Na,K-ATPase: comparison of the cellular localization of alpha-subunit mRNA and polypeptide in mouse cerebellum, retina, and kidney.
A clone encoding mouse brain Na,K-ATPase alpha-subunit was isolated from a mouse brain lambda gt11 cDNA library by using antisera to mouse and bovine brain alpha-subunit. A comparison of the nucleotide sequence of this clone with published sequences of rat brain alpha-subunit isoform clones showed it to be most similar to rat brain alpha 1. 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 alpha 1-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 alpha-subunit mRNA (alpha-mRNA) was compared with that of alpha-subunit polypeptide (alpha-subunit) detected by immunofluorescence in similar sections. In kidney, alpha-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 alpha-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 alpha-subunit but little mRNA. Light labeling for both was seen in the outer nuclear layer. In cerebellum, alpha-mRNA and alpha-subunit were associated with soma of granule cells, basket cells, and stellate cells. Glomeruli and basket terminals contained abundant alpha-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 alpha-subunit epitopes may be co-expressed in cell bodies and/or transported to distant sites in cell-specific patterns.
Hieber
V
V
Department of Neurology, University of Michigan Medical Center, Ann Arbor 48104-1687.
Siegel
G J
GJ
Desmond
T
T
Liu
J L
JL
Ernst
S A
SA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002531:Cerebellum / Q000166:cytology / Q000201:enzymology*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D015342:DNA Probes; D007668:Kidney / Q000166:cytology / Q000201:enzymology*; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009928:Organ Specificity; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*; D012160:Retina / Q000166:cytology / Q000201:enzymology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis / Q000235:genetics*
D015342:DNA Probes; D046911:Macromolecular Substances; D012333:RNA, Messenger; D004247:DNA; D000254:Sodium-Potassium-Exchanging ATPase