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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.

No MeSH data available.


Intracellular Cd2+ inhibition of the I225W/V490W double mutant of CLC-0. (A) Original recording traces (left), steady-state voltage-dependent activation curve (middle), and the normalized steady-state I-V curve (right) of the I225W/V490W mutant. Data were from recording traces with p/4 leak subtraction. The dotted line in the left panel represents the zero-current level. (B) Effects of various intracellular [Cd2+] on the steady-state current of the I225W/V490W mutant. A test pulse of −100 mV was given every 2 s, and the current was measured at the end of the pulse to monitor the Cd2+ inhibition. Dotted line represents the zero-current level. Cd2+ of various concentrations was applied as indicated by the arrows.
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fig4: Intracellular Cd2+ inhibition of the I225W/V490W double mutant of CLC-0. (A) Original recording traces (left), steady-state voltage-dependent activation curve (middle), and the normalized steady-state I-V curve (right) of the I225W/V490W mutant. Data were from recording traces with p/4 leak subtraction. The dotted line in the left panel represents the zero-current level. (B) Effects of various intracellular [Cd2+] on the steady-state current of the I225W/V490W mutant. A test pulse of −100 mV was given every 2 s, and the current was measured at the end of the pulse to monitor the Cd2+ inhibition. Dotted line represents the zero-current level. Cd2+ of various concentrations was applied as indicated by the arrows.

Mentions: The hypothesis that the intracellular Cd2+ inhibition of CLC-0 is mediated by modulating the channel’s slow gating is consistent with the findings that the inhibition and recovery processes are slow (see Fig. 2 D, top). The slow kinetics of the inhibition rendered a precise characterization of the Cd2+ effect technically challenging because of relatively short durations of stable excised inside-out patch recordings of WT CLC-0. The problem was circumvented by an accidental finding that the mutations of I225W and V490W accelerated the inhibition kinetics. Mutations of the corresponding residues in CLC-ec1 (I201W/I422W) have been shown previously to disrupt the subunit interaction, resulting in functional monomeric CLC-ec1 proteins (Robertson et al., 2010). However, the I225W/V490W mutant of CLC-0 remains as a dimer when expressed in HEK293 cells, as shown by the FRET experiment in which the FRET efficiency between a FRET donor and a FRET acceptor attached to the I225W/V490W mutant is no less than that observed in the WT construct (Fig. 3, C and D). Nevertheless, the voltage dependence of the opening of the I225W/V490W mutant channel is opposite to that of the WT channel: the mutant channel’s open probability (Po) increases as the membrane voltage hyperpolarizes (Fig. 4 A). Moreover, Cd2+ inhibition is now faster and more potent: even 1 µM Cd2+ significantly inhibits the current (Fig. 4 B).


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

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

Intracellular Cd2+ inhibition of the I225W/V490W double mutant of CLC-0. (A) Original recording traces (left), steady-state voltage-dependent activation curve (middle), and the normalized steady-state I-V curve (right) of the I225W/V490W mutant. Data were from recording traces with p/4 leak subtraction. The dotted line in the left panel represents the zero-current level. (B) Effects of various intracellular [Cd2+] on the steady-state current of the I225W/V490W mutant. A test pulse of −100 mV was given every 2 s, and the current was measured at the end of the pulse to monitor the Cd2+ inhibition. Dotted line represents the zero-current level. Cd2+ of various concentrations was applied as indicated by the arrows.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: Intracellular Cd2+ inhibition of the I225W/V490W double mutant of CLC-0. (A) Original recording traces (left), steady-state voltage-dependent activation curve (middle), and the normalized steady-state I-V curve (right) of the I225W/V490W mutant. Data were from recording traces with p/4 leak subtraction. The dotted line in the left panel represents the zero-current level. (B) Effects of various intracellular [Cd2+] on the steady-state current of the I225W/V490W mutant. A test pulse of −100 mV was given every 2 s, and the current was measured at the end of the pulse to monitor the Cd2+ inhibition. Dotted line represents the zero-current level. Cd2+ of various concentrations was applied as indicated by the arrows.
Mentions: The hypothesis that the intracellular Cd2+ inhibition of CLC-0 is mediated by modulating the channel’s slow gating is consistent with the findings that the inhibition and recovery processes are slow (see Fig. 2 D, top). The slow kinetics of the inhibition rendered a precise characterization of the Cd2+ effect technically challenging because of relatively short durations of stable excised inside-out patch recordings of WT CLC-0. The problem was circumvented by an accidental finding that the mutations of I225W and V490W accelerated the inhibition kinetics. Mutations of the corresponding residues in CLC-ec1 (I201W/I422W) have been shown previously to disrupt the subunit interaction, resulting in functional monomeric CLC-ec1 proteins (Robertson et al., 2010). However, the I225W/V490W mutant of CLC-0 remains as a dimer when expressed in HEK293 cells, as shown by the FRET experiment in which the FRET efficiency between a FRET donor and a FRET acceptor attached to the I225W/V490W mutant is no less than that observed in the WT construct (Fig. 3, C and D). Nevertheless, the voltage dependence of the opening of the I225W/V490W mutant channel is opposite to that of the WT channel: the mutant channel’s open probability (Po) increases as the membrane voltage hyperpolarizes (Fig. 4 A). Moreover, Cd2+ inhibition is now faster and more potent: even 1 µM Cd2+ significantly inhibits the current (Fig. 4 B).

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.