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Expression and localization of CLC chloride transport proteins in the avian retina.

McMains E, Krishnan V, Prasad S, Gleason E - PLoS ONE (2011)

Bottom Line: Furthermore, knock-out of ClC3 and ClC7 in the mouse result in the degeneration of the hippocampus and the retina.Previous work in our lab has shown that in chicken amacrine cells, internal Cl(-) can be dynamic.We observed a high level of variation in the retinal expression levels and patterns among the different CLC proteins examined.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana, United States of America.

ABSTRACT
Members of the ubiquitously expressed CLC protein family of chloride channels and transporters play important roles in regulating cellular chloride and pH. The CLCs that function as Cl(-)/H(+) antiporters, ClCs 3-7, are essential in particular for the acidification of endosomal compartments and protein degradation. These proteins are broadly expressed in the nervous system, and mutations that disrupt their expression are responsible for several human genetic diseases. Furthermore, knock-out of ClC3 and ClC7 in the mouse result in the degeneration of the hippocampus and the retina. Despite this evidence of their importance in retinal function, the expression patterns of different CLC transporters in different retinal cell types are as yet undescribed. Previous work in our lab has shown that in chicken amacrine cells, internal Cl(-) can be dynamic. To determine whether CLCs have the potential to participate, we used PCR and immunohistochemical techniques to examine CLC transporter expression in the chicken retina. We observed a high level of variation in the retinal expression levels and patterns among the different CLC proteins examined. These findings, which represent the first systematic investigation of CLC transporter expression in the retina, support diverse functions for the different CLCs in this tissue.

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CLC H+/Cl− antiporters have diverse expression patterns in mature chicken retina.A–F, Images of vertical sections of chicken retina. Images were acquired and processed identically except for F, which was acquired at a lower PMT gain to avoid saturation. A, Secondary antibody only-labeled section shows minimal background fluorescence. Signal in photoreceptor outer segments and in oil droplets is autofluorescence. B, Labeling of retinal tissue by ClC3 antibody is barely detectable. C, The ClC4 antibody labels all three nuclear layers of the retina and the IPL. D, ClC5 antibody labeling in nuclear and synaptic layers. Arrow indicates ClC5 expression in the OLM. E, The ClC6 antibody labeled vertical elements in photoreceptors (arrowheads). F, Punctate ClC7 signal in nuclear and synaptic layers. Asterisks and yellow arrows indicate ClC7+ cells at INL/IPL border. White arrow indicates INL band lacking ClC7. G–L, Zoom of CLC antibody labeling in the OPL from the same sections shown in A–F. Images have been individually adjusted to highlight immunoreactive features. ClC5 expression is widespread and diffuse and in scattered processes (arrowheads) whereas ClC6 antibodies label discrete puncta. ClC4 and ClC7 antibody signals are fainter, and ClC3 signal is absent from the OPL. M–R, Zoomed images of anti-CLC labeling in the IPL. Images have been individually adjusted. IPL labeling by ClC3 antibody was minimal. ClC4 antibody labels horizontal processes in outer IPL (arrows) and three distinct bands (1–3). ClC5 is expressed in vertical processes projecting to central IPL (arrows) and four (1–4) to five (arrowhead) bands. Three bands of ClC6 immunoreactivity (1–3) are distinguishable. ClC7 antibody labels one band in outer IPL (1) and one band in inner IPL (2). 3 indicates a thin IPL band lacking ClC7. (Scale bar in A is 50 µm and applies to B–F. Scale bar in G is 5 µm and applies to H–L. Scale bar in M is 50 µm and applies to N–R.)
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pone-0017647-g003: CLC H+/Cl− antiporters have diverse expression patterns in mature chicken retina.A–F, Images of vertical sections of chicken retina. Images were acquired and processed identically except for F, which was acquired at a lower PMT gain to avoid saturation. A, Secondary antibody only-labeled section shows minimal background fluorescence. Signal in photoreceptor outer segments and in oil droplets is autofluorescence. B, Labeling of retinal tissue by ClC3 antibody is barely detectable. C, The ClC4 antibody labels all three nuclear layers of the retina and the IPL. D, ClC5 antibody labeling in nuclear and synaptic layers. Arrow indicates ClC5 expression in the OLM. E, The ClC6 antibody labeled vertical elements in photoreceptors (arrowheads). F, Punctate ClC7 signal in nuclear and synaptic layers. Asterisks and yellow arrows indicate ClC7+ cells at INL/IPL border. White arrow indicates INL band lacking ClC7. G–L, Zoom of CLC antibody labeling in the OPL from the same sections shown in A–F. Images have been individually adjusted to highlight immunoreactive features. ClC5 expression is widespread and diffuse and in scattered processes (arrowheads) whereas ClC6 antibodies label discrete puncta. ClC4 and ClC7 antibody signals are fainter, and ClC3 signal is absent from the OPL. M–R, Zoomed images of anti-CLC labeling in the IPL. Images have been individually adjusted. IPL labeling by ClC3 antibody was minimal. ClC4 antibody labels horizontal processes in outer IPL (arrows) and three distinct bands (1–3). ClC5 is expressed in vertical processes projecting to central IPL (arrows) and four (1–4) to five (arrowhead) bands. Three bands of ClC6 immunoreactivity (1–3) are distinguishable. ClC7 antibody labels one band in outer IPL (1) and one band in inner IPL (2). 3 indicates a thin IPL band lacking ClC7. (Scale bar in A is 50 µm and applies to B–F. Scale bar in G is 5 µm and applies to H–L. Scale bar in M is 50 µm and applies to N–R.)

Mentions: To determine the specificity of the CLC antibodies for CLC proteins in intact chicken retinal tissue, we labeled vertical sections of retina with CLC antibodies that had been pre-incubated with their respective antigenic peptides. Figure 2 shows that, in comparison to sections not exposed to immunogenic peptide, pre-incubation with antigenic peptide greatly reduced the labeling intensity of antibodies against ClC4–7. An assessment of ClC3 antibody labeling block by ClC3 peptide was not done because of the very low labeling intensity of ClC3 antibody in the chicken retina (see Fig. 3B). It is important to note that in the case of ClC5, pre-incubation with antigenic peptide did not reduce antibody labeling of ganglion cells as much as in other retinal cell types (Fig. 2B right, arrowheads) suggesting that some of this labeling might be nonspecific. Interpretation of ClC5 antibody labeling results in ganglion cells must therefore be made with caution.


Expression and localization of CLC chloride transport proteins in the avian retina.

McMains E, Krishnan V, Prasad S, Gleason E - PLoS ONE (2011)

CLC H+/Cl− antiporters have diverse expression patterns in mature chicken retina.A–F, Images of vertical sections of chicken retina. Images were acquired and processed identically except for F, which was acquired at a lower PMT gain to avoid saturation. A, Secondary antibody only-labeled section shows minimal background fluorescence. Signal in photoreceptor outer segments and in oil droplets is autofluorescence. B, Labeling of retinal tissue by ClC3 antibody is barely detectable. C, The ClC4 antibody labels all three nuclear layers of the retina and the IPL. D, ClC5 antibody labeling in nuclear and synaptic layers. Arrow indicates ClC5 expression in the OLM. E, The ClC6 antibody labeled vertical elements in photoreceptors (arrowheads). F, Punctate ClC7 signal in nuclear and synaptic layers. Asterisks and yellow arrows indicate ClC7+ cells at INL/IPL border. White arrow indicates INL band lacking ClC7. G–L, Zoom of CLC antibody labeling in the OPL from the same sections shown in A–F. Images have been individually adjusted to highlight immunoreactive features. ClC5 expression is widespread and diffuse and in scattered processes (arrowheads) whereas ClC6 antibodies label discrete puncta. ClC4 and ClC7 antibody signals are fainter, and ClC3 signal is absent from the OPL. M–R, Zoomed images of anti-CLC labeling in the IPL. Images have been individually adjusted. IPL labeling by ClC3 antibody was minimal. ClC4 antibody labels horizontal processes in outer IPL (arrows) and three distinct bands (1–3). ClC5 is expressed in vertical processes projecting to central IPL (arrows) and four (1–4) to five (arrowhead) bands. Three bands of ClC6 immunoreactivity (1–3) are distinguishable. ClC7 antibody labels one band in outer IPL (1) and one band in inner IPL (2). 3 indicates a thin IPL band lacking ClC7. (Scale bar in A is 50 µm and applies to B–F. Scale bar in G is 5 µm and applies to H–L. Scale bar in M is 50 µm and applies to N–R.)
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3049779&req=5

pone-0017647-g003: CLC H+/Cl− antiporters have diverse expression patterns in mature chicken retina.A–F, Images of vertical sections of chicken retina. Images were acquired and processed identically except for F, which was acquired at a lower PMT gain to avoid saturation. A, Secondary antibody only-labeled section shows minimal background fluorescence. Signal in photoreceptor outer segments and in oil droplets is autofluorescence. B, Labeling of retinal tissue by ClC3 antibody is barely detectable. C, The ClC4 antibody labels all three nuclear layers of the retina and the IPL. D, ClC5 antibody labeling in nuclear and synaptic layers. Arrow indicates ClC5 expression in the OLM. E, The ClC6 antibody labeled vertical elements in photoreceptors (arrowheads). F, Punctate ClC7 signal in nuclear and synaptic layers. Asterisks and yellow arrows indicate ClC7+ cells at INL/IPL border. White arrow indicates INL band lacking ClC7. G–L, Zoom of CLC antibody labeling in the OPL from the same sections shown in A–F. Images have been individually adjusted to highlight immunoreactive features. ClC5 expression is widespread and diffuse and in scattered processes (arrowheads) whereas ClC6 antibodies label discrete puncta. ClC4 and ClC7 antibody signals are fainter, and ClC3 signal is absent from the OPL. M–R, Zoomed images of anti-CLC labeling in the IPL. Images have been individually adjusted. IPL labeling by ClC3 antibody was minimal. ClC4 antibody labels horizontal processes in outer IPL (arrows) and three distinct bands (1–3). ClC5 is expressed in vertical processes projecting to central IPL (arrows) and four (1–4) to five (arrowhead) bands. Three bands of ClC6 immunoreactivity (1–3) are distinguishable. ClC7 antibody labels one band in outer IPL (1) and one band in inner IPL (2). 3 indicates a thin IPL band lacking ClC7. (Scale bar in A is 50 µm and applies to B–F. Scale bar in G is 5 µm and applies to H–L. Scale bar in M is 50 µm and applies to N–R.)
Mentions: To determine the specificity of the CLC antibodies for CLC proteins in intact chicken retinal tissue, we labeled vertical sections of retina with CLC antibodies that had been pre-incubated with their respective antigenic peptides. Figure 2 shows that, in comparison to sections not exposed to immunogenic peptide, pre-incubation with antigenic peptide greatly reduced the labeling intensity of antibodies against ClC4–7. An assessment of ClC3 antibody labeling block by ClC3 peptide was not done because of the very low labeling intensity of ClC3 antibody in the chicken retina (see Fig. 3B). It is important to note that in the case of ClC5, pre-incubation with antigenic peptide did not reduce antibody labeling of ganglion cells as much as in other retinal cell types (Fig. 2B right, arrowheads) suggesting that some of this labeling might be nonspecific. Interpretation of ClC5 antibody labeling results in ganglion cells must therefore be made with caution.

Bottom Line: Furthermore, knock-out of ClC3 and ClC7 in the mouse result in the degeneration of the hippocampus and the retina.Previous work in our lab has shown that in chicken amacrine cells, internal Cl(-) can be dynamic.We observed a high level of variation in the retinal expression levels and patterns among the different CLC proteins examined.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana, United States of America.

ABSTRACT
Members of the ubiquitously expressed CLC protein family of chloride channels and transporters play important roles in regulating cellular chloride and pH. The CLCs that function as Cl(-)/H(+) antiporters, ClCs 3-7, are essential in particular for the acidification of endosomal compartments and protein degradation. These proteins are broadly expressed in the nervous system, and mutations that disrupt their expression are responsible for several human genetic diseases. Furthermore, knock-out of ClC3 and ClC7 in the mouse result in the degeneration of the hippocampus and the retina. Despite this evidence of their importance in retinal function, the expression patterns of different CLC transporters in different retinal cell types are as yet undescribed. Previous work in our lab has shown that in chicken amacrine cells, internal Cl(-) can be dynamic. To determine whether CLCs have the potential to participate, we used PCR and immunohistochemical techniques to examine CLC transporter expression in the chicken retina. We observed a high level of variation in the retinal expression levels and patterns among the different CLC proteins examined. These findings, which represent the first systematic investigation of CLC transporter expression in the retina, support diverse functions for the different CLCs in this tissue.

Show MeSH
Related in: MedlinePlus