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. 2019 Aug 1;317(2):C375-C389.
doi: 10.1152/ajpcell.00444.2018. Epub 2019 Jun 5.

Pressure-dependent modulation of inward-rectifying K+ channels: implications for cation homeostasis and K+ dynamics in glaucoma

Rachel A Fischer  1 , Abigail L Roux  2 , Lauren K Wareham  2 , Rebecca M Sappington  1   2   3
Affiliations

Pressure-dependent modulation of inward-rectifying K+ channels: implications for cation homeostasis and K+ dynamics in glaucoma

Rachel A Fischer et al. Am J Physiol Cell Physiol. .

Abstract

Glaucoma is the leading cause of blindness worldwide, resulting from degeneration of retinal ganglion cells (RGCs), which form the optic nerve. Prior to structural degeneration, RGCs exhibit physiological deficits. Müller glia provide homeostatic regulation of ions that supports RGC physiology through a process called K+ siphoning. Recent studies suggest that several retinal conditions, including glaucoma, involve changes in the expression of K+ channels in Müller glia. To clarify whether glaucoma-related stressors directly alter expression and function of K+ channels in Müller glia, we examined changes in the expression of inwardly rectifying K+ (Kir) channels and two-pore domain (K2P) channels in response to elevated intraocular pressure (IOP) in vivo and in vitro in primary cultures of Müller glia exposed to elevated hydrostatic pressure. We then measured outcomes of cell health, cation homeostasis, and cation flux in Müller glia cultures. Transcriptome analysis in a murine model of microbead-induced glaucoma revealed pressure-dependent downregulation of Kir and K2P channels in vivo. Changes in the expression and localization of Kir and K2P channels in response to elevated pressure were also found in Müller glia in vitro. Finally, we found that elevated pressure compromises the plasma membrane of Müller glia and induces cation dyshomeostasis that involves changes in ion flux through cation channels. Pressure-induced changes in cation flux precede both cation dyshomeostasis and membrane compromise. Our findings have implications for Müller glia responses to pressure-related conditions, i.e., glaucoma, and identify cation dyshomeostasis as a potential contributor to electrophysiological impairment observed in RGCs of glaucomatous retina.

Keywords: K channels; K siphoning; K2P; Kir; Müller glia; glaucoma; microbead; retina.

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Conflict of interest statement

No conflicts of interest, financial or otherwise, are declared by the authors.

Figures

Fig. 1.
Fig. 1.
Ocular hypertension alters RNA expression of K+ channels in vivo. RNA sequencing was performed on whole retina from saline- and microbead-injected eyes. A: box plots of fold change (log scale) of expression change between saline and microbead retinas from the Kcnj (left) and Kcnk (right) gene families. B: box plots of percentage of expression change between saline and microbead retinas from the Kcnj (left) and Kcnk (right) gene families. n(PBS) = 5 retinas; n(MOM) = 5 retinas. C: quantitative RT-PCR was performed on RNAs isolated from primary, purified cultures of Müller glia exposed to ambient or elevated pressure for 48 h. Bar graphs of mean threshold cycle (Ct) values normalized to Gapdh, compared between ambient and elevated pressure for Kcnk1 (left) and Kcnk3 (right). n = 3 for all. *P < 0.05. MOM, microbead occlusion model.
Fig. 2.
Fig. 2.
Intensity of inwardly rectifying K+ (Kir) channel staining in glaucomatous retinal sections. A: representative fluorescent micrographs of retinal sections from saline- or microbead-injected eyes. Immunolabeling of Kir2.1 (green), Kir4.1 (green), and CD44 (red) was performed. B: bar graphs of the mean intensity of Kir2.1 and Kir4.1 staining compared between saline and microbead retina. Kir2.1: n(PBS)= 7 retina sections; n(MOM)= 7; Kir4.1: n(PBS)= 6; n(MOM) = 7. Images taken at ×40; scale bar = 20 µm. Student’s t-test was used to analyze statistical significance. *P < 0.05, **P < 0.005. MOM, microbead occlusion model.
Fig. 3.
Fig. 3.
Intensity of inwardly rectifying K+ (Kir) channel staining in Müller glia cultures following pressure elevation. Primary, purified cultures of Müller glia were exposed to ambient (Amb) or elevated (Elev) pressure for 48 h. A: representative fluorescent micrographs of Müller glia from each condition. Immunolabeling of Kir2.1 (green), Kir4.1 (green), and CD44 (red) was performed. Panels directly below the fluoromicrographs are a z-plane view of the image, with associated plot of fluorescent intensity. B: bar graphs of the mean intensity of Kir2.1 and Kir4.1 staining compared between ambient and elevated pressure. Kir2.1: n(Amb) = 29 cells; n(Elev) = 18; Kir4.1: n(Amb) = 19; n(Elev) = 20. Images taken at ×60; scale bar = 40 µm. Inset taken at ×60 + ×2.5 zoom; scale bar = 10 µm. Student’s t-test was used to analyze statistical significance. **P < 0.005.
Fig. 4.
Fig. 4.
Intensity of TWIK-1 and TASK-1 channel staining in glaucomatous retinal sections. A: representative fluorescent micrographs of retinal sections from saline- or microbead-injected eyes. Immunolabeling of TWIK-1 (green), TASK-1 (green), and CD44 (red) was performed. B: bar graphs of the mean intensity of TWIK-1 and TASK-1 staining compared between saline and microbead retina. TWIK-1: n(PBS) = 6 retina sections; n(MOM) = 7; TASK-1: n(PBS) = 7; n(MOM) = 7. Images taken at ×40; scale bar = 20 µm. Student’s t-test was used to analyze statistical significance. **P < 0.005. MOM, microbead occlusion model.
Fig. 5.
Fig. 5.
Intensity of TWIK-1 and TASK-1 channel staining in Müller glia cultures following pressure elevation. Primary, purified cultures of Müller glia were exposed to ambient (Amb) or elevated (Elev) pressure for 48 h. A: representative fluorescent micrographs of Müller glia from each condition. Immunolabeling of TWIK-1 (green), TASK-1 (green), and CD44 (red) was performed. Panels directly below the fluoromicrographs are a z-plane view of the image, with associated plot of fluorescent intensity. B: bar graphs of the mean intensity of TWIK-1 and TASK-1 staining compared between ambient and elevated pressure. TWIK-1: n(Amb) = 22 cells; n(Elev) = 13; TASK-1: n(Amb) = 21; n(Elev) = 22. Images taken at ×60; scale bar = 40 µm. Inset taken at ×60 + ×2.5 zoom; scale bar = 10 µm. Student’s t-test was used to analyze statistical significance. **P < 0.005.
Fig. 6.
Fig. 6.
Intensity of TREK-2, TRAAK, and TRESK channel staining in glaucomatous retinal sections. A: representative fluorescent micrographs of retinal sections from saline- or microbead-injected eyes. Immunolabeling of TREK-2 (green), TRAAK (green), TRESK (green), and CD44 (red) was performed. B: bar graphs of the mean intensity of TREK-2, TRAAK, and TRESK staining compared between saline and microbead retina. TREK-2: n(PBS) = 7 retina sections; n(MOM) = 7; TRAAK: n(PBS)= 6; n(MOM)= 7; TRESK: n(PBS) = 6; n(MOM) = 6. Images taken at ×40; scale bar = 20 µm. Student’s t-test was used to analyze statistical significance. *P < 0.05. MOM, microbead occlusion model.
Fig. 7.
Fig. 7.
Intensity of TREK-2, TRAAK, and TRESK channel staining in Müller glia cultures following pressure elevation. Primary, purified cultures of Müller glia were exposed to ambient (Amb) or elevated (Elev) pressure for 48 h. A: representative fluorescent micrographs of Müller glia from each condition. Immunolabeling of TREK-2 (green), TRAAK (green), TRESK (green), and CD44 (red) was performed. Panels directly below the fluoromicrographs are a z-plane view of the image, with associated plot of fluorescent intensity. B: bar graphs of the mean intensity of TREK-2, TRAAK, and TRESK staining compared between ambient and elevated pressure. TREK-2: n(Amb) = 24 cells; n(Elev) =21; TRAAK, n(Amb) = 19; n(Elev) = 21; TRESK: n(Amb) = 12; n(Elev) = 22. Images taken at ×60; scale bar = 40 µm. Inset taken at ×60 + ×2.5 zoom; scale bar = 10 µm. Student’s t-test was used to analyze statistical significance. *P < 0.05, **P < 0.005.
Fig. 8.
Fig. 8.
Effect of short- and long-term pressure elevation on Müller glia cell health. Primary, purified cultures of Müller glia were exposed to ambient (Amb) or elevated Elev) pressure for 48 h. A: representative DIC micrographs of Müller glia from each condition. B: bar graphs of the percentage of TUNEL-positive Müller glia compared between each condition. n = 4 wells per condition (combined 5 images at ×20/well). C: bar graphs of the amount of lactate dehydrogenase (LDH) released in the Müller glia culture media compared between each condition. n(Amb 4 h) = 5 wells; n(Elev 4 h) = 5; n(Amb 48 h) = 15; n(Elev 48 h) = 17. Student’s t-test was used to analyze statistical significance. **P < 0.001.
Fig. 9.
Fig. 9.
Elevated pressure alters Na+ and K+ homeostasis in Müller glia. Primary, purified cultures of Müller glia were exposed to ambient (Amb) or elevated (Elev) pressure for 4 or 48 h. A: box plots of the concentration of K+ ions in the culture media of Müller glia at each condition. One-way ANOVA with pairwise comparison by Tukey’s method was used to analyze statistical significance. B: box plots of the concentration of Na+ ions in the culture media of Müller glia compared between each condition. One-way ANOVA with pairwise comparison by Dunn’s method was used to analyze statistical significance. n(Amb 4 h) = 5 wells; n(Elev 4 h) = 4; n(Amb 48 h) = 12; n(Elev 48 h) = 12. **P < 0.001.
Fig. 10.
Fig. 10.
Cation channel activity in Müller glia exposed to elevated pressure. Primary, purified cultures of Müller glia were exposed to ambient (Amb) or elevated (Elev) pressure for 4 or 48 h. A: representative heat map showing the fluorescent signal of Thallos dye in Müller glia from each condition. Images were taken after the addition of thallium. B: line graphs displaying the normalized fluorescence intensity of Thallos dyes over time for each condition. Student’s t-test was used to analyze statistical significance. C: box plots of the average fluorescence intensity following addition of thallium compared between each condition. One-way ANOVA with pairwise comparison by Dunn’s method was used to analyze statistical significance. n(Amb 4 h) = 5 cells; n(Elev 4 h) = 12; n(Amb 48 h) = 5; n(Elev 48 h) = 18. *P < 0.05.
Fig. 11.
Fig. 11.
Inhibition of cation channel activity in Müller glia by fluoxetine (Fluox) treatment. Primary, purified cultures of Müller glia were exposed to ambient (Amb) or elevated (Elev) pressure for 4 h, in the presence of vehicle or 100 µM fluoxetine. A: representative heat map showing the fluorescent signal of Thallos dye in Müller glia from each condition. Images were taken after the addition of thallium. B: line graphs displaying the normalized fluorescence intensity of Thallos dyes over time for each condition. Student’s t-test was used to analyze statistical significance. C: box plots of the average fluorescence intensity following the addition of thallium compared between each condition. One-way ANOVA with pairwise comparison by Dunn’s method was used to analyze statistical significance. n(Amb) = 5 cells; n(Elev) = 12; n(Amb+Fluox) = 28; n(Elev+Fluox) = 22. *P < 0.05.
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References

    1. Bolz S, Schuettauf F, Fries JE, Thaler S, Reichenbach A, Pannicke T. K+ currents fail to change in reactive retinal glial cells in a mouse model of glaucoma. Graefes Arch Clin Exp Ophthalmol 246: 1249–1254, 2008. doi: 10.1007/s00417-008-0872-x. - DOI - PubMed
    1. Bringmann A, Francke M, Pannicke T, Biedermann B, Kodal H, Faude F, Reichelt W, Reichenbach A. Role of glial K+ channels in ontogeny and gliosis: a hypothesis based upon studies on Müller cells. Glia 29: 35–44, 2000. doi: 10.1002/(SICI)1098-1136(20000101)29:1<35:AID-GLIA4>3.0.CO;2-A. - DOI - PubMed
    1. Butt AM, Kalsi A. Inwardly rectifying potassium channels (Kir) in central nervous system glia: a special role for Kir4.1 in glial functions. J Cell Mol Med 10: 33–44, 2006. doi: 10.1111/j.1582-4934.2006.tb00289.x. - DOI - PMC - PubMed
    1. Calkins DJ. Critical pathogenic events underlying progression of neurodegeneration in glaucoma. Prog Retin Eye Res 31: 702–719, 2012. doi: 10.1016/j.preteyeres.2012.07.001. - DOI - PMC - PubMed
    1. Chatelain FC, Bichet D, Douguet D, Feliciangeli S, Bendahhou S, Reichold M, Warth R, Barhanin J, Lesage F. TWIK1, a unique background channel with variable ion selectivity. Proc Natl Acad Sci USA 109: 5499–5504, 2012. doi: 10.1073/pnas.1201132109. - DOI - PMC - PubMed

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