Expression and localization of the inwardly rectifying potassium channel Kir7.1 in native bovine retinal pigment epithelium.
OBJECTIVE
The purpose of this study was to identify the molecular basis of the apical membrane K(+) conductance in native bovine retinal pigment epithelium (RPE).


METHODS
RT-PCR, Northern blot, and Western blot analyses were used to detect the expression of the inwardly rectifying K(+) (Kir) channel subunits Kir7.1 and Kir4.1 in native bovine RPE and neural retina. The distribution of Kir7.1 protein was determined in frozen sections of bovine retina-RPE-choroid by indirect immunofluorescence analysis.


RESULTS
RT-PCR analysis revealed Kir7.1 transcript in both RPE and neural retina, but Kir4.1 transcript only in the neural retina. In Northern blot analysis, 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 approximately 1.5 kb in both RPE and neural retina, but with greater expression in RPE. In Western blot analysis, Kir7.1 antibody recognized a major monomer of approximately 53 kDa in RPE, whereas Kir4.1 antibody recognized a monomer of approximately 60 kDa in neural retina but not in RPE. 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 colocalized with Kir7.1.


CONCLUSIONS
These results indicate that the Kir7.1 channel subunit, but not Kir4.1, is a major component of the apical K(+) conductance in bovine RPE. Kir7.1 is distributed over the length of apical processes, where it probably functions in the regulation of K(+) transport and the electrical response of the RPE to light-evoked changes in subretinal K(+) concentration.
Yang
Dongli
D
Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor, Michigan 48015-0714, USA.
Pan
Aihua
A
Swaminathan
Anuradha
A
Kumar
Gyanendra
G
Hughes
Bret A
BA
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002417:Cattle; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D012160:Retina / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D014162:Transfection
D024661:Potassium Channels, Inwardly Rectifying; D012333:RNA, Messenger; D000254:Sodium-Potassium-Exchanging ATPase