Kir4.1 and AQP4 associate with Dp71‐ and utrophin‐DAPs complexes in specific and defined microdomains of Müller retinal glial cell membrane

Abstract

The dystrophin-associated proteins (DAPs) complex consisting of dystroglycan, syntrophin, dystrobrevin, and sarcoglycans in muscle cells is associated either with dystrophin or its homolog utrophin. In rat retina, a similar complex was found associated with dystrophin-Dp71 that serves as an anchor for the inwardly rectifying potassium channel Kir4.1 and the aqueous pore, aquaporin-4 (AQP4). Here, using immunofluorescence imaging of isolated retinal Müller glial cells and co-immunoprecipitation experiments performed on an enriched Müller glial cells end-feet fraction, we investigated the effect of Dp71 deletion on the composition, anchoring, and membrane localization of the DAPs-Kir4.1 and/or -AQP4 complex. Two distinct complexes were identified in the end-feet fraction associated either with Dp71 or with utrophin. Upon Dp71 deletion, the corresponding DAPs complex was disrupted and a compensating utrophin upregulation was observed, accompanied by diffuse overall staining of Kir4.1 along the Müller glial cells and redistribution of the K(+) conductance. Dp71 deficiency was also associated with a marked reduction of AQP4 and beta-dystroglycan expression. Furthermore, it was observed that the Dp71-DAPs dependent complex could be, at least partially, associated with a specific membrane fraction. These results demonstrate that Dp71 has a central role in the molecular scaffold responsible for anchoring AQP4 and Kir4.1 in Müller cell end-feet membranes. They also show that despite its close relationship to the dystrophin proteins and its correlated upregulation, utrophin is only partially compensating for the absence of Dp71 in Müller glial cells.

Glial dystrophin-associated proteins, laminin and agrin, are downregulated in the brain of mdx mouse

“…54 Moreover, we have observed that even if AQP4 and Kir 4.1 reduced their protein content, they maintained a polarized expression on the perivascular glial endfeet, suggesting that a similar molecular mechanism is involved in membrane clustering for both proteins. Otherwise, in the retina of Dp71-null mice, AQP4 expression was reduced, whereas Kir 4.1 expression was unaffected, 11 and the deletion of a-syntrophin gene caused AQP4 reduction of AQP4 in Muller cells, but not of Kir 4.1. 55 These discrepancies could be explained by the fact that, in the mdx brain, 427 full-length dystrophin alone was completely lost, whereas the other isoforms, including Dp71 and syntrophin, were decreased, but preserved their link with AQP4 and Kir 4.1.…”

“…They express in a polarized way, on their perivascular endfeet, carrier membranes and channel proteins, including aquaporin-4 (AQP4) water channel and potassium channel Kir 4.1, which are responsible for the water flow rate and spatial K þ buffering control. [4][5][6][7][8][9] In the retina, AQP4 colocalizes with Kir 4.1, [9][10][11] suggesting a tight cooperation between the water flux and K þ siphoning generated by neuronal activity at the BBB interfaces. Moreover, AQP4 and Kir 4.1 rearrangement has been described in BBB alterations, coupled with cytotoxic edema and K þ -delayed buffering capacity.…”

Interdependence of Laminin-mediated Clustering of Lipid Rafts and the Dystrophin Complex in Astrocytes

“…Although such a distribution is functionally important in potassium ion and water homeostasis, the mechanisms regulating the formation of these Kir4.1-and AQP4-enriched domains remain to be elucidated. AQP4 is associated with lipid raft-containing fractions in both brain and retina (27,35) as is another aquaporin, AQP5, that participates in fluid secretion in parotid gland cells (36). We therefore undertook a study to evaluate the role of lipid rafts in laminin-DG-dependent AQP4 clustering, and our data show that laminin induced a dramatic redistribution of GM1 into large cell surface clusters or macrodomains that colocalized extensively with ␤-DG and AQP4.…”

HSF1 functions as a transcription regulator for Dp71 expression

“…The cellular processes HSF1 regulates in cancer including energy metabolism, cell cycle signaling, DNA repair, apoptosis, cell adhesion, and extracellular matrix (Marc et al 2012). As the most ubiquitously expressed isoforms of dystrophin, Dp71 participates in various cellular processes, including cell adhesion, water homeostasis, and cell division in PC12 cells (Fort et al 2008;Acosta et al 2004). Compelling evidence supporting Dp71 and DAPC components in the nucleus suggests its possible biological function via the continuous network linking the plasma membrane to the nuclear envelope (Villarreal-Silva et al 2011).…”