Influence of intramembrane electric charge on Na,K-ATPase

Abstract

Effects of lipophilic ions, tetraphenylphosphonium (TPP+) and tetraphenylboron (TPB-), on interactions of Na+ and K+ with Na,K-ATPase were studied with membrane-bound enzyme from bovine brain, pig kidney, and shark rectal gland. Na+ and K+ interactions with the inward-facing binding sites, monitored by eosin fluorescence and phosphorylation, were not influenced by lipophilic ions. Phosphoenzyme interactions with extracellular cations were evaluated through K+-, ADP-, and Na+-dependent dephosphorylation. TPP+ decreased: 1) the rate of transition of ADP- insensitive to ADP-sensitive phosphoenzyme, 2) the K+ affinity and the rate coefficient for dephosphorylation of the K-sensitive phosphoenzyme, 3) the Na+ affinity and the rate coefficient for Na+-dependent dephosphorylation. Pre-steady state phosphorylation experiments indicate that the subsequent occlusion of extracellular cations was prevented by TPP+. TPB- had opposite effects. Effects of lipophilic ions on the transition between phosphoenzymes were significantly diminished when Na+ was replaced by N-methyl-n-glucamine or Tris+, but were unaffected by the replacement of Cl- by other unions. Lipophilic ions affected Na-ATPase, Na,K-ATPase, and p-nitrophenylphosphatase activities in accordance with their effects on the partial reactions. Effects of lipophilic ions appear to be due to their charge indicating that Na+ and K+ access to their extracellular binding sites is modified by the intramembrane electric field.