Abstract
The Na(+),K(+)-ATPase plays key roles in brain function. Recently, missense mutations in the Na(+),K(+)-ATPase were found associated with familial rapid-onset dystonia parkinsonism (FRDP). Here, we have characterized the functional consequences of FRDP mutations Phe785Leu and Thr618Met. Both mutations lead to functionally altered, but active, Na(+),K(+)-pumps, that display reduced apparent affinity for cytoplasmic Na(+), but the underlying mechanism differs between the mutants. In Phe785Leu, the interaction of the E(1) form with Na(+) is defective, and the E(1)-E(2) equilibrium is not displaced. In Thr618Met, the Na(+) affinity is reduced because of displacement of the conformational equilibrium in favor of the K(+)-occluded E(2)(K(2)) form. In both mutants, K(+) interaction at the external activating sites of the E(2)P phosphoenzyme is normal. The change of cellular Na(+) homeostasis is likely a major factor contributing to the development of FRDP in patients carrying the Phe785Leu or Thr618Met mutation. Phe785Leu moreover interferes with Na(+) interaction on the extracellular side and reduces the affinity for ouabain significantly. Analysis of two additional Phe(785) mutants, Phe785Leu/Leu786Phe and Phe785Tyr, demonstrated that the aromatic function of the side chain, as well as its exact position, is critical for Na(+) and ouabain binding. The effects of substituting Phe(785) could be explained by structural modeling, demonstrating that Phe(785) participates in a hydrophobic network between three transmembrane segments. Thr(618) is located in the cytoplasmic part of the molecule near the catalytic site, and the structural modeling indicates that the Thr618Met mutation interferes with the bonding pattern in the catalytic site in the E(1) form, thereby destabilizing E(1) relative to E(2)(K(2)).
Highlights
Familial hemiplegic migraine type 2 (FHM)3 (OMIM 602481) [3, 4] and the movement disorder familial rapid-onset dystonia parkinsonism (FRDP) (OMIM 128235) [5]
The Mutation Phe785Leu Reduces the Affinity for Naϩ and the Vmax of Phosphorylation from ATP—The activation of phosphorylation from ATP requires binding of Naϩ at cytoplasmically facing high affinity sites of the E1 form
To determine the maximal phosphorylation rate and the apparent affinity for ATP of the E1Na3 form, the enzyme was further analyzed in phosphorylation experiments carried out in the presence of various ATP concentrations under conditions otherwise identical to those described for Fig. 1B
Summary
REACTION 1 describes how the phosphorylation and dephosphorylation of the Naϩ,Kϩ-ATPase protein and transitions between the major conformational states E1 and E2 lead to ATP hydrolysis coupled with ion translocation across the membrane [1, 2]. In the present study of Naϩ,Kϩ-ATPase, we have focused on the function of a phenylalanine, Phe785, near the extracellular end of transmembrane segment M5. To understand the pathophysiological mechanism, it is essential to characterize functionally the disease-causing Naϩ,Kϩ-ATPase mutants This phenylalanine is fully conserved in all known Naϩ,Kϩ-ATPases, encompassing different species and isoforms, as well as in the non-gastric Hϩ,Kϩ-ATPase, whereas it is conservatively replaced by tyrosine in the gastric Hϩ,Kϩ-ATPase and by isoleucine in the sarco(endo)plasmic reticulum Ca2ϩ-ATPases. In addition to Phe785Leu, we have expressed and characterized mutant Phe785Tyr, as well as a mutant in which the two residues Phe785 and Leu786 are swapped In this way, we have been able to examine the importance of the exact position of the aromatic side chain and its functionality. Like Phe785Leu, the Thr618Met mutation affects the Naϩ affinity, but by a different mechanism, which involves a displacement of the conformational equilibrium in favor of E2(K2)
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