Mechanism of Rescue of Na+,K+-ATPase Function in Neurological Disease Revealed by New Super-Efficient Second-Site Mutations.

Serious neurological disorders are caused by mutation of Na+,K+-ATPase α1-α3 isoforms. Several disease mutations reduce the affinity for Na+, which in vivo results in an increase in the intracellular Na+ concentration with a secondary rise in intracellular Ca2+. Current treatments are ineffective, as they do not address Na+,K+-ATPase function. Hence, a platform for the development of a rational therapeutic approach that restores Na+ affinity is needed. Using the COS-1 cell expression system, one of the most conspicuous reductions in Na+ affinity of Na+,K+-ATPase has been reported for the neurological disease mutation α3-D923N of the α3 isoform. We describe here second-site mutations ("rescue mutations") that fully restore Na+ affinity of α3-D923N and the corresponding rat α1-D928N and reveal the underlying molecular mechanism. Paradoxically, this insight evolved from investigation of a rapid-onset ataxia Na+,K+-ATPase mutant, α3-G316S, that exhibits an increased affinity for Na+. When applied as second-site mutations, α3-G316S and the corresponding rat α1-G321S are super-efficient rescuers of the reduced Na+ affinity of α3-D923N and rat α1-D928N. Replacement of the glycine with serine leads to a clash between transmembrane helices in the K+-bound E2 state of Na+,K+-ATPase but not in the Na+-bound E1 state. A similarly strong rescuing power is observed for second-site mutations breaking a salt bridge present only in the E2 state. The rescue is due to a structural change resembling the rearrangement of the ion binding sites occurring during the normal Na+,K+-ATPase transport cycle as part of the E2-to-E1 conformational transition.