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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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Comparison of gating in 2 mM Ca2+o vs. Ba2+o. Values are from all control recordings (n = 52 in Ca2+o, n = 47 in Ba2+o). (A) Effects of Ca2+ (▪) and Ba2+ (○) on inactivation of inward currents. Time constants were averaged over a 20–30-mV range, centered ∼30 mV negative to the reversal potential (A). (B) I-V relations in 2 mM Ca2+o vs. 2 mM Ba2+o. The same symbols were used in B and D–F (defined in B). Outward currents for the most positive voltage steps (up to +100 mV) are offscale. (C) Voltage shifts, 2 mM Ba2+ minus 2 mM Ca2+. Experimental values (“Data”) and theoretical values (“Model”) are from the analysis shown in D–F. Voltage shifts were measured as in Fig. 5, for both experimental data and model output. (D) PO,r in 2 mM Ca2+o or Ba2+o (from Fig. 2, C and D). (E) τIIV (from Fig. 3, A and B). (F) TPeak (from Fig. 3, E and F). Smooth curves in D–F are calculated from a fit to the Serrano et al. (1999) model (see text). Parameter values for Ca2+ (named as in Serrano et al., 1999) were kV = 2521.1 s−1, k-V = 7.7 s−1, kO = 3601.1 s−1, k-O = 23.1 s−1, kI = 70.1 s−1, k-I = 1.4 s−1, f = 0.7555, h = 0.0999. kV, k-V, and k-O changed with depolarization e-fold for 24.8062, −17.0591, and −34.114 mV (respectively). Parameters were identical in Ba2+, except kI = 122.5 s−1, thus kI,Ca/ kI,Ba = 0.57.
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fig12: Comparison of gating in 2 mM Ca2+o vs. Ba2+o. Values are from all control recordings (n = 52 in Ca2+o, n = 47 in Ba2+o). (A) Effects of Ca2+ (▪) and Ba2+ (○) on inactivation of inward currents. Time constants were averaged over a 20–30-mV range, centered ∼30 mV negative to the reversal potential (A). (B) I-V relations in 2 mM Ca2+o vs. 2 mM Ba2+o. The same symbols were used in B and D–F (defined in B). Outward currents for the most positive voltage steps (up to +100 mV) are offscale. (C) Voltage shifts, 2 mM Ba2+ minus 2 mM Ca2+. Experimental values (“Data”) and theoretical values (“Model”) are from the analysis shown in D–F. Voltage shifts were measured as in Fig. 5, for both experimental data and model output. (D) PO,r in 2 mM Ca2+o or Ba2+o (from Fig. 2, C and D). (E) τIIV (from Fig. 3, A and B). (F) TPeak (from Fig. 3, E and F). Smooth curves in D–F are calculated from a fit to the Serrano et al. (1999) model (see text). Parameter values for Ca2+ (named as in Serrano et al., 1999) were kV = 2521.1 s−1, k-V = 7.7 s−1, kO = 3601.1 s−1, k-O = 23.1 s−1, kI = 70.1 s−1, k-I = 1.4 s−1, f = 0.7555, h = 0.0999. kV, k-V, and k-O changed with depolarization e-fold for 24.8062, −17.0591, and −34.114 mV (respectively). Parameters were identical in Ba2+, except kI = 122.5 s−1, thus kI,Ca/ kI,Ba = 0.57.

Mentions: For measurement of I-V relations, peak inward currents were <2.5 nA in all conditions (except 100 nM Ca2+o or Ba2+o), so errors should be <2 mV (with 0.75 MΩ of uncompensated series resistance). Errors in TPeak should also be small, since that value was also measured from inward currents with the I-V protocol. Activation curves were calculated from the I-V/IIV ratio, and the error can be significant for the IIV. In the worst case (100 nM Ca2+o or Ba2+o), at the measured 50% activation voltage (−50 mV), there would be ∼7 mV of error for the IIV. The error would be less than half that value at 10 μM Ca2+o or Ba2+o, and even smaller in all other conditions (e.g., for 110 mM Ca2+o, 2 mV error near half activation). Errors where τIIV = 2 ms will be significant (∼15 mV) for the large inward tail currents at 0.1–10 μM Ca2+o or Ba2+o, ∼7 mV in 110 mM Ca2+o, but <5 mV in other conditions. Overall, activation measurements should be reliable (except perhaps at 100 nM), although small shifts (<5 mV) should be interpreted with caution. For the relatively small currents with 2 mM Ca2+o or Ba2+o the estimated errors in our voltage shift measurements are <1 mV, so the small effects in Fig. 12 (C–F) should not be affected by series resistance error (which would be similar in Ca2+o vs. Ba2+o in any case). Voltage shifts shown in the figures are not corrected for series resistance errors.


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)

Comparison of gating in 2 mM Ca2+o vs. Ba2+o. Values are from all control recordings (n = 52 in Ca2+o, n = 47 in Ba2+o). (A) Effects of Ca2+ (▪) and Ba2+ (○) on inactivation of inward currents. Time constants were averaged over a 20–30-mV range, centered ∼30 mV negative to the reversal potential (A). (B) I-V relations in 2 mM Ca2+o vs. 2 mM Ba2+o. The same symbols were used in B and D–F (defined in B). Outward currents for the most positive voltage steps (up to +100 mV) are offscale. (C) Voltage shifts, 2 mM Ba2+ minus 2 mM Ca2+. Experimental values (“Data”) and theoretical values (“Model”) are from the analysis shown in D–F. Voltage shifts were measured as in Fig. 5, for both experimental data and model output. (D) PO,r in 2 mM Ca2+o or Ba2+o (from Fig. 2, C and D). (E) τIIV (from Fig. 3, A and B). (F) TPeak (from Fig. 3, E and F). Smooth curves in D–F are calculated from a fit to the Serrano et al. (1999) model (see text). Parameter values for Ca2+ (named as in Serrano et al., 1999) were kV = 2521.1 s−1, k-V = 7.7 s−1, kO = 3601.1 s−1, k-O = 23.1 s−1, kI = 70.1 s−1, k-I = 1.4 s−1, f = 0.7555, h = 0.0999. kV, k-V, and k-O changed with depolarization e-fold for 24.8062, −17.0591, and −34.114 mV (respectively). Parameters were identical in Ba2+, except kI = 122.5 s−1, thus kI,Ca/ kI,Ba = 0.57.
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Related In: Results  -  Collection

License 1 - License 2
Show All Figures
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fig12: Comparison of gating in 2 mM Ca2+o vs. Ba2+o. Values are from all control recordings (n = 52 in Ca2+o, n = 47 in Ba2+o). (A) Effects of Ca2+ (▪) and Ba2+ (○) on inactivation of inward currents. Time constants were averaged over a 20–30-mV range, centered ∼30 mV negative to the reversal potential (A). (B) I-V relations in 2 mM Ca2+o vs. 2 mM Ba2+o. The same symbols were used in B and D–F (defined in B). Outward currents for the most positive voltage steps (up to +100 mV) are offscale. (C) Voltage shifts, 2 mM Ba2+ minus 2 mM Ca2+. Experimental values (“Data”) and theoretical values (“Model”) are from the analysis shown in D–F. Voltage shifts were measured as in Fig. 5, for both experimental data and model output. (D) PO,r in 2 mM Ca2+o or Ba2+o (from Fig. 2, C and D). (E) τIIV (from Fig. 3, A and B). (F) TPeak (from Fig. 3, E and F). Smooth curves in D–F are calculated from a fit to the Serrano et al. (1999) model (see text). Parameter values for Ca2+ (named as in Serrano et al., 1999) were kV = 2521.1 s−1, k-V = 7.7 s−1, kO = 3601.1 s−1, k-O = 23.1 s−1, kI = 70.1 s−1, k-I = 1.4 s−1, f = 0.7555, h = 0.0999. kV, k-V, and k-O changed with depolarization e-fold for 24.8062, −17.0591, and −34.114 mV (respectively). Parameters were identical in Ba2+, except kI = 122.5 s−1, thus kI,Ca/ kI,Ba = 0.57.
Mentions: For measurement of I-V relations, peak inward currents were <2.5 nA in all conditions (except 100 nM Ca2+o or Ba2+o), so errors should be <2 mV (with 0.75 MΩ of uncompensated series resistance). Errors in TPeak should also be small, since that value was also measured from inward currents with the I-V protocol. Activation curves were calculated from the I-V/IIV ratio, and the error can be significant for the IIV. In the worst case (100 nM Ca2+o or Ba2+o), at the measured 50% activation voltage (−50 mV), there would be ∼7 mV of error for the IIV. The error would be less than half that value at 10 μM Ca2+o or Ba2+o, and even smaller in all other conditions (e.g., for 110 mM Ca2+o, 2 mV error near half activation). Errors where τIIV = 2 ms will be significant (∼15 mV) for the large inward tail currents at 0.1–10 μM Ca2+o or Ba2+o, ∼7 mV in 110 mM Ca2+o, but <5 mV in other conditions. Overall, activation measurements should be reliable (except perhaps at 100 nM), although small shifts (<5 mV) should be interpreted with caution. For the relatively small currents with 2 mM Ca2+o or Ba2+o the estimated errors in our voltage shift measurements are <1 mV, so the small effects in Fig. 12 (C–F) should not be affected by series resistance error (which would be similar in Ca2+o vs. Ba2+o in any case). Voltage shifts shown in the figures are not corrected for series resistance errors.

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