Abstract
The highly conserved amino acids of rat Na,K-ATPase, Thr-774 in the transmembrane helices M5, Val-920 and Gln-923 in M8, and Glu-953 and Glu-954 in M9, the side chains of which appear to be in close proximity, were mutated, and the resulting proteins, T774A, E953A/K, and E954A/K, V920E and Q923N/E/D/L, were expressed in HeLa cells. Ouabain-resistant cell lines were obtained from T774A, V920E, E953A, and E954A, whereas Q923N/E/D/L, E953K, and E954K could only be transiently expressed as fusion proteins with an enhanced green fluorescent protein. The apparent K0.5 values for Na+, as estimated by the Na+-dependent phosphoenzyme formation (K0.5(Na,EP)) or Na,K-ATPase activity (K(0.5)(Na,ATPase)), were increased by around 2 approximately 8-fold in the case of T774A, V920E, and E954A. The apparent K0.5 values for K+, as estimated by the Na,K-ATPase (K0.5(K,ATPase)) or p-nitrophenylphosphatase activity (K0.5(K,pNPPase)), were affected only slightly by the 3 mutations, except that V920E showed a 1.7-fold increase in the K0.5(K,ATPase). The apparent K0.5 values for ATP (K0.5(EP)), as estimated by phosphorylation (a high affinity ATP effect), were increased by 1.6 approximately 2.6-fold in the case of T774A, V920E, and E954A. Those estimated by Na,K-ATPase activity (K0.5(ATPase)) and ATP-induced inhibition (K(i,0.5)(pNPPase)) of K-pNPPase activity (low affinity ATP effects) were, respectively, increased by 1.8-fold and unchanged in the case of T774A but decreased by 2- and 4.8-fold in the case of V920E and were slightly changed and increased by 1.7-fold in the case of E954A. The E953A showed little significant change in the apparent affinities. These results suggest that Gln-923 in M8 is crucial for the active transport of Na+ and/or K+ across membranes and that the side chain oxygen atom of Thr-774 in M5, the methyl group(s) of Val-920 in M8, and the carboxyl oxygen(s) of Glu-954 in M9 mainly play some role in the transport of Na+ and also in the high and low affinity ATP effects rather than the transport of K+.
Highlights
Na,K-ATPase, a member of the P-type ATPase family that includes a gastric H,K-ATPase and sarcoplasmic reticulum
The highly conserved amino acids of rat Na,K-ATPase, Thr-774 in the transmembrane helices M5, Val-920 and Gln-923 in M8, and Glu-953 and Glu-954 in M9, the side chains of which appear to be in close proximity, were mutated, and the resulting proteins, T774A, E953A/K, and E954A/K, V920E and Q923N/E/D/L, were expressed in HeLa cells
Ouabain-resistant cell lines were obtained from T774A, V920E, E953A, and E954A, whereas Q923N/ E/D/L, E953K, and E954K could only be transiently expressed as fusion proteins with an enhanced green fluorescent protein
Summary
Na,K-ATPase, a member of the P-type ATPase family that includes a gastric H,K-ATPase and sarcoplasmic reticulum. The amino acid residues, in which the side-chain oxygen atoms contribute to Ca2ϩ binding in SERCA, which include Glu-309 in M4 (Glu-327 or Glu-344 in Na,KATPase or H,K-ATPase, respectively), Asn-768 (Asn-775 or Asn-793) and Glu-771 in M5 (Glu-779 or Glu-796), Asn-796 (Asp-804 or Glu-821), Thr-799 (Thr-807 or Thr-824) and Asp800 in M6 (Asp-808 or Asp-825), and Glu-908 in M8 (Val-920 or Glu-940) are highly conserved among these P-type ATPases These data and other findings suggest the presence of a cationoccluded structure in Na/K-, H/K- and SERCA-Ca/H-ATPase that is shared by all molecules in this class, the structures required for cation selectivity could be different. Naϩ and Kϩ, respectively; K0.5EP, ATP concentration giving half-maximum EP formation; K0.5ATPase, ATP concentration giving half-maximum activation of Na,K-ATPase activity; Ki,0.5, ATP concentration giving half-maximum inhibition of K-pNPPase activity; EGFP, enhanced green fluorescent protein
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
