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

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.