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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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The instantaneous current–voltage protocol (IIV) used to examine permeation and block. (A and C) Currents recorded in 2 mM Ca2+o, a test solution (100 nM Ca2+o in A, 110 mM Ca2+o in C), and following return to 2 mM Ca2+o (shown from left to right, in 40-mV increments). For this and all figures illustrating current records, dashed lines indicate zero current following leak subtraction. 3 kHz digital Gaussian filter. (B and D) IIV relations measured from the initial amplitude of single-exponential fits (see Materials and methods), following a brief strong depolarization (2 ms to +60 mV) to produce maximal activation with minimal inactivation. Values in 2 mM Ca2+o recorded before and after application of the test solution are shown as upward and downward triangles (respectively), with a line drawn through the average. Voltages in 110 mM Ca2+o (D) are corrected for a junction potential (see Materials and methods).
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fig1: The instantaneous current–voltage protocol (IIV) used to examine permeation and block. (A and C) Currents recorded in 2 mM Ca2+o, a test solution (100 nM Ca2+o in A, 110 mM Ca2+o in C), and following return to 2 mM Ca2+o (shown from left to right, in 40-mV increments). For this and all figures illustrating current records, dashed lines indicate zero current following leak subtraction. 3 kHz digital Gaussian filter. (B and D) IIV relations measured from the initial amplitude of single-exponential fits (see Materials and methods), following a brief strong depolarization (2 ms to +60 mV) to produce maximal activation with minimal inactivation. Values in 2 mM Ca2+o recorded before and after application of the test solution are shown as upward and downward triangles (respectively), with a line drawn through the average. Voltages in 110 mM Ca2+o (D) are corrected for a junction potential (see Materials and methods).

Mentions: The classical method for separating gating from permeation is the instantaneous current–voltage relationship (IIV) (Hodgkin and Huxley, 1952). Channels are first activated by a fixed depolarizing pulse, followed by repolarization to a wide range of voltages. The current measured “instantaneously” at each new voltage reflects the current through the population of channels opened by the prior depolarizing pulse (Fig. 1). Thus, the number of channels open is constant, so any variation in current must reflect the effect of voltage on permeation through those open channels. The IIV method makes several assumptions, most critically that currents can be accurately measured before gating processes affect the number of channels open (see Materials and methods).


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)

The instantaneous current–voltage protocol (IIV) used to examine permeation and block. (A and C) Currents recorded in 2 mM Ca2+o, a test solution (100 nM Ca2+o in A, 110 mM Ca2+o in C), and following return to 2 mM Ca2+o (shown from left to right, in 40-mV increments). For this and all figures illustrating current records, dashed lines indicate zero current following leak subtraction. 3 kHz digital Gaussian filter. (B and D) IIV relations measured from the initial amplitude of single-exponential fits (see Materials and methods), following a brief strong depolarization (2 ms to +60 mV) to produce maximal activation with minimal inactivation. Values in 2 mM Ca2+o recorded before and after application of the test solution are shown as upward and downward triangles (respectively), with a line drawn through the average. Voltages in 110 mM Ca2+o (D) are corrected for a junction potential (see Materials and methods).
© Copyright Policy
Related In: Results  -  Collection

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

fig1: The instantaneous current–voltage protocol (IIV) used to examine permeation and block. (A and C) Currents recorded in 2 mM Ca2+o, a test solution (100 nM Ca2+o in A, 110 mM Ca2+o in C), and following return to 2 mM Ca2+o (shown from left to right, in 40-mV increments). For this and all figures illustrating current records, dashed lines indicate zero current following leak subtraction. 3 kHz digital Gaussian filter. (B and D) IIV relations measured from the initial amplitude of single-exponential fits (see Materials and methods), following a brief strong depolarization (2 ms to +60 mV) to produce maximal activation with minimal inactivation. Values in 2 mM Ca2+o recorded before and after application of the test solution are shown as upward and downward triangles (respectively), with a line drawn through the average. Voltages in 110 mM Ca2+o (D) are corrected for a junction potential (see Materials and methods).
Mentions: The classical method for separating gating from permeation is the instantaneous current–voltage relationship (IIV) (Hodgkin and Huxley, 1952). Channels are first activated by a fixed depolarizing pulse, followed by repolarization to a wide range of voltages. The current measured “instantaneously” at each new voltage reflects the current through the population of channels opened by the prior depolarizing pulse (Fig. 1). Thus, the number of channels open is constant, so any variation in current must reflect the effect of voltage on permeation through those open channels. The IIV method makes several assumptions, most critically that currents can be accurately measured before gating processes affect the number of channels open (see Materials and methods).

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