A basolateral sorting signal is encoded in the alpha-subunit of Na-K-ATPase

Am J Physiol. 1998 Mar;274(3):C688-96. doi: 10.1152/ajpcell.1998.274.3.C688.

Abstract

Na-K-ATPase and H-K-ATPase are highly homologous ion pumps that exhibit distinct plasma membrane distributions in epithelial cells. We have studied the alpha-subunits of these heterodimeric pumps to identify the protein domains responsible for their polarized sorting. A chimeric alpha-subunit construct (N519H) was generated in which the first 519 amino acid residues correspond to the Na-K-ATPase sequence and the remaining 500 amino acids are derived from the H-K-ATPase sequence. In stably transfected LLC-PK1 cell lines, we found that the N519H chimera is restricted to the basolateral surface under steady-state conditions, suggesting that residues within the NH2-terminal 519 amino acids of the Na-K-ATPase alpha-subunit contain a basolateral sorting signal. H-K-ATPase beta-subunit expressed alone in LLC-PK1 cells accumulates at the apical surface. When coexpressed with N519H, the H-K-ATPase beta-subunit assembles with this chimera and accompanies it to the basolateral surface. Thus the NH2-terminal basolateral signal in the Na-K-ATPase alpha-subunit masks or is dominant over any apical sorting information present in the beta-polypeptide. In gastric parietal cells, the H-K-ATPase beta-subunit targets the H-K-ATPase to an intracellular vesicular compartment which fuses with the plasma membrane in response to secretagogue stimulation. To test whether the chimera-H-K-ATPase beta-subunit complex is directed to a similar compartment in LLC-PK1 cells, we treated transfected cells with drugs that raise intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels. Elevation of cytosolic cAMP increased the surface expression of both the N519H chimera and the H-K-ATPase beta-subunit. This increase in surface expression, however, appears to be the result of transcriptional upregulation and not recruitment of chimera to the surface from a cAMP-inducible compartment.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 1-Methyl-3-isobutylxanthine / pharmacology
  • Animals
  • Cell Polarity
  • Colforsin / pharmacology
  • Cyclic AMP / metabolism
  • H(+)-K(+)-Exchanging ATPase / metabolism
  • LLC-PK1 Cells
  • Protein Conformation
  • Protein Sorting Signals / chemistry
  • Protein Sorting Signals / genetics
  • Protein Sorting Signals / metabolism*
  • RNA, Messenger / metabolism
  • Recombinant Fusion Proteins / metabolism
  • Sodium-Potassium-Exchanging ATPase / chemistry
  • Sodium-Potassium-Exchanging ATPase / genetics
  • Sodium-Potassium-Exchanging ATPase / metabolism*
  • Surface Properties
  • Swine
  • Transfection

Substances

  • Protein Sorting Signals
  • RNA, Messenger
  • Recombinant Fusion Proteins
  • Colforsin
  • Cyclic AMP
  • H(+)-K(+)-Exchanging ATPase
  • Sodium-Potassium-Exchanging ATPase
  • 1-Methyl-3-isobutylxanthine