A Highly Conserved Glycine in a Hotspot for Neurological Disease Mutations in Na+,K+-ATPase Is Critical to Na+ and K+ Occlusion

Na+,K+-ATPase possesses a highly conserved glycine (G358 in the α3 isoform) that—together with a nearby isoleucine (I363 in α3)—is targeted by mutations causing some of the most severe neurological phenotypes of the clinical spectrum of α3-Na+,K+-ATPase mutations. The disease mutations α3-G358V and α3-I363N affect Na+ and K+ transport to an extent incompatible with cell growth. However, alanine replacement of the corresponding glycine G363 in the α1 isoform is compatible with cell growth, allowing the effects on Na+,K+-ATPase function to be addressed using enzymatic assays on plasma membranes isolated from transfected cells. Occlusion of Na+ appears to be defective in mutant G363A, resulting in a reduced rate of phosphorylation from ATP. Furthermore, the mutation displaces the major conformational equilibrium of Na+,K+-ATPase such that the K+-occluded state is destabilized and occluded K+ is released faster, thereby leading to accumulation of a non-productive state without bound Na+ or K+. The critical function of the glycine can be ascribed to a strategic location at the bending point between an α helix and a β strand, where it connects the catalytic ATP hydrolysis site in the cytoplasmic P domain with the ion-binding region in the membrane and coordinates important intramolecular domain movements during the Na+,K+-ATPase transport cycle.