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Mads Schak Toustrup-Jensen

Importance of Residues Outside the Cation Binding Pocket for Na+ and K+ Binding to the Na+/K+-ATPase

Publikation: KonferencebidragPosterForskning

Mutagenesis studies have identified several oxygen-containing residues in the transmembrane region which are important for the coordination of Na+ and/or K+. These were later confirmed by the high-resolution crystal structures of the Na+/K+-ATPase with bound Na+ or K+. However, more information is needed to reveal the translocation pathway through the Na+/K+-ATPase. Mutagenesis studies have shown that several other transmembrane residues located outside the cation binding pocket, including Phe785 and Phe788, affect the ion binding properties of the Na+/K+-ATPase (1,2).
It is well-established that K+ antagonizes ouabain binding, and vice versa. Furthermore, recent crystal structures have shown that ouabain binds in an extracellular cavity created by residues of transmembrane helices 4, 5, and 6 (3). This cavity, which is lined by Phe785 and Phe788, as well as Phe318, Arg882, and Asp886, may be involved in the extracellular handling of K+ and Na+. Site-directed mutagenesis and functional studies of Phe318, Arg882, and Asp886 were used to investigate the role of these residues on the extracellular ion selectivity.
Phe318 was replaced by tryptophan to investigate the effects of an additional aromatic ring, while Arg882 and Asp886 were mutated to leucine and alanine, respectively, to investigate the importance of charge and size of the residues.
All three mutants could sustain growth and proliferation under ouabain pressure. However, the mutants exhibited a reduced turnover number. All three mutants displayed an increased apparent K+ affinity at the external binding sites in measurements of ATPase activity: for Phe318Trp, Arg882Leu, and Asp886Ala 2.2-, 5.1-, and 1.8-fold increases compared to the wild type, respectively. Similarly the three mutants exhibited 10-, 6.4-, and 4.1-fold decreases in the apparent ouabain affinity, respectively.
The three mutations affected the apparent Na+ affinity at the internal binding sites differently: Phe318Trp exhibited a 6.7-fold decrease, not caused by a shift in the conformational equilibrium towards the E2 forms, while mutations Arg882Leu and Asp886Ala did not significantly affect the Na+ binding properties of the Na+/K+-ATPase.

1. Vilsen, B. (1999) Biochemistry 38, 11389
2. Rodacker, V. et al (2006) JBC 281, 18539
3. Laursen M et al. (2013) PNAS 110, 10958
StatusUdgivet - 2014
Begivenhed14th International ATPase Conference “Na,K‐ATPase and related transport ATPases: Structure, mechanism, cell biology, health and disease” - Conference Center De Werelt, Lunteren, Holland
Varighed: 30 aug. 20145 sep. 2014


Konference14th International ATPase Conference “Na,K‐ATPase and related transport ATPases: Structure, mechanism, cell biology, health and disease”
LokationConference Center De Werelt

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