2+ channel blocker) did not affect α7 currents and inhibited α3β4 currents by only 15%.
  4. L-type Ca2+ channel blockers furnidipine, verapamil and, particularly, diltiazem exhibited a preferential blocking activity on α3β4 nicotinic AChRs.
  5. The mechanism of α3β4 currents blockade by ω-conotoxins and diltiazem differed in the following aspects: (i) the onset and reversal of the blockade was faster for toxins; (ii) the blockade by the peptides was voltage-dependent, while that exerted by diltiazem was not; (iii) diltiazem promoted the inactivation of the current while ω-toxins did not.
  6. These data show that, at concentrations currently employed as Ca2+ channel blockers, some of these compounds also inhibit certain subtypes of nicotinic AChR currents. Our data calls for caution when interpreting many of the results obtained in neurons and other cell types, where nicotinic receptor and Ca2+ channels coexist.
Keywords: ω-conotoxin MVIIC, ω-conotoxin GVIA, ω-agatoxin IVA, diltiazem, α3β4 nicotinic AChRs, α7 nicotinic AChRs, Xenopus oocytes


In addition to specific interactions with the α1 subunit of the voltage-dependent L-type Ca2+ channels, an increasing number of other molecular targets for the different subgroups of organic Ca2+ channel antagonist drugs have been recognized (Zernig, 1990). Thus, blocking effects exerted by dihydropyridines, verapamil and diltiazem on 45Ca2+ uptake, intracellular Ca2+ signal, catecholamine secretion, whole-cell inward currents, and 86Rb+ efflux, upon neuronal nicotinic acetylcholine receptor (nicotinic AChR) stimulation have been described in bovine chromaffin cells (López et al., 1993; Gandía et al., 1996; Villarroya et al., 1997) and in a human neuroblastoma cell line (Donnelly-Roberts et al., 1995). However, contradictory data have been obtained with ω-toxins traditionally employed as selective blockers of non L-type Ca2+ channels. For instance, the N-type Ca2+ channel blocker ω-conotoxin GVIA (1 μM) and the P-type Ca2+ channel blocker ω-agatoxin IVA (100 nM) reduce by 80 and 70% respectively, the nicotinic currents in bovine chromaffin cells (Fernández et al., 1995; Granja et al., 1995). In contrast, other authors found no significant effects of these two toxins on 45Ca2+ uptake (Villarroya et al., 1997) or 86Rb+ efflux (Donnelly-Roberts et al., 1995) induced by nicotinic activation of chromaffin cells and human neuroblastoma cells. No data on nicotinic receptors are available with the N/P/Q-type Ca2+ channel blocker ω-conotoxin MVIIC.

Recently, several nicotinic subunits from bovine chromaffin cells which resemble the brain α3, α5, α7 and β4 neuronal nicotinic AChR subunits with homologies above 90%, have been cloned (Criado et al., 1992; García-Guzmán et al., 1995; Campos-Caro et al., 1997). Moreover, α3, α5 and β4 subunits are expressed both in adrenergic and noradrenergic chromaffin cells, while the α7 subunit is preferentially expressed in adrenergic cells (Criado et al., 1997). Therefore, it is possible that a wide variety of nicotinic AChR subtypes could be expressed in each cell type. Since previous experiments were not able to distinguish between the blockade exerted by Ca2+ channel blockers on different subtypes of nicotinic AChRs, discrepancies concerning the effects of ω-toxins on nicotinic-mediated responses could be explained by differences in the chromaffin cell type tested and/or the nicotinic receptor subtype(s) expressed in each cell assayed.

In this study we have taken advantage of the oocyte as a receptor expression model, to determine the sensitivity of pure populations of α3β4 or α7 neuronal nicotinic AChRs to ω-toxins and non-peptide molecules, at concentrations in the range of those used as Ca2+ channel blockers. Our results show that whereas α7 nicotinic AChRs are scarcely affected by most of the compounds tested, α3β4 nicotinic AChRs are sensitive to all of them, particularly to ω-conotoxin MVIIC and diltiazem. We present here a thorough study on the differential inhibitory effects of peptide and non-peptide Ca2+ channel blockers, on rat brain nicotinic AChRs of the α3β4 and α7 subtypes expressed in Xenopus laevis oocytes. Additionally, we present a study on the mechanism of blockade exerted by ω-conotoxin MVIIC and by diltiazem, the two most potent blockers of α3β4 receptors.


Techniques for the in vitro transcription of nicotinic AChR subunits cDNAs, oocytes injection and electrophysiological recordings of the expressed foreign receptors have been described previously (Miledi et al., 1989; Montiel et al., 1997; López et al., 1998).

Preparation of RNA and injection of Xenopus oocytes

The plasmids pPCA48E, pZPC13, PCX49 and pHIP306 containing the entire coding regions of rat brain nicotinic AChR α3, β4, β2 and α7 subunits were linearized with the restriction enzymes EcoRI, XhoI, BamHI and SmaI, respectively. Linearized plasmids were transcribed with SP6 (α3), T3 (β4) and T7 (β2, α7) RNA polymerases using a mCAP RNA capping Kit (Stratagene C.S. La Jolla, CA, U.S.A.).

Mature female Xenopus laevis frogs obtained from a commercial supplier (CRBM du CNRS, Montpellier, France) were anaesthetized with tricaine solution (0.125%) and ovarian lobes were dissected out. Then, follicle-enclosed oocytes were manually stripped from the ovary membranes and incubated overnight at 16°C in a modified Barth's solution containing (in mM): NaCl 88, KCl 1, NaHCO3 2.4, MgSO4 0.82, Ca(NO3)2 0.33, CaCl2 0.41, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid) 10, buffered to pH 7.4 and supplemented with gentamycin (0.1 mg ml−1) and sodium pyruvate (5 mM). Next day, healthy follicle-enclosed oocytes were injected with 50 nl (50 ng) of α7 RNA or 50 nl (25 : 25 ng) of α3 : β4 or α3 : β2 RNAs using a nanoject automatic injector (Drummond Scientific Co., Broomall, PA, U.S.A.). Electrophysiological recordings were made 2–5 days after RNA injections.

Electrophysiological recordings

Experiments were carried out at room temperature (22–25°C) in Ringer's solution containing (in mM): NaCl 115, KCl 2, CaCl2 1.8, HEPES 5, buffered to pH 7.4 with NaOH. Membrane currents were recorded with a two-electrode voltage clamp amplifier (OC-725-B Warner Instrument Corporation, Hamden, CT, U.S.A.) using microelectrodes with resistances of 0.5–5 MΩ made from borosilicate glass (GC100TF-15, Clark Electromedical, Pangbourne, U.K.) and filled with KCl (3 M). The holding potential in all experiments was −60 mV, except in those carried out to study the voltage-dependent effects of Ca2+ antagonist compounds (see Results). Single oocytes were held in a 0.3 ml volume chamber and constantly superfused with Ringer's solution by gravity (4 ml.min−1). The volume in the chamber was maintained constant using the reverse suction of one air pump. Solutions containing the nicotinic agonist dimethylphenylpiperazinium (DMPP), or the nicotinic blockers were applied with the use of a set of 2-mm diameter glass tubes located close to the oocyte. Voltage protocols, DMPP pulses and data adquisition were controlled using a Digidata 1200 Interface and CLAMPEX software (Axon Instruments, Foster City, CA, U.S.A.).

Materials and solutions

All products not specified were purchased from SIGMA (Madrid, Spain). Furnidipine was supplied by Laboratorios Alter (Madrid, Spain). Diltiazem and verapamil were purchased from Research Biochemical International (Natick, MA, U.S.A.). ω-Conotoxin MVIIC and ω-agatoxin IVA were purchased from Peptide Institute (Osaka, Japan) and ω-conotoxin GVIA from Bachem Feinchemikalien (Bubendorf, Switzerland). Furnidipine was dissolved in dimethylsulphoxide (DMSO) at 10−2 M and diluted in Ringer's solution to the desired concentrations. Toxins were prepared in distilled water at 10−4 M. Concentrate stock solutions of toxins were aliquoted and stored at −20°C until use. Final concentrations of toxins were prepared in Ringer's solution.

Statistical analysis

Values of agonist concentration eliciting half maximal current EC50 and antagonist concentration eliciting 50% blockade of maximal current IC50 were estimated through non-linear regression analysis of ISI software, for a PC computer from the concentration-response curves for agonist (DMPP) and antagonists (diltiazem, ω-conotoxin MVIIC). To calculate the time constant (τ) for blockade and recovery of nicotinic currents, records were fitted to a single exponential curve. Differences between groups were compared by Student's t-test with the statistical program Statworks TM; a value of P[less-than-or-eq, slant]0.05 was taken as the limit of statistical significance.


Blockade by ω-toxins of α3β4 and α7 currents

Oocytes expressing α3β4 or α7 nicotinic AChRs were stimulated with DMPP (100 μM, 20 s) at a holding potential of −60 mV. As previously described (Papke & Heinemann, 1991; López et al., 1998), important differences in the kinetics of currents (IDMPP) between both receptor subtypes were seen. Figure 1a shows normalized IDMPP for α3β4 or α7 nicotinic AChRs; α7 current exhibited a faster activation and inactivation kinetics. With the purpose of studying the effect of ω-toxins on α3β4 or α7-activated currents, protocols with brief pulses of DMPP (100 μM, 1 s) applied at 1 min intervals were selected; this interval was chosen in order to avoid nicotinic receptor desensitization.

Figure 1
Effects of ω-conotoxin MVIIC on α7 and α3β4 nicotinic AChRs expressed in oocytes. Oocytes were voltage-clamped at a holding potential of −60 mV and stimulated with 100 μM DMPP. (a) Normalized ...

After a few initial DMPP pulses, α3β4 and α7-mediated peak currents were quite reproducible over a 30 min period. The peak amplitude of the stabilized agonist-induced current, just preceding the addition of toxin, was used as control response (100%); then toxin was added 1 min before the next DMPP pulse. Figure 1 (b and c) show two examples of original traces of control currents obtained in two different oocytes expressing α7 and α3β4 nicotinic AChRs, and the inhibitory effects exerted by 1 μM ω-conotoxin MVIIC. Whereas α7 nicotinic AChRs were unaffected, ω-conotoxin MVIIC blocked by 50% the α3β4 current. This protocol was repeated but using increasing concentrations of either ω-conotoxin MVIIC or ω-conotoxin GVIA, in different oocytes expressing α3β4 or α7 nicotinic AChRs. Figure 2 (a and b) shows averaged results of the inhibition curve obtained using these two toxins. ω-Conotoxin MVIIC had little effect on α7 current, whereas α3β4 currents were very sensitive to blockade (IC50, 1.3 μM). Thus, at a concentration as low as 0.3 μM, this toxin produced a significant inhibition of IDMPP evoked by activation of α3β4 nicotinic AChRs (20±2%; P[less-than-or-eq, slant]0.01). Moreover, concentrations of ω-conotoxin MVIIC usually employed to block N/P/Q-type Ca2+ channels (1 and 3 μM), inhibited significantly (P[less-than-or-eq, slant]0.001) by 44±3 and 55±5% respectively, the α3β4 currents. At 10 μM, the α7 current was inhibited by 17±6% (P[less-than-or-eq, slant]0.05) whereas the α3β4 current was blocked by 75±6% (P[less-than-or-eq, slant]0.001). In all cases, the reversibility of blockade upon washout of the toxin was fast and complete (not shown). This contrasts with the long-lasting blockade exerted by this toxin on N/P/Q-type Ca2+ channels (Albillos et al., 1996; Gandía et al., 1997).

Figure 2
Concentration-response blockade of α3β4 and α7 nicotinic AChRs by different peptides and non-peptide Ca2+ channel antagonists. Currents (IDMPP) were induced by DMPP pulses (100 μM, 1 s) applied at ...

Figure 2b shows the effects of increasing concentrations of ω-conotoxin GVIA; at the concentration usually employed as Ca2+ channel blocker (1 μM), the toxin inhibited α3β4 and α7 currents by 24% (P[less-than-or-eq, slant]0.05) and 20% (P[less-than-or-eq, slant]0.05), respectively. This inhibition was fully reversible upon washout (not shown). Once again, this finding contrasts with the long-lasting blockade of N-type Ca2+ channels exerted by this toxin (Albillos et al., 1996; Gandía et al., 1997).

ω-Agatoxin IVA was unable to block significantly α7 currents in spite of the use of 1 μM, a concentration higher than that considered selective to block P-type Ca2+ channels (low nanomolar range) and in the same range of that used to block Q-type channels (Figure 2c). Due to the expense of this toxin, it was not used at concentrations above 1 μM. At this concentration, ω-agatoxin IVA blocked the α3β4 current by 15±4% (P[less-than-or-eq, slant]0.05).

Inhibition by diltiazem, furnidipine and verapamil of α3β4 or α7 currents

The same experimental protocol described for ω-toxins was employed to assay the effects of diltiazem (a benzothiazepine derivative), furnidipine (a 1,4-dihydropyridine and verapamil (a phenylalkylamine) on nicotinic AChR currents. The α7 current was the most resistant to blockade in all cases. Diltiazem was the most potent on α3β4 nicotinic AChRs (IC50 3 μM; Figure 2d). At the higher concentration tested (10 μM), diltiazem blocked by 75±4 and 32±3% (P[less-than-or-eq, slant]0.001) the IDMPP induced by activation of α3β4 and α7 nicotinic AChRs, respectively. Figure 2d also shows the α3β4 or α7 current inhibition by furnidipine and verapamil at the highest concentration used (10 μM). At this concentration, the inhibition by furnidipine of α3β4 and α7 currents was 62±4% (P[less-than-or-eq, slant]0.001) and 24±3% (P[less-than-or-eq, slant]0.05), respectively; whereas verapamil reduced by 55±2% (P[less-than-or-eq, slant]0.001) and 32±3% (P[less-than-or-eq, slant]0.01) the currents elicited by the activation of these two receptors. At 3 μM, furnidipine inhibited significantly (P[less-than-or-eq, slant]0.01) α3β4 currents by 39±4% and α7 currents by 21±3%; while verapamil reduced α3β4 and α7 currents by 32±3% (P[less-than-or-eq, slant]0.001) and 26±2% (P[less-than-or-eq, slant]0.01), respectively.

Nicotinic currents measured in oocytes clamped at a holding potential of −60 mV, were a combination of a direct Na+ influx current through the nicotinic ionophore plus an indirect chloride efflux generated upon the activation of chloride channels by Ca2+ entry through the nicotinic pore; hence, the blockade of the current by peptide and non-peptide compounds in this study could be attributed to a direct effect on these chloride channels more than a nicotinic receptor inhibition. However, this did not seem to be the case since when chloride channels were directly recruited by photoreleased Ca2+, currents were not affected by any of the non-peptide (data not shown) and peptide blockers used in this study (Lomax et al., 1998). Furthermore, diltiazem inhibited in the same extent α3β4 or α7 currents in the presence of external Ca2+ (current experiments) or 1.8 mM Ba2+ (not shown). Additionally, it is well known that α7 nicotinic AChRs are highly permeable to Ca2+ (Seguela et al., 1993), which imply a higher component of Ca2+-activated chloride current in the α7 mediated response. Since most of the compounds used in this study had little or no effect on the α7 current, it seems unlikely that they were affecting the chloride current itself.

Effect of ω-toxins and diltiazem on the kinetics of nicotinic receptor currents: relevance of the β-subunit

Diltiazem, the most potent blocker among all the organic Ca2+ channel antagonists assayed, was selected for further comparative studies with ω-toxins. To study current kinetics, oocytes expressing α3β4 nicotinic AChRs were stimulated with DMPP for 20 s. To avoid receptor desensitization and current inactivation, the concentration of DMPP was reduced to 10 μM, and the pulses applied every 3 min. Under these experimental conditions, the control currents were quite reproducible and they exhibited practically no desensitization.

Figure 3 shows typical records of α3β4 control currents, and their inhibition by ω-conotoxin MVIIC (1 μM), ω-conotoxin GVIA (1 μM), ω-agatoxin IVA (1 μM) and diltiazem (3 μM), added 1 min before and during the DMPP pulse. Whereas the three toxins blocked IDMPP without affecting the kinetics of the current (a–c), diltiazem promoted a clear current inactivation (see different blockade of peak and late IDMPP in Figure 3d). This inactivating effect exerted by diltiazem did not occur in oocytes expressing α3β2 receptors. The blocking effects of diltiazem and ω-conotoxin MVIIC on α3β2 currents are shown in Figure 4. Note the specially marked blockade induced by the toxin on this receptor subtype. Because desensitization of control α3β2 currents was higher than that obtained with α3β4 receptors, a lower concentration of DMPP (3 μM) was employed for α3β2 experiments.

Figure 3
Effects of ω-toxins and diltiazem on the kinetics of α3β4 currents induced by long DMPP pulses. Currents (IDMPP) were elicited by pulses of DMPP (10 μM, 20 s) applied every 3 min in oocytes expressing ...
Figure 4
Effects of ω-conotoxin MVIIC and diltiazem on the kinetics of α3β2 currents induced by long DMPP pulses. Currents (IDMPP) were induced by 3 μM DMPP applied during 20 s every 3 min. Blockers were ...

Quantitative averaged blockade of peak and late IDMPP upon the activation of α3β4 and α3β2 nicotinic AChRs exerted by diltiazem and ω-conotoxin MVIIC are plotted in Figure 5. Diltiazem blocked significantly more the late IDMPP than the peak α3β4 currents (63 versus 43%). However, a similar degree of blockade of peak and late IDMPP by diltiazem, in oocytes expressing α3β2 nicotinic AChRs, was observed (34 versus 33%). Note the significant higher blockade by diltiazem of late IDMPP in oocytes expressing α3β4 receptors compared with those expressing α3β2 receptors. ω-Conotoxin MVIIC, at a concentration of 1 μM, blocked to a similar extent the peak and late IDMPP in oocytes expressing α3β4 and α3β2 nicotinic AChRs; however, this time the β2 subunit conferred the receptor a higher sensitivity to the toxin (64±3% blockade of peak IDMPP in oocytes expressing α3β2 nicotinic AChRs versus 47±3% inhibition of peak IDMPP in oocytes expressing α3β4 receptors; P[less-than-or-eq, slant]0.01).

Figure 5
Blockade by ω-conotoxin MVIIC and diltiazem of peak and late DMPP currents in oocytes expressing α3β4 or α3β2 nicotinic AChRs. Protocols and concentrations were similar to those described in Figures 3 and ...

Time-course of α3β4 current blockade and recovery induced by ω-conotoxin MVIIC, ω-conotoxin GVIA and diltiazem

These experiments were designed to study the rates of blockade and recovery of nicotinic α3β4 currents following the application of ω-toxins and diltiazem. Two different experimental protocols were used. In the first, two DMPP pulses (10 μM, 80 s), 5 min apart, were applied to the same oocyte. This concentration and time interval were selected because currents induced in this way were reproducible (not shown) and do not present run-down upon successive pulses. After a first DMPP pulse, in which a stable current response was obtained in most of the oocytes tested, a second DMPP pulse was applied 5 min later. During this second pulse, when current stabilized (20 s after starting DMPP stimulation), ω-conotoxin MVIIC (1 μM), ω-conotoxin GVIA (3 μM) or diltiazem (3 μM) were added along with DMPP. Typical examples of this protocol are illustrated in Figure 6 (a–c). ω-Conotoxin MVIIC produced an inhibition of the current following a time-course curve that could be fitted to a single exponential with a τon of 3±1 s (n=6); blockade of current amounted to 40–50%. The same was observed with ω-conotoxin GVIA assayed at a higher concentration (3 μM) to observe better the blockade; this blockade also fitted to a single exponential with a τon of 1.9±0.1 s (n=4). Diltiazem (3 μM) inhibited the α3β4 current, although with a slower time-course than ω-toxins (τon, 10±1 s; n=4; P[less-than-or-eq, slant]0.05). Differences in the time required to remove the blockade were also found between toxins and diltiazem using a second experimental protocol. Now, oocytes were stimulated with two DMPP pulses (80 s), 5 min apart; toxins or diltiazem were superfused during the first 40 s of the second DMPP pulse, and then the blockers were removed. Typical records corresponding to ω-conotoxin MVIIC, ω-conotoxin GVIA or diltiazem washouts are shown in Figure 6 (d–f). After removing ω-conotoxin MVIIC or ω-conotoxin GVIA, IDMPP blockade recovered faster (τoff, 1.6±0.2 and 2.4±0.3 s, respectively; n=4) than diltiazem (τoff 9.3±0.9 s; n=4; P[less-than-or-eq, slant]0.01).

Figure 6
Onset and offset of blockade produced by ω-conotoxin MVIIC, ω-conotoxin GVIA or diltiazem on α3β4 currents. (a, b and c) show original records of control currents obtained in three different oocytes (each out of 4–6) ...

Voltage-dependence and use-dependence of the blockade of α3β4 currents exerted by ω-toxins and diltiazem

The possible voltage-dependence of the blocking effects of ω-conotoxin MVIIC, ω-conotoxin GVIA and diltiazem on α3β4 currents were explored at different holding potentials (from −100 to +20 mV), using 20 mV steps. At each holding potential, DMPP current (IDMPP) was activated by two pulses of DMPP (100 μM 1 s, 1 min apart). When all potentials were tested, the protocol was repeated in the same oocyte, but this time in the presence of ω-conotoxin MVIIC (1 μM), ω-conotoxin GVIA (3 μM) or diltiazem (3 μM); blockers were present throughout the experiment, including the minute before and during the DMPP pulse. Figure 7 shows that whereas the blockade by diltiazem did not exhibit voltage-dependence (Figure 7c), the inhibition by ω-toxins was higher at hyperpolarizing potentials. For instance, at −100 mV ω-conotoxin MVIIC blocked IDMPP by 74±2%, while at −40 mV blockade was significantly lower (P[less-than-or-eq, slant]0.001) and amounted to 37±3% (n=4). Similar difference in the blockade (P [less-than-or-eq, slant]0.001) were obtained in the case of ω-conotoxin GVIA, 77±4 and 30±2% blockade (n=4) at −100 mV and −40 mV, respectively (Figure 7, right parts of a and b).

Figure 7
Effects of membrane potential on the blockade of IDMPP elicited by ω-conotoxin MVIIC (MVIIC), ω-conotoxin GVIA (GVIA) and diltiazem in α3β4-injected oocytes. Oocytes were clamped at different holding potentials (from −100 ...

To address the question of whether toxins and diltiazem produce a use-dependent receptor blockade, the effects of these compounds on successive DMPP pulses, applied at different frequencies, were assayed. Oocytes expressing α3β4 nicotinic AChRs were stimulated with DMPP pulses (10 μM, 500 ms) every 20 s. Once a stable current was reached (IDMPPmax), ω-conotoxin MVIIC (1 μM), ω-conotoxin GVIA (3 μM), or diltiazem (3 μM) were superfused during the next 5–6 DMPP pulses. After washing out the blocker and a complete recovery of current was obtained, the protocol was repeated in the same oocyte, but using 10 s intervals between DMPP pulses (up to ten pulses). Once more, upon washout of the blockers, a third stimulation train with 15 successive DMPP pulses, at 5 s intervals, was applied in the absence or presence of the blocker. Figure 8 shows the results obtained from a typical oocyte (out of three) for each blocker Figure 8a–c show the experimental protocol for ω-conotoxin MVIIC, ω-conotoxin GVIA, or diltiazem, using 10 s intervals between DMPP pulses. Figure 8 (d–f) summarizes the IDMPP/IDMPPmax results obtained with the blockers during the first 40 s, using the three frequencies. Both ω-toxins and diltiazem inhibited the current equally well, and in a single step. No statistical differences in the blockade exerted by each of the compounds, at the three frequencies studied, were found. Therefore, IDMPP/IDMPPmax values obtained during the last DMPP pulse, for the three frequencies assayed (5, 10 and 20 s) were respectively, 0.45±0.04, 0.48±0.05 and 0.46±0.05 with ω-conotoxin MVIIC; 0.38±0.02, 0.39±0.03 and 0.44±0.04 with ω-conotoxin GVIA; and 0.56±0.05, 0.60±0.04 and 0.59±0.04 with diltiazem.

Figure 8
Blocking effects of ω-conotoxin MVIIC, ω-conotoxin GVIA and diltiazem on α3β4 currents induced by DMPP pulses applied at different frequencies. Oocytes expressing α3β4 nicotinic AChRs were stimulated with ...

Blockade by ω-conotoxin MVIIC and diltiazem of α3β4 currents generated by increasing DMPP concentrations

To know more about the nature of blockers interaction with the neuronal nicotinic AChR, attempts were made to define whether the blockade of IDMPP was competitive or non-competitive. Oocytes expressing α3β4 receptors were stimulated with increasing concentrations of DMPP applied as brief pulses (1 s) every 1 min, in the absence and later on, in the presence of ω-conotoxin MVIIC (1 μM) or diltiazem (3 μM). Figure 9 shows that a maximal current amplitude was evoked by 100 μM of DMPP; higher concentrations of the nicotinic agonist produced an important desensitization of the receptor. The inhibition produced by ω-conotoxin MVIIC or diltiazem could not be overcome by increasing concentrations of DMPP as it is clearly shown in the two inserts of the figure. The calculated EC50 values for DMPP in the absence or presence of ω-conotoxin MVIIC were 17 and 12 μM respectively; and 26 and 17 μM in the absence or the presence of diltiazem. All these results suggest a non-competitive mechanism of action between both blockers and the nicotinic agonist for the receptor.

Figure 9
Non-competitive effect of ω-conotoxin MVIIC and diltiazem on α3β4 nicotinic currents. Currents were evoked by successive pulses (1 s) of increasing concentrations of DMPP, applied every 1 min, in the absence or ...


The results of this study are relevant in two aspects: (i) an emerging pharmacology of nicotinic AChR subtypes; (ii) the limited selectivity of agents available to block L-, N- or P/Q-subtypes of Ca2+ channels. We report, for the first time, that ω-conotoxin MVIIC is a selective blocker of heteromeric α3β2 and α3β4 nicotinic AChRs, but did not recognize homomeric α7 receptors. This finding contrasts with the fact that α7 currents are highly sensitive to other toxins such as α-conotoxin ImI, methyllycaconitine or α-bungarotoxin (López et al., 1998). Another interesting finding is the scarce activity of diltiazem and other non-peptide Ca2+ channel antagonists on α7 receptors in contrast to their blocking effects on α3β4 nicotinic AChRs.

Blockade of neuronal nicotinic AChR by ω-conotoxins and non-peptide compounds described above does not seem to be the result of a non-specific indiscriminate interaction with ion-activated or ligand-gated ion channels. Thus, ω-toxins do not inhibit the Ca2+-activated chloride channels in oocytes (Lomax et al., 1998) and present results show that both organic and peptide molecules affected little if at all, α7 nicotinic AChRs. Moreover, this study point out that these compounds were capable of discriminating between nicotinic AChRs containing β2 or β4 subunits; i.e. ω-conotoxin MVIIC blocked more α3β2 than α3β4 receptors whereas diltiazem promoted the inactivation of IDMPP elicited by the activation of α3β4 but not by α3β2 receptors. These findings agree with the recent view that β subunits are involved in determining the physical structure and the pharmacological and kinetic properties of nicotinic AChRs (Duvoisin et al., 1989; Cachelin & Jaggi, 1991; Luetje & Patrick, 1991, Papke & Heinemann, 1991; Harvey & Luetje, 1996).

Our data shows that we are dealing with selective blocking effects of peptide and non-peptide molecules, traditionally considered as Ca2+ channel antagonists, on nicotinic AChRs. Also we have observed, at least, three pronounced differences between non-peptide drugs and ω-conotoxins regarding their mechanism of nicotinic blockade. Firstly, the onset and offset of α3β4 current blockade are quite different. So, ω-conotoxins MVIIC and GVIA had a τon for blockade of only 2–3 s, while that of diltiazem was 10 s. Also, the τoff for reversal of blockade exhibited a similar pattern. These effects might simply reflect the different degrees of hydrophylicity of Ca2+ channels ω-toxins blockers (water soluble, polar compounds) and diltiazem (a lipophilic molecule); but it is also plausible that more selective mechanisms (i.e. different dissociation equilibrium constants, KD, to specific receptor sites) might also contribute to the observed differences. Secondly, another interesting difference is the promotion of α3β4 current inactivation by diltiazem, but not by ω-conotoxins. This keeps pace with previous observations that organic Ca2+ antagonist molecules directly interact with the muscle nicotinic receptor channel to enhance its autodesensitization (Chang et al., 1990). Thirdly, we have observed differences in the voltage-dependence of blockade between ω-conotoxins and diltiazem. Thus, whereas diltiazem blocked in a similar extent α3β4 currents at all membrane potentials, ω-conotoxins exerted stronger inhibition of the current at hyperpolarized potentials. This finding, along with the non-competitive blockade, would indicate that ω-conotoxins behave as open-channel blockers of α3β4 nicotinic AChRs. However, the lack of use-dependent blockade does not agree with such mechanism (Buisson & Bertrand, 1998). Our results suggest that ω-conotoxins and diltiazem should bind to a different receptor site; in the case of toxins such binding-site should be in a receptor region located deeply enough in the membrane to detect the changes of potential.

The findings of this study have clinical and methodological implications. From the clinical point of view, concerning the wide use of L-type Ca2+ channel blockers in the therapy of cardiovascular diseases, it might very well be that, at therapeutic doses, when plasma levels around 0.5–1 μM could be reached (Yeung et al., 1996), a mild blockade of nicotinic receptors in adrenal medulla and in sympathetic ganglia (where nicotinic AChRs containing α3 and β2 subunits are present) might mitigate the surge of catecholamines to the circulation during stressful conflicts. Interestingly, the methodological implications of this study are even more relevant since the action of these compounds on nicotinic AChRs might obscure the conclusions related to the involvement of certain Ca2+ channel subtypes in the regulation of various central and peripheral functions. This is the case for the physiologically mediated catecholamine release response to ACh stimulation of adrenal chromaffin cells. We have recently demonstrated that nicotinic receptors containing α3β4 or α7 subunits participate in the ACh-mediated catecholamine release responses in chromaffin cells (López et al., 1998). Therefore, in trying to determine the Ca2+ channel subtypes that control the ACh-evoked catecholamine release, a judicious use of Ca2+ channel blockers and activators should be made.

From the results of the present study it seems that ω-agatoxin IVA can be safely used to irreversibly block the P/Q-type Ca2+ channels (Olivera et al., 1994; García et al., 1996), without much interference with nicotinic AChR functions. This is not the case for ω-conotoxin MVIIC, that caused a pronounced blockade of α3β2 and α3β4 currents at concentrations currently used to block the N- and P/Q-type channels. However, while blockade of nicotinic AChR currents was fully reversible in only a few seconds after toxin washout, blockade by this toxin of P/Q-type Ca2+ channels in chromaffin cells and in neurons is long-lasting (Albillos et al., 1996; Gandía et al., 1997; Lara et al., 1998; McDonough et al., 1996). Thus, a selective stable blockade of non- L-type Ca2+ channels can be achieved by preincubation of the cells with ω-conotoxin MVIIC, followed by a few minutes washout; under these conditions the nicotinic AChRs are unlikely to be affected. Although to a smaller extent, caution should also be taken with ω-conotoxin GVIA when using it to block N-type Ca2+ channels; α3β4 and α7 nicotinic AChRs were moderately affected by this toxin at concentrations currently employed for Ca2+ channel blockade. Once again, differences in the duration of blockade of neuronal Ca2+ channels (Olivera et al., 1994; Kasai et al., 1987) and nicotinic AChRs (present study) by ω-conotoxin GVIA, might discriminate between both potential toxin targets. Concerning L-channels, it seems that all non-peptide blockers will affect to some extent the nicotinic AChRs, particularly diltiazem. It could be that peptide blockers of L-type channels recently available (i.e. calcicludine, calciseptine; Schweitz et al., 1994; De Weille et al., 1991) might preserve the functionality of nicotinic AChRs and, hence, can be more adequate tools than organic molecules. This hypothesis needs experimental testing.

In conclusion, our data calls for caution when interpreting data related with the regulation of various central and peripheral functions in neurons and other cell types, where nicotinic receptor and Ca2+ channels coexist.


We thank Professor Stephen F. Heinemann (from Salk Institute, La Jolla, CA, U.S.A.) for providing the plasmids pPCA48E, pZPC13 and pHIP306 containing the coding regions of the rat brain nicotinic AChR α3, β4 and α7 subunits) and to Professor Patrick (from Baylor College of Medicine, Houston, TX, U.S.A.) for the PCX49 plasmid containing the rat nicotinic AChR β2 subunit. Supported by grants from DGES (no. PB97-0047) to C. Montiel; from DGICYT (no. PB94-0150) to A. G. García, and from grant Plan Nacional de Investigación I+D (no. 2FD97-0388-C02-01) to A. G. García. Also, the support of Fundacion Teófilo Hernando, Madrid, Spain, is greatly acknowledged.


acethylcholine receptors
dimethyl sulphoxide
4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid


  • ALBILLOS A., GARCÍA A.G., OLIVERA B., GANDÍA L. Re-evaluation of the P/Q Ca2+ channel components of Ba2+ currents in bovine chromaffin cells superfused with solutions containing low and high Ba2+ concentrations. Pflügers. Arch. 1996;432:1030–1038. [PubMed]
  • BUISSON B., BERTRAND D. Open-channel blockers at the human α4β2 neuronal nicotinic acetylcholine receptor. Mol. Pharmacol. 1998;53:555–563. [PubMed]
  • CACHELIN A.B., JAGGI R. Beta subunits determine the time course of desensitization in rat alpha 3 neuronal nicotinic acetylcholine receptors. Pflügers. Arch. 1991;19:579–582. [PubMed]
  • CAMPOS-CARO A., SMILLIE F.I., DOMÍNGUEZ DEL TORO E., ROVIRA J.C., VICENTE-AGULLÓ F., CHAPULI J., JUÍZ J.M., SALA S., SALA F., BALLESTA J.J., CRIADO M. Neuronal nicotinic acetylcoline receptor on bovine chromaffin cells: Cloning, expression and genomic organization of receptor subunits. J. Neurochem. 1997;68:488–497. [PubMed]
  • CHANG C.C., HUANG C.Y., HONG S.J. Organic calcium channel antagonists provoke acetylcholine receptor autodesensitization on train stimulation of motor nerve. Neuroscience. 1990;38:731–742. [PubMed]
  • CRIADO M., ALAMO L., NAVARRO A. Primary structure of an agonist binding subunit of the nicotinic acetylcholine receptor from bovine adrenal chromaffin cells. Neurochem. Res. 1992;17:281–287. [PubMed]
  • CRIADO M., DOMÍNGUEZDETORO E, , CARRASCO-SERRANO C., SMILLIE F.I., JUÍZ J.M., VINIEGRA S., BALLESTA J.J. Differential expression of α-bungarotoxin-sensitive neuronal nicotinic receptors in adrenergic chromaffin cells: A role for transcription factor Egr-1. J. Neurosci. 1997;17:6554–6564. [PubMed]
  • DE WEILLE J.R., SCHWEITZ H., MAES P., TARTAR A., LAZDUNSKI M. Calciseptine, a peptide isolated from black mamba venom, is a specific blocker of the L-type calcium channel. Proc. Natl. Acad. Sci. U.S.A. 1991;88:2437–2440. [PMC free article] [PubMed]
  • DONNELLY-ROBERTS D.L., ARNERIC S.P., SULLIVAN J.P. Functional modulation of human ‘ganglionic-like' neuronal nicotinic acetylcholine receptors (nAChRs) by L-Type calcium channel antagonists. Biochem. Biophys. Res. Comm. 1995;213:657–662. [PubMed]
  • DUVOISIN R.M., DENERIS E.S., PATRICK J., HEINEMANN S. The functional diversity of the neuronal nicotinic acetylcholine receptors is increased by a novel subunit: beta 4. Neuron. 1989;3:487–496. [PubMed]
  • FERNÁNDEZ J.M., GRANJA R., IZAGUIRRE V., GONZÁLEZ-GARCÍA C., CEÑA V. ω-Conotoxin GVIA blocks nicotine-induced catecholamine secretion by blocking the nicotinic receptor-activated inward currents in bovine chromaffin cells. Neurosci. Lett. 1995;191:59–62. [PubMed]
  • GANDÍA L., LARA B., IMPERIAL J.S., VILLARROYA M., ALBILLOS A., MAROTO R., GARCÍA A.G., OLIVERA B.M. Analogies and differences between ω-conotoxins MVIIC and MVIID: binding sites and functions in bovine chromaffin cells. Pflügers. Arch. 1997;435:55–64. [PubMed]
  • GANDÍA L., VILLARROYA M., SALA F., REIG J.A., VINIEGRA S., QUINTANAR J.L., GARCÍA A.G., GUTIÉRREZ L.M. Inhibition of nicotinic receptor-mediated responses in bovine chromaffin cells by diltiazem. Br. J. Pharmacol. 1996;118:1301–1307. [PMC free article] [PubMed]
  • GARCÍA A.G., ALBILLOS A., GANDÍA L., LÓPEZ M.G., MICHELENA P., MONTIEL C. ω-Toxins, calcium channels and neurosecretion Cellular and Molecular Mechanisms of Toxin Action: Toxins and Signal Transduction 1996. Switzerland: Harwood Academic Publishers; 155–209.209eds. Gutman, Y. & Lazarorovici, P. pp
  • GARCÍA-GUZMÁN B.M., SALA F., SALA S., CAMPOS-CARO A,. , STÜHMER W., GUTIÉRREZ L.M., CRIADO M. α-Bungarotoxin-sensitive nicotinic receptors on bovine chromaffin cells: Molecular cloning, functional expression and alternative splicing of the α7 subunit. Eur. J. Neurosci. 1995;7:647–655. [PubMed]
  • GRANJA R., FERNÁNDEZ-FERNÁNDEZ J.M., IZAGUIRRE V., GONZÁLEZ-GARCÍA C., CEÑA V. Omega-Agatoxin IVA blocks nicotinic receptor channels in bovine chromaffin cells. FEBS Lett. 1995;362:15–18. [PubMed]
  • HARVEY S.C., LUETJE C.W. Determinants of competitive antagonist sensitivity on neuronal nicotinic receptor beta subunits. J. Neurosci. 1996;16:3798–3806. [PubMed]
  • KASAI H., AOSAKI T., FUKUDA J. Presynaptic Ca-antagonist ω-conotoxin irreversibly blocks N-type Ca channels in chick sensory neurons. Neurosci. Res. 1987;4:228–235. [PubMed]
  • LARA B., GANDÍA L., MARTÍNEZ-SIERRA R., TORRES A., GARCÍA A.G. Q-type Ca2+ channels are located closer to secretory sites than L-type channels: functional evidence in chromaffin cells. Pflügers. Arch. 1998;435:472–478. [PubMed]
  • LOMAX R.B., HERRERO C.J., GARCÍA-PALOMERO E., GARCÍA A.G., MONTIEL C. Capacitative Ca2+ entry into Xenopus oocytes is sensitive to ω-conotoxins GVIA, MVIIA and MVIIC. Cell Calcium. 1998;23:229–239. [PubMed]
  • LÓPEZ M.G., FONTERÍZ R.I., GANDÍA L., DE LA FUENTE M.T., VILLARROYA M., GARCÍA-SANCHO J., GARCÍA A.G. The nicotinic acetylcholine receptor of the bovine chromaffin cell, a new target for dihydropyridines. Eur. J. Pharmacol. 1993;247:199–207. [PubMed]
  • LÓPEZ M.G., MONTIEL C., HERRERO C.J., GARCÍA-PALOMERO E., MAYORGAS I., HERNÁNDEZ-GUIJO J.M., VILLARROYA M., OLIVARES R., GANDÍA L., MCINTOSH J.M., OLIVERA B.M., GARCÍA A.G. Unmasking the functions of the chromaffin cell α7 nicotinic receptor by using short pulses of acetylcholine and novel selective blockers. Proc. Natl. Acad. Sci. U.S.A. 1998;95:14184–14189. [PMC free article] [PubMed]
  • LUETJE C.W., PATRICK J. Both alpha- and beta-subunits contribute to the agonist sensitivity of neuronal nicotinic acetylcholine receptors. J. Neurosci. 1991;11:837–845. [PubMed]
  • MCDONOUGH S.I., SWARTZ K., MINTZ I.M., BOLAND L.M., BEAN B.P. Inhibition of calcium channels in rat central and peripheral neurons by ω-conotoxin MVIIC. J. Neurosci. 1996;16:2612–2623. [PubMed]
  • MILEDI R., PARKER I., SUMIKAWA K. Properties of acetylcholine receptors translated by cat muscle mRNA in Xenopus oocytes. EMBO. J. 1989;1:1307–1312. [PMC free article] [PubMed]
  • MONTIEL C., HERRERO C.J., GARCÍA-PALOMERO E., RENART J., GARCÍA A.G., LOMAX R.B. Serotonergic effects of dotarizine in coronary artery and in oocytes expressing 5-HT2 receptors. Eur. J. Pharmacol. 1997;332:183–193. [PubMed]
  • OLIVERA B.M., MILJANICH G.P., RAMACHANDRAN J., ADAMS M.E. Calcium channel diversity and neurotransmitter release: the ω-conotoxins and ω-agatoxins. Annu. Rev. Biochem. 1994;63:823–867. [PubMed]
  • PAPKE R.L., HEINEMANN S.F. The role of the beta 4-subunit in determining the kinetic properties of rat neuronal nicotinic acetylcholine alpha 3-receptors. J. Physiol. (Lond). 1991;440:95–112. [PMC free article] [PubMed]
  • SEGUELA P., WADICHE J., DINELEY-MILLER K., DANI J.A., PATRICK J.W. Molecular cloning, functional expression and distribution of rat brain α7. A nicotinic cation channel highly permeable to calcium. J. Neurosci. 1993;13:596–604. [PubMed]
  • SCHWEITZ H., HEURTEAUX C., BOIX P., MOINIER D., ROMEY G., LAZDUNSKI M. Calcicludine, a venom peptide of the Kunitz-type protease inhibitor family, is a potent blocker of high-threshold Ca2+ channels with a high affinity for L-type channels in cerebellar granule neurons. Proc. Natl. Acad. Sci. U.S.A. 1994;91:878–882. [PMC free article] [PubMed]
  • VILLARROYA M., DE LA FUENTE M.T., LÓPEZ M.G., GANDÍA L., GARCÍA A.G. Distinct effects of ω-conotoxins and various groups of Ca2+-entry inhibitors on nicotinic acetylcholine receptor and Ca2+ channels of chromaffin cells. Eur. J. Pharmacol. 1997;320:249–257. [PubMed]
  • YEUNG P.K., BUCKLEY S.J., HUNG O.R., POLLAK P.T., BARCLAY K.D., FENG J.D., FARMER P.S., KLASSEN G.A. Steady-state plasma concentraton of diltiazem and its metabolites in patients and healthy volunteers. Ther. Drug Monit. 1996;18:40–45. [PubMed]
  • ZERNIG G. Widening potential for Ca2+ antagonists: non-L-type Ca2+ channel interaction. Trends. Pharmacol. Sci. 1990;11:38–44. [PubMed]

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