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Quantitative analysis of the voltage-dependent gating of mouse parotid ClC-2 chloride channel.

de Santiago JA, Nehrke K, Arreola J - J. Gen. Physiol. (2005)

Bottom Line: However, we demonstrate in this work that the nearly ubiquitous ClC-2 shows significant differences in gating when compared with ClC-0 and ClC-1.To test these models, we mutated conserved residues that had been previously shown to eliminate or alter P(f) or P(s) in other ClC channels.These data provide a new perspective on ClC-2 gating, suggesting that the protopore gate contributes to both fast and slow gating and that gating relies strongly on the E213 residue.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Física, Universidad Autonóma de San Luis Potosí, San Luis Potosí, SLP 78290, México.

ABSTRACT
Various ClC-type voltage-gated chloride channel isoforms display a double barrel topology, and their gating mechanisms are thought to be similar. However, we demonstrate in this work that the nearly ubiquitous ClC-2 shows significant differences in gating when compared with ClC-0 and ClC-1. To delineate the gating of ClC-2 in quantitative terms, we have determined the voltage (V(m)) and time dependence of the protopore (P(f)) and common (P(s)) gates that control the opening and closing of the double barrel. mClC-2 was cloned from mouse salivary glands, expressed in HEK 293 cells, and the resulting chloride currents (I(Cl)) were measured using whole cell patch clamp. WT channels had I(Cl) that showed inward rectification and biexponential time course. Time constants of fast and slow components were approximately 10-fold different at negative V(m) and corresponded to P(f) and P(s), respectively. P(f) and P(s) were approximately 1 at -200 mV, while at V(m) > or = 0 mV, P(f) approximately 0 and P(s) approximately 0.6. Hence, P(f) dominated open kinetics at moderately negative V(m), while at very negative V(m) both gates contributed to gating. At V(m) > or = 0 mV, mClC-2 closes by shutting off P(f). Three- and two-state models described the open-to-closed transitions of P(f) and P(s), respectively. To test these models, we mutated conserved residues that had been previously shown to eliminate or alter P(f) or P(s) in other ClC channels. Based on the time and V(m) dependence of the two gates in WT and mutant channels, we constructed a model to explain the gating of mClC-2. In this model the E213 residue contributes to P(f), the dominant regulator of gating, while the C258 residue alters the V(m) dependence of P(f), probably by interacting with residue E213. These data provide a new perspective on ClC-2 gating, suggesting that the protopore gate contributes to both fast and slow gating and that gating relies strongly on the E213 residue.

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Kinetic model for mClC-2. A kinetic model developed to explain ClC-0 and ClC-1 was modified to account for the three states of the protopore gate. Scheme 1 describes the common gate. This resulted in a 12-state model consisting of seven nonconductive (C1–C7) and five conductive (O1–O5) states. Left column: states representing the common gate closed. Right column: states representing the common gate in the open position. Transitions between states are controlled by the indicated rate constants, these were assumed to be exponentially related to Vm. Transitions leading to the opening of the common gate (left column to right column) were assumed to follow the same kinetics and to be controlled by rate constants λ and μ. Likewise, we assumed that transitions of the protopore gate were independent of the common gate state. Free parameters were α1, β1, α2, β2, λ, and μ (see Table I).
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fig8: Kinetic model for mClC-2. A kinetic model developed to explain ClC-0 and ClC-1 was modified to account for the three states of the protopore gate. Scheme 1 describes the common gate. This resulted in a 12-state model consisting of seven nonconductive (C1–C7) and five conductive (O1–O5) states. Left column: states representing the common gate closed. Right column: states representing the common gate in the open position. Transitions between states are controlled by the indicated rate constants, these were assumed to be exponentially related to Vm. Transitions leading to the opening of the common gate (left column to right column) were assumed to follow the same kinetics and to be controlled by rate constants λ and μ. Likewise, we assumed that transitions of the protopore gate were independent of the common gate state. Free parameters were α1, β1, α2, β2, λ, and μ (see Table I).

Mentions: WT mClC-2 channels expressed in HEK 293 cells. (A) Raw traces elicited by voltage steps between +60 and −130 mV given in 10-mV increments. (B) I-V relationship (n = 9). ICl at each Vm was normalized to the corresponding value obtained at −200 mV and the resulting relative currents were averaged and plotted. (C) Apparent P0 at each test pulse was estimated as the magnitude of the tail current recorded at +60 mV divided by Imax estimated from a Boltzmann fit (see description in MATERIALS AND METHODS; n = 7). Continuous line is the fit of Eq. 1. (D) Fast (τf) and slow (τs) time constants as function of Vm. Time constants were obtained by fitting raw data with Eq. 2. (E) Relative weight of fast, slow, and instantaneous components. D and E show that our model displayed in Fig. 6 was also able to account for the double exponential behavior of whole cell currents. Simulated currents, like those depicted in Fig. 7 B, were fit with Eq. 2 to determine τf, τs, Af, As, and C. Those are plotted as continuous lines in D and E.


Quantitative analysis of the voltage-dependent gating of mouse parotid ClC-2 chloride channel.

de Santiago JA, Nehrke K, Arreola J - J. Gen. Physiol. (2005)

Kinetic model for mClC-2. A kinetic model developed to explain ClC-0 and ClC-1 was modified to account for the three states of the protopore gate. Scheme 1 describes the common gate. This resulted in a 12-state model consisting of seven nonconductive (C1–C7) and five conductive (O1–O5) states. Left column: states representing the common gate closed. Right column: states representing the common gate in the open position. Transitions between states are controlled by the indicated rate constants, these were assumed to be exponentially related to Vm. Transitions leading to the opening of the common gate (left column to right column) were assumed to follow the same kinetics and to be controlled by rate constants λ and μ. Likewise, we assumed that transitions of the protopore gate were independent of the common gate state. Free parameters were α1, β1, α2, β2, λ, and μ (see Table I).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2266594&req=5

fig8: Kinetic model for mClC-2. A kinetic model developed to explain ClC-0 and ClC-1 was modified to account for the three states of the protopore gate. Scheme 1 describes the common gate. This resulted in a 12-state model consisting of seven nonconductive (C1–C7) and five conductive (O1–O5) states. Left column: states representing the common gate closed. Right column: states representing the common gate in the open position. Transitions between states are controlled by the indicated rate constants, these were assumed to be exponentially related to Vm. Transitions leading to the opening of the common gate (left column to right column) were assumed to follow the same kinetics and to be controlled by rate constants λ and μ. Likewise, we assumed that transitions of the protopore gate were independent of the common gate state. Free parameters were α1, β1, α2, β2, λ, and μ (see Table I).
Mentions: WT mClC-2 channels expressed in HEK 293 cells. (A) Raw traces elicited by voltage steps between +60 and −130 mV given in 10-mV increments. (B) I-V relationship (n = 9). ICl at each Vm was normalized to the corresponding value obtained at −200 mV and the resulting relative currents were averaged and plotted. (C) Apparent P0 at each test pulse was estimated as the magnitude of the tail current recorded at +60 mV divided by Imax estimated from a Boltzmann fit (see description in MATERIALS AND METHODS; n = 7). Continuous line is the fit of Eq. 1. (D) Fast (τf) and slow (τs) time constants as function of Vm. Time constants were obtained by fitting raw data with Eq. 2. (E) Relative weight of fast, slow, and instantaneous components. D and E show that our model displayed in Fig. 6 was also able to account for the double exponential behavior of whole cell currents. Simulated currents, like those depicted in Fig. 7 B, were fit with Eq. 2 to determine τf, τs, Af, As, and C. Those are plotted as continuous lines in D and E.

Bottom Line: However, we demonstrate in this work that the nearly ubiquitous ClC-2 shows significant differences in gating when compared with ClC-0 and ClC-1.To test these models, we mutated conserved residues that had been previously shown to eliminate or alter P(f) or P(s) in other ClC channels.These data provide a new perspective on ClC-2 gating, suggesting that the protopore gate contributes to both fast and slow gating and that gating relies strongly on the E213 residue.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Física, Universidad Autonóma de San Luis Potosí, San Luis Potosí, SLP 78290, México.

ABSTRACT
Various ClC-type voltage-gated chloride channel isoforms display a double barrel topology, and their gating mechanisms are thought to be similar. However, we demonstrate in this work that the nearly ubiquitous ClC-2 shows significant differences in gating when compared with ClC-0 and ClC-1. To delineate the gating of ClC-2 in quantitative terms, we have determined the voltage (V(m)) and time dependence of the protopore (P(f)) and common (P(s)) gates that control the opening and closing of the double barrel. mClC-2 was cloned from mouse salivary glands, expressed in HEK 293 cells, and the resulting chloride currents (I(Cl)) were measured using whole cell patch clamp. WT channels had I(Cl) that showed inward rectification and biexponential time course. Time constants of fast and slow components were approximately 10-fold different at negative V(m) and corresponded to P(f) and P(s), respectively. P(f) and P(s) were approximately 1 at -200 mV, while at V(m) > or = 0 mV, P(f) approximately 0 and P(s) approximately 0.6. Hence, P(f) dominated open kinetics at moderately negative V(m), while at very negative V(m) both gates contributed to gating. At V(m) > or = 0 mV, mClC-2 closes by shutting off P(f). Three- and two-state models described the open-to-closed transitions of P(f) and P(s), respectively. To test these models, we mutated conserved residues that had been previously shown to eliminate or alter P(f) or P(s) in other ClC channels. Based on the time and V(m) dependence of the two gates in WT and mutant channels, we constructed a model to explain the gating of mClC-2. In this model the E213 residue contributes to P(f), the dominant regulator of gating, while the C258 residue alters the V(m) dependence of P(f), probably by interacting with residue E213. These data provide a new perspective on ClC-2 gating, suggesting that the protopore gate contributes to both fast and slow gating and that gating relies strongly on the E213 residue.

Show MeSH
Related in: MedlinePlus