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Interactions among toxins that inhibit N-type and P-type calcium channels.

McDonough SI, Boland LM, Mintz IM, Bean BP - J. Gen. Physiol. (2002)

Bottom Line: On N-type channels, results are consistent with blockade of the channel pore by omega-CgTx-GVIA, omega-Aga-IIIA, and omega-CTx-MVIIC, whereas grammotoxin likely binds to a separate region coupled to channel gating. omega-Aga-IIIA produces partial channel block by decreasing single-channel conductance.On P-type channels, omega-CTx-MVIIC and omega-Aga-IIIA both likely bind near the mouth of the pore. omega-Aga-IVA and grammotoxin each bind to distinct regions associated with channel gating that do not overlap with the binding region of pore blockers.For both N- and P-type channels, omega-CTx-MVIIC binding produces complete channel block, but is prevented by previous partial channel block by omega-Aga-IIIA, suggesting that omega-CTx-MVIIC binds closer to the external mouth of the pore than does omega-Aga-IIIA.

View Article: PubMed Central - PubMed

Affiliation: Marine Biological Laboratory, Woods Hole, MA 02543, USA. smcdonough@mbl.edu

ABSTRACT
A number of peptide toxins from venoms of spiders and cone snails are high affinity ligands for voltage-gated calcium channels and are useful tools for studying calcium channel function and structure. Using whole-cell recordings from rat sympathetic ganglion and cerebellar Purkinje neurons, we studied toxins that target neuronal N-type (Ca(V)2.2) and P-type (Ca(V)2.1) calcium channels. We asked whether different toxins targeting the same channels bind to the same or different sites on the channel. Five toxins (omega-conotoxin-GVIA, omega-conotoxin MVIIC, omega-agatoxin-IIIA, omega-grammotoxin-SIA, and omega-agatoxin-IVA) were applied in pairwise combinations to either N- or P-type channels. Differences in the characteristics of inhibition, including voltage dependence, reversal kinetics, and fractional inhibition of current, were used to detect additive or mutually occlusive effects of toxins. Results suggest at least two distinct toxin binding sites on the N-type channel and three on the P-type channel. On N-type channels, results are consistent with blockade of the channel pore by omega-CgTx-GVIA, omega-Aga-IIIA, and omega-CTx-MVIIC, whereas grammotoxin likely binds to a separate region coupled to channel gating. omega-Aga-IIIA produces partial channel block by decreasing single-channel conductance. On P-type channels, omega-CTx-MVIIC and omega-Aga-IIIA both likely bind near the mouth of the pore. omega-Aga-IVA and grammotoxin each bind to distinct regions associated with channel gating that do not overlap with the binding region of pore blockers. For both N- and P-type channels, omega-CTx-MVIIC binding produces complete channel block, but is prevented by previous partial channel block by omega-Aga-IIIA, suggesting that omega-CTx-MVIIC binds closer to the external mouth of the pore than does omega-Aga-IIIA.

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ω-CTx-MVIIC blocks P-type channels exposed to saturating GTx. (top) Inward and outward currents evoked by a 15-ms test pulse to −10 and +150 mV from a holding potential of −80 mV, followed by tail currents at −60 mV, in control (left), after maximal inhibition by 800 nM GTx (middle), and 160 s after addition of 5 μM ω-CTx-MVIIC, in the continual presence of GTx (right). (bottom) Test currents (left) and tail currents at −60 mV (right) after a 15-ms step to the indicated test voltage. Open circles, control; closed circles, +800 nM GTx; open squares, GTx + 5 μM ω-CTx-MVIIC. External solution: 2 mM BaCl2, 160 mM TEACl, 10 mM HEPES (pH adjusted to 7.4 with TEAOH), 0.6 μM tetrodotoxin, 5 μM nimodipine, 1 μM ω-conotoxin GVIA, and 1 mg/ml cytochrome C. Internal solution (in mM): 56 CsCl, 68 CsF, 2.2 MgCl2, 4.5 EGTA, 9 HEPES, 4 MgATP, 14 creatine phosphate (Tris salt), and 0.3 GTP (Tris salt), pH adjusted to 7.4 with CsOH. To correct for small noncalcium channel currents, currents remaining in 600 μM CdCl2 were subtracted. Experiment performed at 10°C to slow tail kinetics.
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fig8: ω-CTx-MVIIC blocks P-type channels exposed to saturating GTx. (top) Inward and outward currents evoked by a 15-ms test pulse to −10 and +150 mV from a holding potential of −80 mV, followed by tail currents at −60 mV, in control (left), after maximal inhibition by 800 nM GTx (middle), and 160 s after addition of 5 μM ω-CTx-MVIIC, in the continual presence of GTx (right). (bottom) Test currents (left) and tail currents at −60 mV (right) after a 15-ms step to the indicated test voltage. Open circles, control; closed circles, +800 nM GTx; open squares, GTx + 5 μM ω-CTx-MVIIC. External solution: 2 mM BaCl2, 160 mM TEACl, 10 mM HEPES (pH adjusted to 7.4 with TEAOH), 0.6 μM tetrodotoxin, 5 μM nimodipine, 1 μM ω-conotoxin GVIA, and 1 mg/ml cytochrome C. Internal solution (in mM): 56 CsCl, 68 CsF, 2.2 MgCl2, 4.5 EGTA, 9 HEPES, 4 MgATP, 14 creatine phosphate (Tris salt), and 0.3 GTP (Tris salt), pH adjusted to 7.4 with CsOH. To correct for small noncalcium channel currents, currents remaining in 600 μM CdCl2 were subtracted. Experiment performed at 10°C to slow tail kinetics.

Mentions: Fig. 8 shows currents from a Purkinje neuron exposed to first 800 nM GTx (Fig. 8 top, middle) and then to GTx + 5 μM ω-CTx-MVIIC (Fig. 8 top, right). For each condition, currents are shown in response to depolarizations to –10 mV and +150 mV, with tail currents measured at –60 mV. The depolarization to +150 mV in control (Fig. 8 top, left) resulted in a transient phase of outward current and in a slow, sigmoidally decaying tail current; these are due not to poor space clamp, but to an additional open state of the native rat P-type channel reached via strong depolarizations (McDonough et al., 1997b; McFarlane, 1997). GTx produced full inhibition of current at −10 mV, but just as for GTx action on N-type currents, channels could still be activated by sufficiently large depolarizations, though with greatly slowed kinetics. Depolarization to +150 mV elicited a slow outward current, followed by a large tail current at −60 mV. Subsequent application of ω-CTx-MVIIC blocked virtually all current. Graphs at bottom display the amplitudes of the test current (Fig. 8, left) and the inward tail current (Fig. 8, right) at test voltages from −70 to +160 mV. Current through GTx-bound channels (Fig. 8, closed circles) was completely removed by ω-CTx-MVIIC (Fig. 8, open squares; records shown were taken 160 s after first application of ω-CTx-MVIIC, not enough time for GTx to unbind appreciably at this temperature). Evidently ω-CTx-MVIIC blocks current through channels after alteration of gating by GTx.


Interactions among toxins that inhibit N-type and P-type calcium channels.

McDonough SI, Boland LM, Mintz IM, Bean BP - J. Gen. Physiol. (2002)

ω-CTx-MVIIC blocks P-type channels exposed to saturating GTx. (top) Inward and outward currents evoked by a 15-ms test pulse to −10 and +150 mV from a holding potential of −80 mV, followed by tail currents at −60 mV, in control (left), after maximal inhibition by 800 nM GTx (middle), and 160 s after addition of 5 μM ω-CTx-MVIIC, in the continual presence of GTx (right). (bottom) Test currents (left) and tail currents at −60 mV (right) after a 15-ms step to the indicated test voltage. Open circles, control; closed circles, +800 nM GTx; open squares, GTx + 5 μM ω-CTx-MVIIC. External solution: 2 mM BaCl2, 160 mM TEACl, 10 mM HEPES (pH adjusted to 7.4 with TEAOH), 0.6 μM tetrodotoxin, 5 μM nimodipine, 1 μM ω-conotoxin GVIA, and 1 mg/ml cytochrome C. Internal solution (in mM): 56 CsCl, 68 CsF, 2.2 MgCl2, 4.5 EGTA, 9 HEPES, 4 MgATP, 14 creatine phosphate (Tris salt), and 0.3 GTP (Tris salt), pH adjusted to 7.4 with CsOH. To correct for small noncalcium channel currents, currents remaining in 600 μM CdCl2 were subtracted. Experiment performed at 10°C to slow tail kinetics.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2311392&req=5

fig8: ω-CTx-MVIIC blocks P-type channels exposed to saturating GTx. (top) Inward and outward currents evoked by a 15-ms test pulse to −10 and +150 mV from a holding potential of −80 mV, followed by tail currents at −60 mV, in control (left), after maximal inhibition by 800 nM GTx (middle), and 160 s after addition of 5 μM ω-CTx-MVIIC, in the continual presence of GTx (right). (bottom) Test currents (left) and tail currents at −60 mV (right) after a 15-ms step to the indicated test voltage. Open circles, control; closed circles, +800 nM GTx; open squares, GTx + 5 μM ω-CTx-MVIIC. External solution: 2 mM BaCl2, 160 mM TEACl, 10 mM HEPES (pH adjusted to 7.4 with TEAOH), 0.6 μM tetrodotoxin, 5 μM nimodipine, 1 μM ω-conotoxin GVIA, and 1 mg/ml cytochrome C. Internal solution (in mM): 56 CsCl, 68 CsF, 2.2 MgCl2, 4.5 EGTA, 9 HEPES, 4 MgATP, 14 creatine phosphate (Tris salt), and 0.3 GTP (Tris salt), pH adjusted to 7.4 with CsOH. To correct for small noncalcium channel currents, currents remaining in 600 μM CdCl2 were subtracted. Experiment performed at 10°C to slow tail kinetics.
Mentions: Fig. 8 shows currents from a Purkinje neuron exposed to first 800 nM GTx (Fig. 8 top, middle) and then to GTx + 5 μM ω-CTx-MVIIC (Fig. 8 top, right). For each condition, currents are shown in response to depolarizations to –10 mV and +150 mV, with tail currents measured at –60 mV. The depolarization to +150 mV in control (Fig. 8 top, left) resulted in a transient phase of outward current and in a slow, sigmoidally decaying tail current; these are due not to poor space clamp, but to an additional open state of the native rat P-type channel reached via strong depolarizations (McDonough et al., 1997b; McFarlane, 1997). GTx produced full inhibition of current at −10 mV, but just as for GTx action on N-type currents, channels could still be activated by sufficiently large depolarizations, though with greatly slowed kinetics. Depolarization to +150 mV elicited a slow outward current, followed by a large tail current at −60 mV. Subsequent application of ω-CTx-MVIIC blocked virtually all current. Graphs at bottom display the amplitudes of the test current (Fig. 8, left) and the inward tail current (Fig. 8, right) at test voltages from −70 to +160 mV. Current through GTx-bound channels (Fig. 8, closed circles) was completely removed by ω-CTx-MVIIC (Fig. 8, open squares; records shown were taken 160 s after first application of ω-CTx-MVIIC, not enough time for GTx to unbind appreciably at this temperature). Evidently ω-CTx-MVIIC blocks current through channels after alteration of gating by GTx.

Bottom Line: On N-type channels, results are consistent with blockade of the channel pore by omega-CgTx-GVIA, omega-Aga-IIIA, and omega-CTx-MVIIC, whereas grammotoxin likely binds to a separate region coupled to channel gating. omega-Aga-IIIA produces partial channel block by decreasing single-channel conductance.On P-type channels, omega-CTx-MVIIC and omega-Aga-IIIA both likely bind near the mouth of the pore. omega-Aga-IVA and grammotoxin each bind to distinct regions associated with channel gating that do not overlap with the binding region of pore blockers.For both N- and P-type channels, omega-CTx-MVIIC binding produces complete channel block, but is prevented by previous partial channel block by omega-Aga-IIIA, suggesting that omega-CTx-MVIIC binds closer to the external mouth of the pore than does omega-Aga-IIIA.

View Article: PubMed Central - PubMed

Affiliation: Marine Biological Laboratory, Woods Hole, MA 02543, USA. smcdonough@mbl.edu

ABSTRACT
A number of peptide toxins from venoms of spiders and cone snails are high affinity ligands for voltage-gated calcium channels and are useful tools for studying calcium channel function and structure. Using whole-cell recordings from rat sympathetic ganglion and cerebellar Purkinje neurons, we studied toxins that target neuronal N-type (Ca(V)2.2) and P-type (Ca(V)2.1) calcium channels. We asked whether different toxins targeting the same channels bind to the same or different sites on the channel. Five toxins (omega-conotoxin-GVIA, omega-conotoxin MVIIC, omega-agatoxin-IIIA, omega-grammotoxin-SIA, and omega-agatoxin-IVA) were applied in pairwise combinations to either N- or P-type channels. Differences in the characteristics of inhibition, including voltage dependence, reversal kinetics, and fractional inhibition of current, were used to detect additive or mutually occlusive effects of toxins. Results suggest at least two distinct toxin binding sites on the N-type channel and three on the P-type channel. On N-type channels, results are consistent with blockade of the channel pore by omega-CgTx-GVIA, omega-Aga-IIIA, and omega-CTx-MVIIC, whereas grammotoxin likely binds to a separate region coupled to channel gating. omega-Aga-IIIA produces partial channel block by decreasing single-channel conductance. On P-type channels, omega-CTx-MVIIC and omega-Aga-IIIA both likely bind near the mouth of the pore. omega-Aga-IVA and grammotoxin each bind to distinct regions associated with channel gating that do not overlap with the binding region of pore blockers. For both N- and P-type channels, omega-CTx-MVIIC binding produces complete channel block, but is prevented by previous partial channel block by omega-Aga-IIIA, suggesting that omega-CTx-MVIIC binds closer to the external mouth of the pore than does omega-Aga-IIIA.

Show MeSH
Related in: MedlinePlus