Specific sites in the cytoplasmic N terminus modulate conformational transitions of the Na,K-ATPase.

The cytoplasmic N terminus of the Na,K-ATPase is a highly charged and flexible structure that comprises three predicted helical regions including H1 spanning residues 27 to 33 and H2 spanning residues 42 to 50. Previous deletion mutagenesis experiments showed that deletion of residues up to and including most of H2 shifts the E(1)/E(2) conformational equilibrium toward E(1). The present study describes a clustered charge-to-alanine mutagenesis approach designed to delineate specific sites within the N terminus that modulate the steady-state E(1) <--> E(2) and E(1)P <--> E(2)P poise.

Alterations in the alpha2 isoform of Na,K-ATPase associated with familial hemiplegic migraine type 2.

A number of missense mutations in the Na,K-ATPase alpha2 catalytic subunit have been identified in familial hemiplegic migraine with aura. Two alleles (L764P and W887R) showed loss-of-function, whereas a third (T345A) is fully functional but with altered Na,K-ATPase kinetics. This study describes two additional mutants, R689Q and M731T, originally identified by Vanmolkot et al.

Kinetic alterations due to a missense mutation in the Na,K-ATPase alpha2 subunit cause familial hemiplegic migraine type 2.

A number of missense mutations in the ATP1A2 gene, which encodes the Na,K-ATPase alpha2 subunit, have been identified in familial hemiplegic migraine with aura. Loss of function and haploinsufficiency have been the suggested mechanisms in mutants for which functional analysis has been reported. This paper describes a kinetic analysis of mutant T345A, recently identified in a detailed genetic analysis of a large Finnish family (Kaunisto, M.

Structural basis for alpha1 versus alpha2 isoform-distinct behavior of the Na,K-ATPase.

We showed earlier that the kinetic behavior of the alpha2 isoform of the Na,K-ATPase differs from the ubiquitous alpha1 isoform primarily by a shift in the steady-state E(1)/E(2) equilibrium of alpha2 in favor of E(1) form(s). The aim of the present study was to identify regions of the alpha chain that confer the alpha1/alpha2 distinct behavior using a mutagenesis and chimera approach. Criteria to assess shifts in conformational equilibrium included (i) K(+) sensitivity of Na-ATPase measured at micromolar ATP, under which condition E(2)(K(+)) --> E(1) + K(+) becomes rate-limiting, (ii) changes in K'(ATP) for low affinity ATP binding, (iii) vanadate sensitivity of Na,K-ATPase activity, and (iv) the rate of the partial reaction E(1)P --> E(2)P.

New insights into the role of the N terminus in conformational transitions of the Na,K-ATPase.

The deletion of 32 residues from the N terminus of the alpha1 catalytic subunit of the rat Na,K-ATPase (mutant alpha1M32) shifts the E(1)/E(2) conformational equilibrium toward E(1), and the combination of this deletion with mutation E233K in the M2-M3 loop acts synergistically to shift the conformation further toward E(1) (Boxenbaum, N., Daly, S. E.