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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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Single-channel analysis of block by Ca2+o. (A) Bursts of channel activity recorded in the cell-attached configuration, selected from long (5–10 s) depolarizations to −70 mV in 0.1, 3, and 10 μM Ca2+o (n = 3–4). No leak subtraction; tick marks at the left indicate zero pipet current. (B) Mean open times (symbols defined in C). (C) Mean closed times, for the fast component (see Fig. S4, D–F). (D) Dependence of the net closing rate (the reciprocal of the mean open time) on [Ca2+]o. For this analysis, open times from −40 to −90 mV were averaged at each concentration. From a fit to bimolecular kinetics (straight line), the zero-Ca2+o closing rate was 1.2 ms−1, and the blocking rate was 5.3 × 108 M−1s−1. The fit minimized the sum of squared errors for all measured time constants (i.e., it is not a simple linear regression).
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fig8: Single-channel analysis of block by Ca2+o. (A) Bursts of channel activity recorded in the cell-attached configuration, selected from long (5–10 s) depolarizations to −70 mV in 0.1, 3, and 10 μM Ca2+o (n = 3–4). No leak subtraction; tick marks at the left indicate zero pipet current. (B) Mean open times (symbols defined in C). (C) Mean closed times, for the fast component (see Fig. S4, D–F). (D) Dependence of the net closing rate (the reciprocal of the mean open time) on [Ca2+]o. For this analysis, open times from −40 to −90 mV were averaged at each concentration. From a fit to bimolecular kinetics (straight line), the zero-Ca2+o closing rate was 1.2 ms−1, and the blocking rate was 5.3 × 108 M−1s−1. The fit minimized the sum of squared errors for all measured time constants (i.e., it is not a simple linear regression).

Mentions: Effects of ions on permeation were assessed using IIV relations, measured by fitting single exponentials to the decay of current following a brief (2-ms) step to +60 mV (Serrano et al., 2000). The exponential fit began when the tail currents reached a peak, as an estimate of the current at the time when accurate voltage clamp was actually achieved, and extended 20 ms into the voltage steps. The amplitude of the fitted exponential at the starting point of the fit was used for the IIV measurements shown. In most of our experimental conditions, the time course of block is fast, i.e., pore occupancy has reached steady state by the time the IIV is measured. The time course of block can be resolved by careful analysis in certain conditions (1 mM Mg2+o in 2 mM Ba2+o, Serrano et al., 2000; 10 μM Ca2+o, Figs. 7–9), but the single exponential fit did not include those very fast (τ = 0.1–0.2 ms) components, so we interpret the IIV measurements as representing steady-state block in all experimental conditions in this study.


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)

Single-channel analysis of block by Ca2+o. (A) Bursts of channel activity recorded in the cell-attached configuration, selected from long (5–10 s) depolarizations to −70 mV in 0.1, 3, and 10 μM Ca2+o (n = 3–4). No leak subtraction; tick marks at the left indicate zero pipet current. (B) Mean open times (symbols defined in C). (C) Mean closed times, for the fast component (see Fig. S4, D–F). (D) Dependence of the net closing rate (the reciprocal of the mean open time) on [Ca2+]o. For this analysis, open times from −40 to −90 mV were averaged at each concentration. From a fit to bimolecular kinetics (straight line), the zero-Ca2+o closing rate was 1.2 ms−1, and the blocking rate was 5.3 × 108 M−1s−1. The fit minimized the sum of squared errors for all measured time constants (i.e., it is not a simple linear regression).
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Single-channel analysis of block by Ca2+o. (A) Bursts of channel activity recorded in the cell-attached configuration, selected from long (5–10 s) depolarizations to −70 mV in 0.1, 3, and 10 μM Ca2+o (n = 3–4). No leak subtraction; tick marks at the left indicate zero pipet current. (B) Mean open times (symbols defined in C). (C) Mean closed times, for the fast component (see Fig. S4, D–F). (D) Dependence of the net closing rate (the reciprocal of the mean open time) on [Ca2+]o. For this analysis, open times from −40 to −90 mV were averaged at each concentration. From a fit to bimolecular kinetics (straight line), the zero-Ca2+o closing rate was 1.2 ms−1, and the blocking rate was 5.3 × 108 M−1s−1. The fit minimized the sum of squared errors for all measured time constants (i.e., it is not a simple linear regression).
Mentions: Effects of ions on permeation were assessed using IIV relations, measured by fitting single exponentials to the decay of current following a brief (2-ms) step to +60 mV (Serrano et al., 2000). The exponential fit began when the tail currents reached a peak, as an estimate of the current at the time when accurate voltage clamp was actually achieved, and extended 20 ms into the voltage steps. The amplitude of the fitted exponential at the starting point of the fit was used for the IIV measurements shown. In most of our experimental conditions, the time course of block is fast, i.e., pore occupancy has reached steady state by the time the IIV is measured. The time course of block can be resolved by careful analysis in certain conditions (1 mM Mg2+o in 2 mM Ba2+o, Serrano et al., 2000; 10 μM Ca2+o, Figs. 7–9), but the single exponential fit did not include those very fast (τ = 0.1–0.2 ms) components, so we interpret the IIV measurements as representing steady-state block in all experimental conditions in this study.

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