Expression,Localization And Development Of Kir Channels In Retina

Purpose: The purpose of this study was to detect expression and localization of kir2.1 and kir4.1 in bovine retina, identify the molecular basis of the apical membrane K+ conductance in native bovine retinal pigment epithelium (RPE), study localization of Kir channels in different species and development of Kir channels in murine retinae.Methods. RT-PCR, Northern blot, and Western blot analyses were used to detect the expression of the inwardly rectifying K+ (Kir) channel subunits Kir2.1, Kir4.1 and Kir7.1 in native bovine RPE and neural retina. The distribution of Kir2.1, Kir4.1 and Kir7.1 protein was determined in frozen sections of bovine, monkey, human and developing murine retinae by immunofluorescence and ABC immunohistochemistry.Results. RT-PCR analysis revealed Kir7.1 transcript in both RPE and neural retina, but Kir2.1and Kir4.1 transcript in the neural retina alone. In Northern blots, Kir2.1 and Kir4.1 probe hybridized to an appropriately sized-transcript in neural retina but not in RPE. Kir7.1 probe hybridized to a major transcript of~1.5 Kb in both RPE and neural retina, but with greater expression in RPE. In Western blots, Kir7.1 antibody recognized a major monomer of ~53KDa in RPE, while Kir2.1and Kir4.1 antibody recognized a monomer of~60 KDa in neural retina but not in RPE. Immunofluorescence revealed Kir2.1 and Kir4.1 immullostaining was expressed at many parts of muller cells, as Kir2.1especially in the membrane domains of muller cells that contact retinal neurons, ie, along the two stem processes over the soma, and in the side branches extending into the synaptil layers. No immunostaining was seen in RPE. Double staining that Kir2.1 and glutamine synthetase, Kir2.1 and glutamine synthetase colocalized well. Intense Kir7.1 immunolabeling was present on the apical surface of all RPE cells and appeared to extend over the length of the apical processes. Na+, K+ -ATPase expression varied among RPE cells, but in highly expressing cells, it co-localized with Kir7.1. Immunoreactivity of Kir2.1?Kir4.1 and Kir7.1 acomplished by ABC immunohistochemistry in monkey and human retinae got the nearly same results as bovine . Expression of Kir2.1 and Kir4.1 channel subunits in retinae got nearly same pattern as adults at P16 murine retinae, for Kir7.1, nearly same property as adults at P7 murine RPE.Conclusions. These results indicate that Kir2.1 and Kir4.1 channel subunits, nearly same property as adults at P16 murine retinae, is in muller cells , not in RPE. There is no difference between species. The Kir7.1 channel subunit, nearly same property as adults at P7 murine RPE, is a major component of the apical K+ conductance in bovine RPE. Kir7.1 is distributed over the length of apical processes, where it likely functions in the regulation of K+ transport and the electrical response of the RPE to light-evoked changes in subretinal K+ concentration.