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J. Biol. Chem., Vol. 283, Issue 41, 27982-27990, October 10, 2008

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Identification and Function of a Cytoplasmic K⁺ Site of the Na⁺, K⁺-ATPase^*

Vivien Rodacker Schack, Jens Preben Morth^¶, Mads S. Toustrup-Jensen, Anne Nyholm Anthonisen, Poul Nissen^¶, Jens Peter Andersen, and Bente Vilsen¹

From the Centre for Membrane Pumps in Cells and Disease (PUMPKIN), Danish National Research Foundation, the Institute of Physiology and Biophysics, and the ^¶Department of Molecular Biology, University of Aarhus, DK-8000 Aarhus C, Denmark

A cytoplasmic nontransport K⁺/Rb⁺ site in the P-domain of the Na⁺, K⁺-ATPase has been identified by anomalous difference Fourier map analysis of crystals of the form of the enzyme. The functional roles of this third K⁺/Rb⁺ binding site were studied by site-directed mutagenesis, replacing the side chain of Asp⁷⁴² donating oxygen ligand(s) to the site with alanine, glutamate, and lysine. Unlike the wild-type Na⁺, K⁺-ATPase, the mutants display a biphasic K⁺ concentration dependence of E₂P dephosphorylation, indicating that the cytoplasmic K⁺ site is involved in activation of dephosphorylation. The affinity of the site is lowered significantly (30-200-fold) by the mutations, the lysine mutation being most disruptive. Moreover, the mutations accelerate the E₂ to E₁ conformational transition, again with the lysine substitution resulting in the largest effect. Hence, occupation of the cytoplasmic K⁺/Rb⁺ site not only enhances E₂P dephosphorylation but also stabilizes the E₂ dephosphoenzyme. These characteristics of the previously unrecognized nontransport site make it possible to account for the hitherto poorly understood trans-effects of cytoplasmic K⁺ by the consecutive transport model, without implicating a simultaneous exposure of the transport sites toward the cytoplasmic and extracellular sides of the membrane. The cytoplasmic K⁺/Rb⁺ site appears to be conserved among Na⁺, K⁺-ATPases and P-type ATPases in general, and its mode of operation may be associated with stabilizing the loop structure at the C-terminal end of the P6 helix of the P-domain, thereby affecting the function of highly conserved catalytic residues and promoting helix-helix interactions between the P- and A-domains in the E₂ state.

Received for publication, May 8, 2008 , and in revised form, July 25, 2008.

^* This study was funded in part by grants from the Lundbeck Foundation, Denmark, the Danish Medical Research Council, the Novo Nordisk Foundation, Denmark, and the Danish National Research Foundation (PUMPKIN Centre). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Institute of Physiology and Biophysics, University of Aarhus, Ole Worms Allé 1185, DK-8000 Aarhus C, Denmark. Fax: 45-86129065; E-mail: bv{at}fi.au.dk.

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