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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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Inhibition of CaV3.2 by 20 μM Ni2+. (A) Sample currents with the IIV protocol. Cell d080508, 3-kHz Gaussian filter. (B and C) IIV relationships for inhibition by Ni2+ in 2 mM Ca2+ (n = 7) and 2 mM Ba2+ (n = 4). Prepulses to +60 mV were 2 ms or 3 ms in different cells. (D and E) Ni2+/control ratios, calculated from chord conductances, from IIV and I-V protocols (see Figs. S8 and S9). (F and G) Time constants from the IIV protocol.
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fig11: Inhibition of CaV3.2 by 20 μM Ni2+. (A) Sample currents with the IIV protocol. Cell d080508, 3-kHz Gaussian filter. (B and C) IIV relationships for inhibition by Ni2+ in 2 mM Ca2+ (n = 7) and 2 mM Ba2+ (n = 4). Prepulses to +60 mV were 2 ms or 3 ms in different cells. (D and E) Ni2+/control ratios, calculated from chord conductances, from IIV and I-V protocols (see Figs. S8 and S9). (F and G) Time constants from the IIV protocol.

Mentions: Fig. 11 demonstrates that high-affinity inhibition of CaV3.2 by 20 μM Ni2+ does not exhibit the key kinetic signatures of pore block. Inhibition of CaV3.2 was nearly independent of voltage over a 350-mV range, and was comparable with the I-V and IIV protocols (Fig. 11, D and E; Fig. S8). Inhibition was not any stronger in 2 mM Ba2+ than in Ca2+ (Fig. 11, B and C; Fig. S9). There was little effect on the kinetics of tail currents (Fig. 11, F and G); tails were ∼20% faster in Ni2+ with Ba2+ (P = 0.02–0.04 from −80 to −120 mV), possibly indicating a small amount of slow pore block. These results suggest that the kinetic and molecular mechanisms of Ni2+ action are fundamentally different for high-affinity inhibition of CaV3.2, vs. pore block of CaV3.1.


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

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

Inhibition of CaV3.2 by 20 μM Ni2+. (A) Sample currents with the IIV protocol. Cell d080508, 3-kHz Gaussian filter. (B and C) IIV relationships for inhibition by Ni2+ in 2 mM Ca2+ (n = 7) and 2 mM Ba2+ (n = 4). Prepulses to +60 mV were 2 ms or 3 ms in different cells. (D and E) Ni2+/control ratios, calculated from chord conductances, from IIV and I-V protocols (see Figs. S8 and S9). (F and G) Time constants from the IIV protocol.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2483332&req=5

fig11: Inhibition of CaV3.2 by 20 μM Ni2+. (A) Sample currents with the IIV protocol. Cell d080508, 3-kHz Gaussian filter. (B and C) IIV relationships for inhibition by Ni2+ in 2 mM Ca2+ (n = 7) and 2 mM Ba2+ (n = 4). Prepulses to +60 mV were 2 ms or 3 ms in different cells. (D and E) Ni2+/control ratios, calculated from chord conductances, from IIV and I-V protocols (see Figs. S8 and S9). (F and G) Time constants from the IIV protocol.
Mentions: Fig. 11 demonstrates that high-affinity inhibition of CaV3.2 by 20 μM Ni2+ does not exhibit the key kinetic signatures of pore block. Inhibition of CaV3.2 was nearly independent of voltage over a 350-mV range, and was comparable with the I-V and IIV protocols (Fig. 11, D and E; Fig. S8). Inhibition was not any stronger in 2 mM Ba2+ than in Ca2+ (Fig. 11, B and C; Fig. S9). There was little effect on the kinetics of tail currents (Fig. 11, F and G); tails were ∼20% faster in Ni2+ with Ba2+ (P = 0.02–0.04 from −80 to −120 mV), possibly indicating a small amount of slow pore block. These results suggest that the kinetic and molecular mechanisms of Ni2+ action are fundamentally different for high-affinity inhibition of CaV3.2, vs. pore block of CaV3.1.

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