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Permeation and gating in CaV3.1 (alpha1G) T-type calcium channels effects of Ca2+, Ba2+, Mg2+, and Na+.

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

Bottom Line: However, analysis of chord conductances found that apparent K(d) values were similar for Ca(2+) and Ba(2+), both for block of currents carried by Na(+) (3 muM for Ca(2+) vs. 4 muM for Ba(2+), at -30 mV; weaker at more positive or negative voltages) and for permeation (3.3 mM for Ca(2+) vs. 2.5 mM for Ba(2+); nearly voltage independent).The accelerated inactivation in Ba(2+)(o) correlated with the transition from Na(+) to Ba(2+) permeation, suggesting that Ba(2+)(o) speeds inactivation by occupying the pore.We conclude that the selectivity of the "surface charge" among divalent cations differs between calcium channel families, implying that the surface charge is channel specific.

View Article: PubMed Central - PubMed

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

ABSTRACT
We examined the concentration dependence of currents through Ca(V)3.1 T-type calcium channels, varying Ca(2+) and Ba(2+) over a wide concentration range (100 nM to 110 mM) while recording whole-cell currents over a wide voltage range from channels stably expressed in HEK 293 cells. To isolate effects on permeation, instantaneous current-voltage relationships (IIV) were obtained following strong, brief depolarizations to activate channels with minimal inactivation. Reversal potentials were described by P(Ca)/P(Na) = 87 and P(Ca)/P(Ba) = 2, based on Goldman-Hodgkin-Katz theory. However, analysis of chord conductances found that apparent K(d) values were similar for Ca(2+) and Ba(2+), both for block of currents carried by Na(+) (3 muM for Ca(2+) vs. 4 muM for Ba(2+), at -30 mV; weaker at more positive or negative voltages) and for permeation (3.3 mM for Ca(2+) vs. 2.5 mM for Ba(2+); nearly voltage independent). Block by 3-10 muM Ca(2+) was time dependent, described by bimolecular kinetics with binding at approximately 3 x 10(8) M(-1)s(-1) and voltage-dependent exit. Ca(2+)(o), Ba(2+)(o), and Mg(2+)(o) also affected channel gating, primarily by shifting channel activation, consistent with screening a surface charge of 1 e(-) per 98 A(2) from Gouy-Chapman theory. Additionally, inward currents inactivated approximately 35% faster in Ba(2+)(o) (vs. Ca(2+)(o) or Na(+)(o)). The accelerated inactivation in Ba(2+)(o) correlated with the transition from Na(+) to Ba(2+) permeation, suggesting that Ba(2+)(o) speeds inactivation by occupying the pore. We conclude that the selectivity of the "surface charge" among divalent cations differs between calcium channel families, implying that the surface charge is channel specific. Voltage strongly affects the concentration dependence of block, but not of permeation, for Ca(2+) or Ba(2+).

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IIV relations at different concentrations of Ca2+o (A and B) and Ba2+o (C and D). Values are from 5, 8, 5, 7, 4, and 3 cells in Ca2+o, and 3, 5, 7, 6, 4, and 3 cells in Ba2+o (respectively, 100 nM, 10 μM, 0.1 mM, 0.5 mM, 8 mM, and 110 mM). Control values at 2 mM Ca2+o or Ba2+o are averaged from all these cells (32 in Ca2+o, 28 in Ba2+o). In each cell, the control value was the average of measurements before and after the test solution, to correct for rundown. Values were normalized for each cell as described in Materials and methods. 3–5 are based on this same dataset.
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fig2: IIV relations at different concentrations of Ca2+o (A and B) and Ba2+o (C and D). Values are from 5, 8, 5, 7, 4, and 3 cells in Ca2+o, and 3, 5, 7, 6, 4, and 3 cells in Ba2+o (respectively, 100 nM, 10 μM, 0.1 mM, 0.5 mM, 8 mM, and 110 mM). Control values at 2 mM Ca2+o or Ba2+o are averaged from all these cells (32 in Ca2+o, 28 in Ba2+o). In each cell, the control value was the average of measurements before and after the test solution, to correct for rundown. Values were normalized for each cell as described in Materials and methods. 3–5 are based on this same dataset.

Mentions: Channel activation was measured as PO,r, the ratio of the peak I-V to the IIV (Serrano et al., 1999). Chord conductances calculated from the I-V protocol are often used as a measure of channel activation, but this assumes that the open-channel IIV relationship is linear, which is far from true for T-channels (Fig. 2). On the other hand, the PO,r is undefined at the reversal potential, and subject to large error nearby. In principle, this singularity can be eliminated by calculating PO,r from chord conductances rather than currents. However, the additional calculation (dividing by driving force) can increase error as well, so we present PO,r values from the method that we judge to be most accurate (as noted in figure legends). The maximal PO,r at strongly depolarized voltages often exceeded 1.0 (in 2 mM Ca2+o, 1.10 ± 0.08, mean ± SD, n = 52), probably because the 2-ms prepulse to +60 mV in the IIV protocol did not precisely match the time of maximal PO. Therefore, PO,r values were normalized to the average values from +70 to +100 mV (except in 110 mM Ca2+o or Ba2+o, where those voltages were near the reversal potential and PO,r was not accurately measurable, so unnormalized values were used).


Permeation and gating in CaV3.1 (alpha1G) T-type calcium channels effects of Ca2+, Ba2+, Mg2+, and Na+.

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

IIV relations at different concentrations of Ca2+o (A and B) and Ba2+o (C and D). Values are from 5, 8, 5, 7, 4, and 3 cells in Ca2+o, and 3, 5, 7, 6, 4, and 3 cells in Ba2+o (respectively, 100 nM, 10 μM, 0.1 mM, 0.5 mM, 8 mM, and 110 mM). Control values at 2 mM Ca2+o or Ba2+o are averaged from all these cells (32 in Ca2+o, 28 in Ba2+o). In each cell, the control value was the average of measurements before and after the test solution, to correct for rundown. Values were normalized for each cell as described in Materials and methods. 3–5 are based on this same dataset.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2483336&req=5

fig2: IIV relations at different concentrations of Ca2+o (A and B) and Ba2+o (C and D). Values are from 5, 8, 5, 7, 4, and 3 cells in Ca2+o, and 3, 5, 7, 6, 4, and 3 cells in Ba2+o (respectively, 100 nM, 10 μM, 0.1 mM, 0.5 mM, 8 mM, and 110 mM). Control values at 2 mM Ca2+o or Ba2+o are averaged from all these cells (32 in Ca2+o, 28 in Ba2+o). In each cell, the control value was the average of measurements before and after the test solution, to correct for rundown. Values were normalized for each cell as described in Materials and methods. 3–5 are based on this same dataset.
Mentions: Channel activation was measured as PO,r, the ratio of the peak I-V to the IIV (Serrano et al., 1999). Chord conductances calculated from the I-V protocol are often used as a measure of channel activation, but this assumes that the open-channel IIV relationship is linear, which is far from true for T-channels (Fig. 2). On the other hand, the PO,r is undefined at the reversal potential, and subject to large error nearby. In principle, this singularity can be eliminated by calculating PO,r from chord conductances rather than currents. However, the additional calculation (dividing by driving force) can increase error as well, so we present PO,r values from the method that we judge to be most accurate (as noted in figure legends). The maximal PO,r at strongly depolarized voltages often exceeded 1.0 (in 2 mM Ca2+o, 1.10 ± 0.08, mean ± SD, n = 52), probably because the 2-ms prepulse to +60 mV in the IIV protocol did not precisely match the time of maximal PO. Therefore, PO,r values were normalized to the average values from +70 to +100 mV (except in 110 mM Ca2+o or Ba2+o, where those voltages were near the reversal potential and PO,r was not accurately measurable, so unnormalized values were used).

Bottom Line: However, analysis of chord conductances found that apparent K(d) values were similar for Ca(2+) and Ba(2+), both for block of currents carried by Na(+) (3 muM for Ca(2+) vs. 4 muM for Ba(2+), at -30 mV; weaker at more positive or negative voltages) and for permeation (3.3 mM for Ca(2+) vs. 2.5 mM for Ba(2+); nearly voltage independent).The accelerated inactivation in Ba(2+)(o) correlated with the transition from Na(+) to Ba(2+) permeation, suggesting that Ba(2+)(o) speeds inactivation by occupying the pore.We conclude that the selectivity of the "surface charge" among divalent cations differs between calcium channel families, implying that the surface charge is channel specific.

View Article: PubMed Central - PubMed

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

ABSTRACT
We examined the concentration dependence of currents through Ca(V)3.1 T-type calcium channels, varying Ca(2+) and Ba(2+) over a wide concentration range (100 nM to 110 mM) while recording whole-cell currents over a wide voltage range from channels stably expressed in HEK 293 cells. To isolate effects on permeation, instantaneous current-voltage relationships (IIV) were obtained following strong, brief depolarizations to activate channels with minimal inactivation. Reversal potentials were described by P(Ca)/P(Na) = 87 and P(Ca)/P(Ba) = 2, based on Goldman-Hodgkin-Katz theory. However, analysis of chord conductances found that apparent K(d) values were similar for Ca(2+) and Ba(2+), both for block of currents carried by Na(+) (3 muM for Ca(2+) vs. 4 muM for Ba(2+), at -30 mV; weaker at more positive or negative voltages) and for permeation (3.3 mM for Ca(2+) vs. 2.5 mM for Ba(2+); nearly voltage independent). Block by 3-10 muM Ca(2+) was time dependent, described by bimolecular kinetics with binding at approximately 3 x 10(8) M(-1)s(-1) and voltage-dependent exit. Ca(2+)(o), Ba(2+)(o), and Mg(2+)(o) also affected channel gating, primarily by shifting channel activation, consistent with screening a surface charge of 1 e(-) per 98 A(2) from Gouy-Chapman theory. Additionally, inward currents inactivated approximately 35% faster in Ba(2+)(o) (vs. Ca(2+)(o) or Na(+)(o)). The accelerated inactivation in Ba(2+)(o) correlated with the transition from Na(+) to Ba(2+) permeation, suggesting that Ba(2+)(o) speeds inactivation by occupying the pore. We conclude that the selectivity of the "surface charge" among divalent cations differs between calcium channel families, implying that the surface charge is channel specific. Voltage strongly affects the concentration dependence of block, but not of permeation, for Ca(2+) or Ba(2+).

Show MeSH
Related in: MedlinePlus