Disease mutations of human α3 Na/K-ATPase define extracellular Na binding/occlusion kinetics at ion binding site III.

Na/K-ATPase, which creates transmembrane electrochemical gradients by exchanging 3 Na for 2 K, is central to the pathogenesis of neurological diseases such as alternating hemiplegia of childhood. Although Na/K-ATPase has 3 distinct ion binding sites I-III, the difficulty of distinguishing ion binding events at each site from the others hinders kinetic study of these transitions. Here, we show that binding of Na at each site in the human α3 Na/K-ATPase can be resolved using extracellular Na-mediated transient currents. When Na/K-ATPase is constrained to bind and release only Na, three kinetic components: fast, medium, and slow, can be isolated, presumably corresponding to the protein dynamics associated with the binding (or release depending on the voltage step direction) and the occlusion (or deocclusion) of each of the 3 Na. Patient-derived mutations of residues which coordinate Na at site III exclusively impact the slow component, demonstrating that site III is crucial for deocclusion and release of the first Na into the extracellular milieu. These results advance understanding of Na/K-ATPase mutation pathogenesis and provide a foundation for study of individual ions' binding kinetics.