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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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Analysis of chord conductances. (A–D) Dependence of chord conductance on Ca2+o or Ba2+o, shown for voltages in 40-mV increments. Positive voltages (A and C) and negative voltages (B and D) are shown for Ca2+o (A and B) and Ba2+o (C and D). Smooth curves are described in Materials and methods. (E and F) Analysis of chord conductances as the sum of Na+ and Ca2+/Ba2+ conductances. (E) Maximal conductances; (F) apparent Kd values for permeation (activation of GCa or Ba) and block (inhibition of GNa).
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fig5: Analysis of chord conductances. (A–D) Dependence of chord conductance on Ca2+o or Ba2+o, shown for voltages in 40-mV increments. Positive voltages (A and C) and negative voltages (B and D) are shown for Ca2+o (A and B) and Ba2+o (C and D). Smooth curves are described in Materials and methods. (E and F) Analysis of chord conductances as the sum of Na+ and Ca2+/Ba2+ conductances. (E) Maximal conductances; (F) apparent Kd values for permeation (activation of GCa or Ba) and block (inhibition of GNa).

Mentions: For most experiments, extracellular solutions were exchanged using a multibarrel perfusion system, with flow driven by gravity, and an Ag/AgCl ground electrode in the bath. This configuration results in a liquid junction potential between the perfusing solution and the bath. The effect of the junction potential in the measurement of the membrane potential (VM) was calculated (Neher, 1992):(1)\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}V_{M}=V-V_{LJ}-V_{2,l},\end{equation*}\end{document}where V is the command potential, VLJ is the liquid junction potential between the 2 mM Ca2+ sealing (also control) solution and the intracellular solution, and V2,1 is the liquid junction potential established between the 2 mM Ca2+ control solution and the test solution. Liquid junction potentials were calculated using a generalized Henderson equation implemented in the JPCalc software (Barry, 1994) that is incorporated into Clampex (Axon Instruments). VLJ in our experimental conditions is 0 mV, but V2,1 ranged from −0.3 in low Ca2+ concentrations to 6.9 mV in isotonic CaCl2 solutions. Junction potentials in isotonic CaCl2 solutions were confirmed experimentally using a 100-μl perfusion chamber grounded with a 3 M KCl 3% agar bridge, in the current clamp configuration of the Axopatch 200 amplifier. Measurements were corrected only for junction potentials calculated to be >1 mV. For analyses where values had to be compared directly at each voltage, values with significant junction potentials were linearly interpolated to the desired voltage (Fig. 5, Fig. S12, E and F, and Fig. S20 E). Original current records in Fig. 1 C, Fig. S5 C, Fig. S11, and Fig. S20 A are not corrected for junction potentials.


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)

Analysis of chord conductances. (A–D) Dependence of chord conductance on Ca2+o or Ba2+o, shown for voltages in 40-mV increments. Positive voltages (A and C) and negative voltages (B and D) are shown for Ca2+o (A and B) and Ba2+o (C and D). Smooth curves are described in Materials and methods. (E and F) Analysis of chord conductances as the sum of Na+ and Ca2+/Ba2+ conductances. (E) Maximal conductances; (F) apparent Kd values for permeation (activation of GCa or Ba) and block (inhibition of GNa).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2483336&req=5

fig5: Analysis of chord conductances. (A–D) Dependence of chord conductance on Ca2+o or Ba2+o, shown for voltages in 40-mV increments. Positive voltages (A and C) and negative voltages (B and D) are shown for Ca2+o (A and B) and Ba2+o (C and D). Smooth curves are described in Materials and methods. (E and F) Analysis of chord conductances as the sum of Na+ and Ca2+/Ba2+ conductances. (E) Maximal conductances; (F) apparent Kd values for permeation (activation of GCa or Ba) and block (inhibition of GNa).
Mentions: For most experiments, extracellular solutions were exchanged using a multibarrel perfusion system, with flow driven by gravity, and an Ag/AgCl ground electrode in the bath. This configuration results in a liquid junction potential between the perfusing solution and the bath. The effect of the junction potential in the measurement of the membrane potential (VM) was calculated (Neher, 1992):(1)\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}V_{M}=V-V_{LJ}-V_{2,l},\end{equation*}\end{document}where V is the command potential, VLJ is the liquid junction potential between the 2 mM Ca2+ sealing (also control) solution and the intracellular solution, and V2,1 is the liquid junction potential established between the 2 mM Ca2+ control solution and the test solution. Liquid junction potentials were calculated using a generalized Henderson equation implemented in the JPCalc software (Barry, 1994) that is incorporated into Clampex (Axon Instruments). VLJ in our experimental conditions is 0 mV, but V2,1 ranged from −0.3 in low Ca2+ concentrations to 6.9 mV in isotonic CaCl2 solutions. Junction potentials in isotonic CaCl2 solutions were confirmed experimentally using a 100-μl perfusion chamber grounded with a 3 M KCl 3% agar bridge, in the current clamp configuration of the Axopatch 200 amplifier. Measurements were corrected only for junction potentials calculated to be >1 mV. For analyses where values had to be compared directly at each voltage, values with significant junction potentials were linearly interpolated to the desired voltage (Fig. 5, Fig. S12, E and F, and Fig. S20 E). Original current records in Fig. 1 C, Fig. S5 C, Fig. S11, and Fig. S20 A are not corrected for junction potentials.

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