Na, K-ATPase: Comparison of the cellular localization of Α-subunit mRNA and polypeptide in mouse cerebellum, retina, and kidney

Abstract

A clone encoding mouse brain Na, K-ATPase Α-subunit was isolated from a mouse brain lambda gt11 cDNA library by using antisera to mouse and bovine brain Α-subunit A comparison of the nucleotide sequence of this clone with published sequences of rat brain Α-subunit isoform clones showed it to be most similar to rat brain Α1. An RNA antisense probe prepared from the cDNA insert of the mouse clone detected a single mRNA of approximately 4.5 kb in Northern blots of mouse brain and kidney RNAs. This probe hybridized only to an Α1-cDNA insert from rat brain under high stringency conditions on Northern blots. The RNA antisense probe was used for in situ hybridization to sections of mouse kidney, cerebellum, and retina, and the cellular distribution of Α-subunit mRNA (Α-mRNA) was compared with that of Α-subunit polypeptide (Α-subunit) detected by immunofluorescence in similar sections. In kidney, Α-mRNA distribution closely paralleled that of the polypeptide with abundant expression in ascending thick limbs and cortical distal tubules and weaker labeling in cortical proximal tubules. The co-distribution of Α-mRNA and polypeptide in kidney where Na, K-ATPase localization is well established is consistent with the specificity of these probes. In the retina, prominent labeling with both probes was seen in photoreceptor inner segments, inner nuclear layer, and ganglion cell bodies. Plexiform layers and optic fibers expressed abundant Α-subunit but little mRNA. Light labeling for both was seen in the outer nuclear layer. In cerebellum, Α-mRNA and Α-subunit were associated with soma of granule cells, basket cells, and stellate cells. Glomeruli and basket terminals contained abundant Α-subunit but exhibited little reactivity with the riboprobe. In Purkinje cell bodies, in contrast, the antibody used to identify the cDNA clone did not resolve significant polypeptide in the somal plasmalemma despite abundant somal mRNA expression. Comparison of distribution of the two probes in cerebellum and retina indicates that message accumulation is primarily in cell bodies, while Α-subunit epitopes may be co-expressed in cell bodies and/or transported to distant sites in cell-specific patterns.