EXPRESSION AND SYNTHESIS OF THE NA,K-ATPASE BETA-2 SUBUNIT IN HUMAN RETINAL-PIGMENT EPITHELIUM

Na,K-ATPase in the retinal pigment epithelium (RPE) is apically locali zed, whereas in most other tissues this pump is found predominantly in the basolateral membrane domain. As part of our investigations into t he molecular aspects of this pump in the RPE, we have cloned the cDNA and characterized the expression of the gene encoding the beta 2 subun it isoform of Na,K-ATPase in human, rat and bovine RPE and in the bovi ne choroid plexus. We have also detected the beta 2 isoform polypeptid e in the human RPE (hRPE). Comparison of complete coding sequences der ived from cloned cDNAs revealed that all beta 2 sequences from RPE, an d the choroid plexus, differed uniformly at positions: P-51/L, M(121)/ I, and L(148)/R from the published sequences for human retina and live r. However, analysis of 10 RT-PCR clones derived from 5 fetal and 2 ad ult human retinas sequenced in our laboratory, revealed that only the P-51/L residue was different with the hRPE beta 2 subunit sequence. No rthern blot analysis indicated a 3.4-kb RNA transcript for the beta 2 subunit, a 4.5-kb RNA for the alpha 1 subunit and a doublet of 2.3 and 2.6 kb for the beta 1 subunit, respectively. alpha 1 (100 kDa), beta 1 (45 kDa) and beta 2 (65 kDa) isoforms were detected in hRPE extracts by immunoblotting. No alpha 2 and alpha 3 RNA transcripts were found in the hRPE. Quantification of beta 2 mRNA by RT-PCR revealed 2.7 x 10 (5) molecules per ng of poly A(+) RNA. This is similar to the beta 1 i soform levels reported previously from our laboratory. These data demo nstrate the coexistence of significant amounts of alpha 1, beta 1 and beta 2 Na,K-ATPase subunits in the RPE. It is therefore reasonable to suggest that both alpha 1 beta 1 and alpha 1 beta 2 heterodimers are p resent in these cells.