Targeted deletion of β1‐syntrophin causes a loss of K_ir4.1 from Müller cell endfeet in mouse retina

Abstract

Proper function of the retina depends heavily on a specialized form of retinal glia called Müller cells. These cells carry out important homeostatic functions that are contingent on their polarized nature. Specifically, the Müller cell endfeet that contact retinal microvessels and the corpus vitreum show a tenfold higher concentration of the inwardly rectifying potassium channel Kir4.1 than other Müller cell plasma membrane domains. This highly selective enrichment of Kir4.1 allows K+ to be siphoned through endfoot membranes in a special form of spatial buffering. Here, we show that Kir4.1 is enriched in endfoot membranes through an interaction with β1‐syntrophin. Targeted disruption of this syntrophin caused a loss of Kir4.1 from Müller cell endfeet without affecting the total level of Kir4.1 expression in the retina. Targeted disruption of α1‐syntrophin had no effect on Kir4.1 localization. Our findings show that the Kir4.1 aggregation that forms the basis for K+ siphoning depends on a specific syntrophin isoform that colocalizes with Kir4.1 in Müller endfoot membranes.

Functional specialization of retinal Müller cell endfeet depends on an interplay between two syntrophin isoforms

“…Here we exploit a newly generated transgenic mouse line [31] to investigate whether functional polarization of Müller cells depends on β1-syntrophin (β1-syn). We have previously used this mouse line to assess the role of β1-syn in anchoring of K ir 4.1 [32]. The present study shows that β1-syn is the most important anchor of AQP4 in Müller cell processes but that α1-syn may partly substitute as anchor if β1-syn is lost.…”

“…All the statistical analyses were carried out in SPSS (SPSS, Chicago, IL, USA). Sample sizes were determined based on previous studies [29,32]. However, same number of animals were used for all the experiments.…”

“…Detailed procedure for quantitative real-time PCR (qPCR) was performed as described in [32]. The primers used to detect Aqp4 were; Forward (5′): TTTGGACCCGCAGT TATCAT; Reverse (3′): GTTGTCCTCCACCTCCATGT.…”

“…The detailed procedure for western blot analysis can be found as described in [32]. Briefly, 20 μg protein from WT, α1-syn KO and β1-syn KO male mouse retinae (n = 5 for each genotype) was separated on 10% Criter-ion™ 18-well TGX gels (BioRad, Hercules, CA, USA) at 160 V and wet blotted to PVDF membranes (BioRad) at 100 V for 45 min.…”

Organisation of extracellular matrix proteins laminin and agrin in pericapillary basal laminae in mouse brain

“…Prior to staining, sections were thawed to room temperature and fixed using 2% formaldehyde for 15 min. Immunofluorescence experiments were performed as previously described (Rao et al 2019). Images of neocortex and cerebellum were acquired using LSM 710 confocal microscope at 20× magnification (Carl Zeiss).…”