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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.


FRET experiments of the cerulean (C)- and eYFP (Y)-tagged CLC0-TM. (A) Fluorescence images of a cell expressing C- and Y-tagged CLC0-TM with I225W/V490W mutations from C (top left) and Y excitation (bottom left). The corresponding spectroscopic images (middle) were obtained by passing the signal through a slit (vertical orange lines). The emission spectra (right) were obtained from the upper edge of the spectroscopic images (arrows). (B; top) Emission spectra from the cell in A by C excitation (blue trace) and Y excitation (yellow trace). Red trace is the standard C emission spectrum from C excitation. Green trace was obtained by subtracting the red trace from the blue trace. (Bottom) Ratio A and Ratio A0 (inset) are plotted against wavelength. (C) Apparent FRET efficiency (Eapp) of the C- and Y-tagged CLC0-TM against the C/Y intensity ratio. Each point represents the Eapp from a cell. (D) Apparent FRET efficiency of the C- and Y-tagged CLC0-TM with I225W/V490W mutations (red dots). Black dots were from a control experiment in which cells were cotransfected with WT CLC-0 and the subunit A2 of the rat olfactory CNG channel. Data points in C and D are fitted (solid curves) to Eqs. 3 and 4 of Bykova et al. (2006). In both C and D, significant FRET efficiency (∼18% for WT and ∼30% for I225W/V490W) was observed compared with the control experiment.
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fig3: FRET experiments of the cerulean (C)- and eYFP (Y)-tagged CLC0-TM. (A) Fluorescence images of a cell expressing C- and Y-tagged CLC0-TM with I225W/V490W mutations from C (top left) and Y excitation (bottom left). The corresponding spectroscopic images (middle) were obtained by passing the signal through a slit (vertical orange lines). The emission spectra (right) were obtained from the upper edge of the spectroscopic images (arrows). (B; top) Emission spectra from the cell in A by C excitation (blue trace) and Y excitation (yellow trace). Red trace is the standard C emission spectrum from C excitation. Green trace was obtained by subtracting the red trace from the blue trace. (Bottom) Ratio A and Ratio A0 (inset) are plotted against wavelength. (C) Apparent FRET efficiency (Eapp) of the C- and Y-tagged CLC0-TM against the C/Y intensity ratio. Each point represents the Eapp from a cell. (D) Apparent FRET efficiency of the C- and Y-tagged CLC0-TM with I225W/V490W mutations (red dots). Black dots were from a control experiment in which cells were cotransfected with WT CLC-0 and the subunit A2 of the rat olfactory CNG channel. Data points in C and D are fitted (solid curves) to Eqs. 3 and 4 of Bykova et al. (2006). In both C and D, significant FRET efficiency (∼18% for WT and ∼30% for I225W/V490W) was observed compared with the control experiment.

Mentions: The CLC-0 and CLC-1 cDNAs used for electrophysiological recordings were constructed in the pcDNA3 vector without any genetic tags of fluorescent proteins. For the constructs used in biochemical experiments, a “Flag” epitope was added to the N terminus (after the first methionine) of different CLC-0 constructs subcloned in the pcDNA3 vector. Mutations were made using the QuikChange site-directed mutagenesis kit (Agilent Technologies) and were confirmed by commercial DNA sequencing services. In the fluorescent resonant energy transfer (FRET) study used to evaluate the interaction of two channel subunits (Fig. 3), the C-terminal cytoplasmic domain of CLC-0 was removed after Ser530 to make a fair comparison with CLC-ec1 because the latter lacks a cytoplasmic domain. This C terminus–truncated mutant of CLC-0, referred to as CLC0-TM, has been described previously (Bykova et al., 2006). It was constructed in the pEGFP-N3 vector, and the C terminus of the CLC0-TM sequence was tagged by a Gly-Ser linker followed by the sequence of cerulean or eYFP. The I225W/V490W mutations of CLC-0 (corresponding to I201W/I422W mutations in CLC-ec1) were made in these two CLC0-TM constructs, and were used in the FRET study. The CNG channel used as the negative control in the FRET experiment was provided by W.N. Zagotta (University of Washington, Seattle, WA), and fluorescent proteins were also tagged to the C terminus of the CNG channel.


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

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

FRET experiments of the cerulean (C)- and eYFP (Y)-tagged CLC0-TM. (A) Fluorescence images of a cell expressing C- and Y-tagged CLC0-TM with I225W/V490W mutations from C (top left) and Y excitation (bottom left). The corresponding spectroscopic images (middle) were obtained by passing the signal through a slit (vertical orange lines). The emission spectra (right) were obtained from the upper edge of the spectroscopic images (arrows). (B; top) Emission spectra from the cell in A by C excitation (blue trace) and Y excitation (yellow trace). Red trace is the standard C emission spectrum from C excitation. Green trace was obtained by subtracting the red trace from the blue trace. (Bottom) Ratio A and Ratio A0 (inset) are plotted against wavelength. (C) Apparent FRET efficiency (Eapp) of the C- and Y-tagged CLC0-TM against the C/Y intensity ratio. Each point represents the Eapp from a cell. (D) Apparent FRET efficiency of the C- and Y-tagged CLC0-TM with I225W/V490W mutations (red dots). Black dots were from a control experiment in which cells were cotransfected with WT CLC-0 and the subunit A2 of the rat olfactory CNG channel. Data points in C and D are fitted (solid curves) to Eqs. 3 and 4 of Bykova et al. (2006). In both C and D, significant FRET efficiency (∼18% for WT and ∼30% for I225W/V490W) was observed compared with the control experiment.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig3: FRET experiments of the cerulean (C)- and eYFP (Y)-tagged CLC0-TM. (A) Fluorescence images of a cell expressing C- and Y-tagged CLC0-TM with I225W/V490W mutations from C (top left) and Y excitation (bottom left). The corresponding spectroscopic images (middle) were obtained by passing the signal through a slit (vertical orange lines). The emission spectra (right) were obtained from the upper edge of the spectroscopic images (arrows). (B; top) Emission spectra from the cell in A by C excitation (blue trace) and Y excitation (yellow trace). Red trace is the standard C emission spectrum from C excitation. Green trace was obtained by subtracting the red trace from the blue trace. (Bottom) Ratio A and Ratio A0 (inset) are plotted against wavelength. (C) Apparent FRET efficiency (Eapp) of the C- and Y-tagged CLC0-TM against the C/Y intensity ratio. Each point represents the Eapp from a cell. (D) Apparent FRET efficiency of the C- and Y-tagged CLC0-TM with I225W/V490W mutations (red dots). Black dots were from a control experiment in which cells were cotransfected with WT CLC-0 and the subunit A2 of the rat olfactory CNG channel. Data points in C and D are fitted (solid curves) to Eqs. 3 and 4 of Bykova et al. (2006). In both C and D, significant FRET efficiency (∼18% for WT and ∼30% for I225W/V490W) was observed compared with the control experiment.
Mentions: The CLC-0 and CLC-1 cDNAs used for electrophysiological recordings were constructed in the pcDNA3 vector without any genetic tags of fluorescent proteins. For the constructs used in biochemical experiments, a “Flag” epitope was added to the N terminus (after the first methionine) of different CLC-0 constructs subcloned in the pcDNA3 vector. Mutations were made using the QuikChange site-directed mutagenesis kit (Agilent Technologies) and were confirmed by commercial DNA sequencing services. In the fluorescent resonant energy transfer (FRET) study used to evaluate the interaction of two channel subunits (Fig. 3), the C-terminal cytoplasmic domain of CLC-0 was removed after Ser530 to make a fair comparison with CLC-ec1 because the latter lacks a cytoplasmic domain. This C terminus–truncated mutant of CLC-0, referred to as CLC0-TM, has been described previously (Bykova et al., 2006). It was constructed in the pEGFP-N3 vector, and the C terminus of the CLC0-TM sequence was tagged by a Gly-Ser linker followed by the sequence of cerulean or eYFP. The I225W/V490W mutations of CLC-0 (corresponding to I201W/I422W mutations in CLC-ec1) were made in these two CLC0-TM constructs, and were used in the FRET study. The CNG channel used as the negative control in the FRET experiment was provided by W.N. Zagotta (University of Washington, Seattle, WA), and fluorescent proteins were also tagged to the C terminus of the CNG channel.

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.