Transcriptomic changes triggered by ouabain in rat cerebellum granule cells: Role of α3- and α1-Na+,K+-ATPase-mediated signaling.

Larisa V Smolyaninova,Aleksey A Moskovtsev,Alexandra A Shiyan,Leonid V Kapilevich,Alexander V Lopachev,Tatiana S Klementieva,Aslan A Kubatiev,Sergei N Orlov,Tatiana N Fedorova,Luis Eduardo M Quintas

doi:10.1371/journal.pone.0222767

Abstract

It was shown previously that inhibition of the ubiquitous α1 isoform of Na+,K+-ATPase by ouabain sharply affects gene expression profile via elevation of intracellular [Na+]i/[K+]i ratio. Unlike other cells, neurons are abundant in the α3 isoform of Na+,K+-ATPase, whose affinity in rodents to ouabain is 104-fold higher compared to the α1 isoform. With these sharp differences in mind, we compared transcriptomic changes in rat cerebellum granule cells triggered by inhibition of α1- and α3-Na+,K+-ATPase isoforms. Inhibition of α1- and α3-Na+,K+-ATPase isoforms by 1 mM ouabain resulted in dissipation of transmembrane Na+ and K+ gradients and differential expression of 994 transcripts, whereas selective inhibition of α3-Na+,K+-ATPase isoform by 100 nM ouabain affected expression of 144 transcripts without any impact on the [Na+]i/[K+]i ratio. The list of genes whose expression was affected by 1 mM ouabain by more than 2-fold was abundant in intermediates of intracellular signaling and transcription regulators, including augmented content of Npas4, Fos, Junb, Atf3, and Klf4 mRNAs, whose upregulated expression was demonstrated in neurons subjected to electrical and glutamatergic stimulation. The role [Na+]i/[K+]i-mediated signaling in transcriptomic changes involved in memory formation and storage should be examined further.

Highlights

Na+,K+-ATPase is an integral plasma membrane protein consisting of α, β- and γ-subunits
Transfection of human endothelial cells with rat α1-Na+,K+-ATPase protected them from the cytotoxic action of high doses of ouabain (3–3000 μM) [36]
Consistent with a previous report [37], we observed that 3 h exposure to ouabain did not affect survival of rat cerebellum granule cells (Fig 1A)

Summary

Introduction

Na+,K+-ATPase is an integral plasma membrane protein consisting of α-, β- and γ-subunits. We used the sharp differences in the affinity of rat α1 and α3-isoforms of Na+,K+-ATPase for CTSs to examine the relative impact of α1 and α3-mediated excitation– transcription coupling by comparing the action of low and high doses of ouabain on the intracellular Na+ and K+ content and gene expression profile in primary culture of rat cerebellar granule cells.

Results

Conclusion