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

Abstract

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 [Rb 2 ]⅐E 2 ⅐MgF 4 2؊ 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 Asp 742 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 E 2 P 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 E 2 to E 1 conformational transition, again with the lysine substitution resulting in the largest effect. Hence, occupation of the cytoplasmic K ؉ /Rb ؉ site not only enhances E 2 P dephosphorylation but also stabilizes the E 2 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 Pand A-domains in the E 2 state.The essential gradients for Na ϩ and K ϩ across the plasma membranes of mammalian cells are created by the Na ϩ ,K ϩ -ATPase, a membranous enzyme that couples ATP hydrolysis to active extrusion of Na ϩ from the cells and uptake of K ϩ at a stoichiometry of three Na ϩ ions exchanged for two K ϩ ions per ATP molecule utilized (1, 2). The Na ϩ ,K ϩ -ATPase belongs to the family of P-type ATPases characterized by the formation of a phosphorylated enzyme intermediate through transfer of the ␥-phosphate of ATP to a conserved aspartate residue in the enzyme. The ATP hydrolysis is tightly coupled to ion translocation, because Na ϩ binding from the cytoplasmic side triggers phosphorylation of the enzyme from ATP, whereas K ϩ binding from the extracellular side leads to rapid dephosphorylation (3). Recently, the structure of the Na ϩ ,K ϩ -ATPase was determined at 3.5 Å resolution (4), revealing two Rb ϩ ions bound as K ϩ congeners close together in a pocket formed by the transmembrane segments M4, 2 M5, M6, and M8 of the catalytic ␣-subunit. The ␣-subunit consists of 10 transmembrane segments, M1-M10, and a large cytoplasmic part containing three distinct domains denoted A (actuator), N (nucleotide-binding), and P (phosphorylation), by analogy to the closely related sarco(end...

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