A number of missense mutations in the ATP1A2 gene, which encodes the Na,K-ATPase ␣2 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. A., Harno, H., Vanmolkot, K. R., Gargus, J. J., Sun, G.,

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... alainen, E., Liukkonen, E., Kallela, M., van den Maagdenberg, A. M., Frants, R. R., Farkkila, M., Palotie, A., and Wessman, M. (2004) Neurogenetics 5, 141-146) . Introducing T345A into the conserved rat ␣2 enzyme does not alter cell growth or catalytic turnover but causes a substantial decrease in apparent K ؉ affinity (2-fold increase in K 0.5(K ؉ ) ). In view of the location of Thr-345 in the cytoplasmic stalk domain adjacent to transmembrane segment 4, the 2-fold increase in K 0.5(K ؉ ) is probably due to T345A replacement altering K ؉ occlusion/deocclusion. Faster K ؉ deocclusion of the mutant via the E 2 (K) ؉ ATP 3 E 1 ⅐ATP ؉ K ؉ partial reaction is evidenced in (i) a marked increase (300%) in K ؉ stimulation of Na-ATPase at micromolar ATP, (ii) a 4-fold decrease in K ATP , and (iii) only a modest increase (ϳ3-fold) in I 50 for vanadate, which was used as a probe of the steady state E 1 /E 2 conformational equilibrium. We suggest that the decreased apparent K ؉ affinity is the basis for a reduced rate of extracellular K ؉ removal, which delays the recovery phase of nerve impulse transmission in the central nervous system and, thereby, the clinical picture of migraine with aura. This is the first demonstration of a mutation that leads to a disease associated with a kinetically altered but fully functional Na,K-ATPase, refining the molecular mechanism of pathogenesis in familial hemiplegic migraine.