Identification and function of a cytoplasmic K+ site of the Na+,K+-ATPase

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] center dot E-2 center dot MgF42-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 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 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 E2P 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 P- and A-domains in the E-2 state.