Distribution and function of polycystin-2 in mouse retinal ganglion cells

Abstract

The polycystin family of transient receptor potential (TRP) channels form Ca2+ regulated cation channels with distinct subcellullar localizations and functions. As part of heteromultimeric channels and multi-protein complexes, polycystins control intracellular Ca2+ signals and more generally the translation of extracellular signals and stimuli to intracellular responses. Polycystin-2 channels have been cloned from retina, but their distribution and function in retinal ganglion cells (RGCs) have not yet been established. In the present study, we determined cellular and subcellular localization as well as functional properties of polycystin-2 channels in RGCs. Polycystin-2 expression and distribution in RGCs was assessed by immunohistochemistry on vertical cryostat section of mouse retina as well as primary cultured mouse RGCs, using fluorescence microscopy. Biophysical and pharmacological properties of polycystin-2 channels isolated from primary cultured RGCs were determined using planar lipid bilayer electrophysiology. We detected polycystin-2 immunoreactivity both in the ganglion cell layer as well as in primary cultured RGCs. Subcellular analysis revealed strong cytosolic localization pattern of polycystin-2. Polycystin-2 channel current was Ca2+ activated, had a maximum slope conductance of 114 pS and could be blocked in a dose-dependent manner by increasing concentrations of Mg2+. The cytosolic localization of polycystin-2 in RGCs is in accordance with its function as intracellular Ca2+ release channel. We conclude that polycystin-2 forms functional channels in RGCs, of which biophysical and pharmacological properties are similar to polycystin-2 channels reported for other tissues and organisms. Our data suggest a potential role for polycystin-2 in RGC Ca2+ signaling.

Differential up-regulation of Vesl-1/Homer 1 protein isoforms associated with decline in visual performance in a preclinical glaucoma model

“…Furthermore, this effect generated by elevated Vesl-1L/Homer 1c levels is potentially exacerbated by increased release of Ca 2+ from intracellular stores following cellular oxidative stress (Andersson, et al, 2011, Kaja, et al, 2011, Lencesova & Krizanova, 2012, Smith, et al, 2005), as occurs during glaucoma and related neurodegenerative pathologies (Chrysostomou et al, 2012, Droge & Schipper, 2007, Smaili, et al, 2009), and likely amplified through polycystin-2 intracellular Ca 2+ release channels present on the ER in the mouse retina (Kaja et al, 2012a). …”

“…Immunhistochemistry was performed essentially as described previously (Kaja, et al, 2012a). Eyecups were fixed in 4% paraformaldehyde in PBS for 15 min, cryprotected in a graded series of sucrose (10–30% in PBS), and sectioned at 16 μm on a CryoCut One cryostat (Vibratome, St. Louis, MO).…”

Plate reader-based cell viability assays for glioprotection using primary rat optic nerve head astrocytes

“…The procedure was carried out essentially as described previously (Kaja et al, 2011; Kaja et al, 2012; Kaja et al, 2015a; Kaja et al, 2015b). Briefly, ONHAs were seeded onto 12 mm round poly-L-lysine coated glass coverslips (BD Biosciences, San Jose, CA) in a 24-well plate at a density of 7,500 cells.…”

Differential subcellular Ca2+ signaling in a highly specialized subpopulation of astrocytes

“…Immunocytochemistry was performed essentially as described previously (Kaja, et al, 2011, Kaja, et al, 2011, Kaja, et al, 2012). Briefly, ONHAs were seeded onto poly-L-lysine coated glass coverslips (BD Biosciences, San Jose, CA) at a density of 25,000 cells.…”