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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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Effect of 3–10 μM Ca2+o on tail current kinetics. (A) Sample records in 2 mM Ca2+o (control and wash, left and right) and 10 μM Ca2+o (middle), shown in 40-mV increments. 3 kHz Gaussian filter. Note biphasic tail currents at −20 and −60 mV (arrows). Tail currents in that voltage range are shown on an expanded time scale for 3 μM Ca2+o (B) and 10 μM Ca2+o (C), from a different cell. (D) Time constants from biexponential fits, from 3 cells in 3 μM Ca2+o (filled circles) and 9 cells in 10 μM Ca2+o (open squares). The dashed lines through the faster time constants are drawn for the 3.3-fold change in time constant expected from bimolecular kinetics. (E) The amplitudes of the fast and slow components to the biexponential fits.
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fig6: Effect of 3–10 μM Ca2+o on tail current kinetics. (A) Sample records in 2 mM Ca2+o (control and wash, left and right) and 10 μM Ca2+o (middle), shown in 40-mV increments. 3 kHz Gaussian filter. Note biphasic tail currents at −20 and −60 mV (arrows). Tail currents in that voltage range are shown on an expanded time scale for 3 μM Ca2+o (B) and 10 μM Ca2+o (C), from a different cell. (D) Time constants from biexponential fits, from 3 cells in 3 μM Ca2+o (filled circles) and 9 cells in 10 μM Ca2+o (open squares). The dashed lines through the faster time constants are drawn for the 3.3-fold change in time constant expected from bimolecular kinetics. (E) The amplitudes of the fast and slow components to the biexponential fits.

Mentions: Close examination of tail currents recorded in 10 μM Ca2+o revealed a rapid component (Fig. 6 A), possibly resulting from time-dependent block by Ca2+o (Lux et al., 1990). Consistent with that interpretation, tail currents were also biphasic in 3 μM Ca2+o (Fig. 6 B), and the fast component was visibly slower in 3 vs. 10 μM Ca2+o (Fig. 6, B and C). Biexponential fits to the tail currents demonstrated a slow component with τ ∼ 10 ms, which did not depend on Ca2+o, and a concentration-dependent fast component (Fig. 6 D). The simplest explanation is that much of the block by Ca2+o is relieved by strong depolarization (the 2-ms step to +60 mV, Fig. 6 A), and then Ca2+o reenters the pore upon repolarization to voltages where steady-state block is stronger (see Figs. 4 and 5). The fast component does not decay completely to zero, suggesting a rapidly equilibrating but incomplete block, followed by normal tail current kinetics for channels that remain unblocked (at −20 to −60 mV most channels inactivate rather than close; Serrano et al., 1999). If so, the relative amplitude of the fast tail current component reflects the fraction of channels blocked (Fig. 6 E).


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)

Effect of 3–10 μM Ca2+o on tail current kinetics. (A) Sample records in 2 mM Ca2+o (control and wash, left and right) and 10 μM Ca2+o (middle), shown in 40-mV increments. 3 kHz Gaussian filter. Note biphasic tail currents at −20 and −60 mV (arrows). Tail currents in that voltage range are shown on an expanded time scale for 3 μM Ca2+o (B) and 10 μM Ca2+o (C), from a different cell. (D) Time constants from biexponential fits, from 3 cells in 3 μM Ca2+o (filled circles) and 9 cells in 10 μM Ca2+o (open squares). The dashed lines through the faster time constants are drawn for the 3.3-fold change in time constant expected from bimolecular kinetics. (E) The amplitudes of the fast and slow components to the biexponential fits.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2483336&req=5

fig6: Effect of 3–10 μM Ca2+o on tail current kinetics. (A) Sample records in 2 mM Ca2+o (control and wash, left and right) and 10 μM Ca2+o (middle), shown in 40-mV increments. 3 kHz Gaussian filter. Note biphasic tail currents at −20 and −60 mV (arrows). Tail currents in that voltage range are shown on an expanded time scale for 3 μM Ca2+o (B) and 10 μM Ca2+o (C), from a different cell. (D) Time constants from biexponential fits, from 3 cells in 3 μM Ca2+o (filled circles) and 9 cells in 10 μM Ca2+o (open squares). The dashed lines through the faster time constants are drawn for the 3.3-fold change in time constant expected from bimolecular kinetics. (E) The amplitudes of the fast and slow components to the biexponential fits.
Mentions: Close examination of tail currents recorded in 10 μM Ca2+o revealed a rapid component (Fig. 6 A), possibly resulting from time-dependent block by Ca2+o (Lux et al., 1990). Consistent with that interpretation, tail currents were also biphasic in 3 μM Ca2+o (Fig. 6 B), and the fast component was visibly slower in 3 vs. 10 μM Ca2+o (Fig. 6, B and C). Biexponential fits to the tail currents demonstrated a slow component with τ ∼ 10 ms, which did not depend on Ca2+o, and a concentration-dependent fast component (Fig. 6 D). The simplest explanation is that much of the block by Ca2+o is relieved by strong depolarization (the 2-ms step to +60 mV, Fig. 6 A), and then Ca2+o reenters the pore upon repolarization to voltages where steady-state block is stronger (see Figs. 4 and 5). The fast component does not decay completely to zero, suggesting a rapidly equilibrating but incomplete block, followed by normal tail current kinetics for channels that remain unblocked (at −20 to −60 mV most channels inactivate rather than close; Serrano et al., 1999). If so, the relative amplitude of the fast tail current component reflects the fraction of channels blocked (Fig. 6 E).

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