Molecular characterization in isolated Müller glial cells of the functional complexes of Dp71 and/or utrophin with Kir4.1 and AQP4.

Abstract

Abstract: : Purpose: To characterize the dystrophin associated proteins (DAPs) complexes associated to Dp71 and utrophin, two members of the dystrophin superfamily. These proteins are the core of a macromolecular complex implicated in retinal homeostasis by stabilizing and clustering the potassium channel Kir4.1 and the aqueous channel AQP4. Methods: The localization of dystrophins, utrophin , dystrophin associated proteins (DAPs), Kir4.1 and AQP4 were determined by immunocytochemistry methods on freshly dissociated Müller cells of wild–type and Dp71–null mice strains with specific antibodies. Double staining were done with glutamine synthetase, a specific Müller cell marker, in order to check the cell morphology and the sub–cellular localization of the DAPs. All fluorescence specimens were viewed by using a confocal microscope. Results: We showed that Dp71 was preferentially expressed in the Müller cell endfeet whereas utrophin was not only in endfeet but also all along the cell. We also observed a differential sub–cellular localization of dystroglycan, syntrophins, dystrobrevins and sarcoglycans. Finally we showed that the absence of Dp71 induced the delocalization of Kir4.1, AQP4 and some of the members of the complex. Conclusions: This work clearly show that there is two distinct complexes that involve Dp71 or utrophin. The DAPs associated are different and exclusively the complex associated to Dp71 is implicated in the localization of Kir4.1 and AQP4.

Keywords: Muller cells • ion channels • microscopy: confocal/tunneling