Low K+ increases Na,K-ATPase abundance in LLC-PK1/Cl4 cells by differentially increasing beta, and not alpha, subunit mRNA.

Abstract

In this paper we establish the response of LLC-PK1/Cl4 cells, a pig kidney cell line, to incubation in medium containing 0.25 mM K+. The amounts of the Na,K-ATPase alpha and beta subunits, determined by Western blot, increase coordinately to greater than 2-fold over control by 24 h in low K+ and remained elevated for the duration of the study period (48 h). Na,K-ATPase activity, measured enzymatically, increased 1.4-fold by 24 h and remained elevated. In order to determine if this response was initiated pretranslationally, alpha and beta subunit mRNA levels were determined by Northern blot analysis. While there was no change in alpha-mRNA levels, beta levels increased significantly, to 1.9-fold over control by 6 h of treatment and remained elevated. This selective increase in beta-mRNA was accompanied by 1.6- and 3.1-fold increases in the respective rates of accumulation of newly synthesized alpha and beta subunits, assessed by immunoprecipitating subunits from pulse-labeled cells. The degradation rates of mature Na,K-ATPase subunits did not change during 16 h of exposure to low K+, but after 16 h there was a selective decrease in the alpha degradation rate, relative to control. These results suggest that increased pretranslational regulation of the beta subunit alone is sufficient to increase accumulation of both alpha and beta subunits. These findings support the notion that in LLC-PK1 cells newly synthesized beta is rate-limiting and thus regulates, through alpha beta assembly, the number of pumps transported to the plasma membrane.