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Modulation of the slow/common gating of CLC channels by intracellular cadmium.

Yu Y, Tsai MF, Yu WP, Chen TY - J. Gen. Physiol. (2015)

Bottom Line: Here, we found that intracellularly applied Cd(2+) reduces the current of CLC-0 because of its inhibition on the slow gating.Our experimental results suggest that mutations of the corresponding residues in CLC-0 change the subunit interaction and alter the slow gating of CLC-0.The effect of these mutations on modulations of slow gating of CLC channels by intracellular Cd(2+) likely depends on their alteration of subunit interactions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Neuroscience and Department of Neurology, University of California, Davis, Davis, CA 95618 Center for Neuroscience and Department of Neurology, University of California, Davis, Davis, CA 95618.

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Voltage dependence of the inhibition and recovery kinetics of the Cd2+ modulation of the I225W/V490W mutant of CLC-0. (A) Time course of current inhibition by various [Cd2+] (in micromolars) at −40 mV. (B) Inverse of the time constant of Cd2+ inhibition (kon = 1/τon) as a function of [Cd2+]. The values of τon were obtained from single-exponential fits as in A. (C) Averaged time course for the inhibition by 30 µM Cd2+ at 0, −40, and −80 mV. Data points were calculated by normalizing the current in various [Cd2+] to the control current ([Cd2+] = 0). (D) Time course of the current recovery from Cd2+ inhibition. Data points were obtained by normalizing the current recovered after Cd2+ was washed out to that before the application of Cd2+. (E) Time constants of the inhibition (τon) and recovery (τoff) as a function of membrane voltage. Time constants were obtained by fitting the data points in C and D with single-exponential functions.
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fig13: Voltage dependence of the inhibition and recovery kinetics of the Cd2+ modulation of the I225W/V490W mutant of CLC-0. (A) Time course of current inhibition by various [Cd2+] (in micromolars) at −40 mV. (B) Inverse of the time constant of Cd2+ inhibition (kon = 1/τon) as a function of [Cd2+]. The values of τon were obtained from single-exponential fits as in A. (C) Averaged time course for the inhibition by 30 µM Cd2+ at 0, −40, and −80 mV. Data points were calculated by normalizing the current in various [Cd2+] to the control current ([Cd2+] = 0). (D) Time course of the current recovery from Cd2+ inhibition. Data points were obtained by normalizing the current recovered after Cd2+ was washed out to that before the application of Cd2+. (E) Time constants of the inhibition (τon) and recovery (τoff) as a function of membrane voltage. Time constants were obtained by fitting the data points in C and D with single-exponential functions.

Mentions: To gain further insights into the Cd2+ modulation mechanism, we studied the kinetics of Cd2+ inhibition on I225W/V490W of CLC-0, taking advantage of the faster kinetics of Cd2+ inhibition in this mutant. Fig. 12 A illustrates a two-pulse protocol used to examine the rate of Cd2+ inhibition by measuring the current of the I225W/V490W mutant elicited by a −100-mV voltage pulse before (first pulse) and after the patch was exposed to Cd2+ for a certain amount of time (second pulse). Combining multiple recording traces from the same patch generates the time course of Cd2+ inhibition (Fig. 12 B). It is worth noticing that there appears to be a delay of the current inhibition because the current recorded after applying Cd2+ for 0.2 s is almost the same as that before the Cd2+ application. This delay has little voltage dependence (Fig. 12 C), but as [Cd2+] is increased, the delay is shortened (Fig. 12 D). To analyze the kinetics of Cd2+ inhibition like that shown in Fig. 12 B, we normalized the current induced by the second voltage pulse to the current before Cd2+ application, and plotted the time course of the current inhibition by Cd2+ (Fig. 13 A). Fitting the Cd2+ inhibition time course with an exponential function gives the inhibition time constant (τon), the inverse of which (1/τon) is defined as the apparent Cd2+ inhibition rate (or apparent on rate), kon. The observation that kon is a saturable function of [Cd2+] (Fig. 13 B) suggests that Cd2+ inhibition is not a simple bimolecular reaction involving only Cd2+ binding. These results suggest that the binding of Cd2+ to the open-state channel may not immediately close the channel, and the delay in current inhibition upon Cd2+ application could result from the conformational change between the open and the closed state of this dimer interface mutant.


Modulation of the slow/common gating of CLC channels by intracellular cadmium.

Yu Y, Tsai MF, Yu WP, Chen TY - J. Gen. Physiol. (2015)

Voltage dependence of the inhibition and recovery kinetics of the Cd2+ modulation of the I225W/V490W mutant of CLC-0. (A) Time course of current inhibition by various [Cd2+] (in micromolars) at −40 mV. (B) Inverse of the time constant of Cd2+ inhibition (kon = 1/τon) as a function of [Cd2+]. The values of τon were obtained from single-exponential fits as in A. (C) Averaged time course for the inhibition by 30 µM Cd2+ at 0, −40, and −80 mV. Data points were calculated by normalizing the current in various [Cd2+] to the control current ([Cd2+] = 0). (D) Time course of the current recovery from Cd2+ inhibition. Data points were obtained by normalizing the current recovered after Cd2+ was washed out to that before the application of Cd2+. (E) Time constants of the inhibition (τon) and recovery (τoff) as a function of membrane voltage. Time constants were obtained by fitting the data points in C and D with single-exponential functions.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4664824&req=5

fig13: Voltage dependence of the inhibition and recovery kinetics of the Cd2+ modulation of the I225W/V490W mutant of CLC-0. (A) Time course of current inhibition by various [Cd2+] (in micromolars) at −40 mV. (B) Inverse of the time constant of Cd2+ inhibition (kon = 1/τon) as a function of [Cd2+]. The values of τon were obtained from single-exponential fits as in A. (C) Averaged time course for the inhibition by 30 µM Cd2+ at 0, −40, and −80 mV. Data points were calculated by normalizing the current in various [Cd2+] to the control current ([Cd2+] = 0). (D) Time course of the current recovery from Cd2+ inhibition. Data points were obtained by normalizing the current recovered after Cd2+ was washed out to that before the application of Cd2+. (E) Time constants of the inhibition (τon) and recovery (τoff) as a function of membrane voltage. Time constants were obtained by fitting the data points in C and D with single-exponential functions.
Mentions: To gain further insights into the Cd2+ modulation mechanism, we studied the kinetics of Cd2+ inhibition on I225W/V490W of CLC-0, taking advantage of the faster kinetics of Cd2+ inhibition in this mutant. Fig. 12 A illustrates a two-pulse protocol used to examine the rate of Cd2+ inhibition by measuring the current of the I225W/V490W mutant elicited by a −100-mV voltage pulse before (first pulse) and after the patch was exposed to Cd2+ for a certain amount of time (second pulse). Combining multiple recording traces from the same patch generates the time course of Cd2+ inhibition (Fig. 12 B). It is worth noticing that there appears to be a delay of the current inhibition because the current recorded after applying Cd2+ for 0.2 s is almost the same as that before the Cd2+ application. This delay has little voltage dependence (Fig. 12 C), but as [Cd2+] is increased, the delay is shortened (Fig. 12 D). To analyze the kinetics of Cd2+ inhibition like that shown in Fig. 12 B, we normalized the current induced by the second voltage pulse to the current before Cd2+ application, and plotted the time course of the current inhibition by Cd2+ (Fig. 13 A). Fitting the Cd2+ inhibition time course with an exponential function gives the inhibition time constant (τon), the inverse of which (1/τon) is defined as the apparent Cd2+ inhibition rate (or apparent on rate), kon. The observation that kon is a saturable function of [Cd2+] (Fig. 13 B) suggests that Cd2+ inhibition is not a simple bimolecular reaction involving only Cd2+ binding. These results suggest that the binding of Cd2+ to the open-state channel may not immediately close the channel, and the delay in current inhibition upon Cd2+ application could result from the conformational change between the open and the closed state of this dimer interface mutant.

Bottom Line: Here, we found that intracellularly applied Cd(2+) reduces the current of CLC-0 because of its inhibition on the slow gating.Our experimental results suggest that mutations of the corresponding residues in CLC-0 change the subunit interaction and alter the slow gating of CLC-0.The effect of these mutations on modulations of slow gating of CLC channels by intracellular Cd(2+) likely depends on their alteration of subunit interactions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Neuroscience and Department of Neurology, University of California, Davis, Davis, CA 95618 Center for Neuroscience and Department of Neurology, University of California, Davis, Davis, CA 95618.

No MeSH data available.


Related in: MedlinePlus