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Ni2+ block of CaV3.1 (alpha1G) T-type calcium channels.

Obejero-Paz CA, Gray IP, Jones SW - J. Gen. Physiol. (2008)

Bottom Line: Na(+)).We conclude that both fast and slow block of Ca(V)3.1 by Ni(2+) are most consistent with occlusion of the pore.The exit rate of Ni(2+) for slow block is reduced at high Ni(2+) concentrations, suggesting that the site responsible for fast block can "lock in" slow block by Ni(2+), at a site located deeper within the pore.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106, USA.

ABSTRACT
Ni(2+) inhibits current through calcium channels, in part by blocking the pore, but Ni(2+) may also allosterically affect channel activity via sites outside the permeation pathway. As a test for pore blockade, we examined whether the effect of Ni(2+) on Ca(V)3.1 is affected by permeant ions. We find two components to block by Ni(2+), a rapid block with little voltage dependence, and a slow block most visible as accelerated tail currents. Rapid block is weaker for outward vs. inward currents (apparent K(d) = 3 vs. 1 mM Ni(2+), with 2 mM Ca(2+) or Ba(2+)) and is reduced at high permeant ion concentration (110 vs. 2 mM Ca(2+) or Ba(2+)). Slow block depends both on the concentration and on the identity of the permeant ion (Ca(2+) vs. Ba(2+) vs. Na(+)). Slow block is 2-3x faster in Ba(2+) than in Ca(2+) (2 or 110 mM), and is approximately 10x faster with 2 vs. 110 mM Ca(2+) or Ba(2+). Slow block is orders of magnitude slower than the diffusion limit, except in the nominal absence of divalent cations ( approximately 3 muM Ca(2+)). We conclude that both fast and slow block of Ca(V)3.1 by Ni(2+) are most consistent with occlusion of the pore. The exit rate of Ni(2+) for slow block is reduced at high Ni(2+) concentrations, suggesting that the site responsible for fast block can "lock in" slow block by Ni(2+), at a site located deeper within the pore. In contrast to the complex pore block observed for Ca(V)3.1, inhibition of Ca(V)3.2 by Ni(2+) was essentially independent of voltage, and was similar in 2 mM Ca(2+) vs. Ba(2+), consistent with inhibition by a different mechanism, at a site outside the pore.

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Effects of Ni2+ on currents carried by Na+. (A) IIV relations, (B) chord conductances, and (C) inhibition by Ni2+, as conductance ratios (Ni2+/control). (D) Effects of Ni2+ on IIV tail currents carried by Na+, from single exponential fits. (E) Pseudo first-order rate constants for Ni2+ block, calculated from single exponential fits. (F) I-V relations, (G) I-V relations on an expanded scale, (H) chord conductances, and (I) inhibition by Ni2+, as conductance ratios (Ni2+/control). The smooth curves in I are the best fit to a Woodhull (1973) model. Symbols and color coding in A apply to all panels in this figure. n = 6 (10 μM Ni2+) or n = 4 (30 μM Ni2+ and 100 μM Ni2+), except n = 5 in 10 μM Ni2+ and n = 3 in 30 μM Ni2+ for D and E.
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fig9: Effects of Ni2+ on currents carried by Na+. (A) IIV relations, (B) chord conductances, and (C) inhibition by Ni2+, as conductance ratios (Ni2+/control). (D) Effects of Ni2+ on IIV tail currents carried by Na+, from single exponential fits. (E) Pseudo first-order rate constants for Ni2+ block, calculated from single exponential fits. (F) I-V relations, (G) I-V relations on an expanded scale, (H) chord conductances, and (I) inhibition by Ni2+, as conductance ratios (Ni2+/control). The smooth curves in I are the best fit to a Woodhull (1973) model. Symbols and color coding in A apply to all panels in this figure. n = 6 (10 μM Ni2+) or n = 4 (30 μM Ni2+ and 100 μM Ni2+), except n = 5 in 10 μM Ni2+ and n = 3 in 30 μM Ni2+ for D and E.

Mentions: Effects of Ni2+ were examined by two basic protocols: the I-V protocol, where currents were recorded directly upon depolarization from a holding potential of −100 mV to the voltages indicated, and the IIV protocol, where the voltage was varied following a 2-ms step to +200 mV to maximally activate channels with minimal inactivation. These two protocols, taken together, help separate effects of voltage and ions on gating vs. permeation. Both protocols were delivered sequentially before, during, and after recovery from Ni2+ exposure, to assess current rundown. Two versions of the I-V protocol were run, brief (5 ms) voltage steps from −90 to +200 mV, and longer (40 ms) steps from −90 to 0 mV (+30 mV in 110 mM Ca2+o or Ba2+o), since channel activation is not complete in 5 ms at some negative voltages. I-V relations shown in the figures are the peak current (from either the 5 or 40 ms protocol), except in nominally Ca2+o -free conditions (Fig. 9), where the current at the end of 5-ms steps was used.


Ni2+ block of CaV3.1 (alpha1G) T-type calcium channels.

Obejero-Paz CA, Gray IP, Jones SW - J. Gen. Physiol. (2008)

Effects of Ni2+ on currents carried by Na+. (A) IIV relations, (B) chord conductances, and (C) inhibition by Ni2+, as conductance ratios (Ni2+/control). (D) Effects of Ni2+ on IIV tail currents carried by Na+, from single exponential fits. (E) Pseudo first-order rate constants for Ni2+ block, calculated from single exponential fits. (F) I-V relations, (G) I-V relations on an expanded scale, (H) chord conductances, and (I) inhibition by Ni2+, as conductance ratios (Ni2+/control). The smooth curves in I are the best fit to a Woodhull (1973) model. Symbols and color coding in A apply to all panels in this figure. n = 6 (10 μM Ni2+) or n = 4 (30 μM Ni2+ and 100 μM Ni2+), except n = 5 in 10 μM Ni2+ and n = 3 in 30 μM Ni2+ for D and E.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC2483332&req=5

fig9: Effects of Ni2+ on currents carried by Na+. (A) IIV relations, (B) chord conductances, and (C) inhibition by Ni2+, as conductance ratios (Ni2+/control). (D) Effects of Ni2+ on IIV tail currents carried by Na+, from single exponential fits. (E) Pseudo first-order rate constants for Ni2+ block, calculated from single exponential fits. (F) I-V relations, (G) I-V relations on an expanded scale, (H) chord conductances, and (I) inhibition by Ni2+, as conductance ratios (Ni2+/control). The smooth curves in I are the best fit to a Woodhull (1973) model. Symbols and color coding in A apply to all panels in this figure. n = 6 (10 μM Ni2+) or n = 4 (30 μM Ni2+ and 100 μM Ni2+), except n = 5 in 10 μM Ni2+ and n = 3 in 30 μM Ni2+ for D and E.
Mentions: Effects of Ni2+ were examined by two basic protocols: the I-V protocol, where currents were recorded directly upon depolarization from a holding potential of −100 mV to the voltages indicated, and the IIV protocol, where the voltage was varied following a 2-ms step to +200 mV to maximally activate channels with minimal inactivation. These two protocols, taken together, help separate effects of voltage and ions on gating vs. permeation. Both protocols were delivered sequentially before, during, and after recovery from Ni2+ exposure, to assess current rundown. Two versions of the I-V protocol were run, brief (5 ms) voltage steps from −90 to +200 mV, and longer (40 ms) steps from −90 to 0 mV (+30 mV in 110 mM Ca2+o or Ba2+o), since channel activation is not complete in 5 ms at some negative voltages. I-V relations shown in the figures are the peak current (from either the 5 or 40 ms protocol), except in nominally Ca2+o -free conditions (Fig. 9), where the current at the end of 5-ms steps was used.

Bottom Line: Na(+)).We conclude that both fast and slow block of Ca(V)3.1 by Ni(2+) are most consistent with occlusion of the pore.The exit rate of Ni(2+) for slow block is reduced at high Ni(2+) concentrations, suggesting that the site responsible for fast block can "lock in" slow block by Ni(2+), at a site located deeper within the pore.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106, USA.

ABSTRACT
Ni(2+) inhibits current through calcium channels, in part by blocking the pore, but Ni(2+) may also allosterically affect channel activity via sites outside the permeation pathway. As a test for pore blockade, we examined whether the effect of Ni(2+) on Ca(V)3.1 is affected by permeant ions. We find two components to block by Ni(2+), a rapid block with little voltage dependence, and a slow block most visible as accelerated tail currents. Rapid block is weaker for outward vs. inward currents (apparent K(d) = 3 vs. 1 mM Ni(2+), with 2 mM Ca(2+) or Ba(2+)) and is reduced at high permeant ion concentration (110 vs. 2 mM Ca(2+) or Ba(2+)). Slow block depends both on the concentration and on the identity of the permeant ion (Ca(2+) vs. Ba(2+) vs. Na(+)). Slow block is 2-3x faster in Ba(2+) than in Ca(2+) (2 or 110 mM), and is approximately 10x faster with 2 vs. 110 mM Ca(2+) or Ba(2+). Slow block is orders of magnitude slower than the diffusion limit, except in the nominal absence of divalent cations ( approximately 3 muM Ca(2+)). We conclude that both fast and slow block of Ca(V)3.1 by Ni(2+) are most consistent with occlusion of the pore. The exit rate of Ni(2+) for slow block is reduced at high Ni(2+) concentrations, suggesting that the site responsible for fast block can "lock in" slow block by Ni(2+), at a site located deeper within the pore. In contrast to the complex pore block observed for Ca(V)3.1, inhibition of Ca(V)3.2 by Ni(2+) was essentially independent of voltage, and was similar in 2 mM Ca(2+) vs. Ba(2+), consistent with inhibition by a different mechanism, at a site outside the pore.

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Related in: MedlinePlus