Alterations in the α2 isoform of Na,K-ATPase associated with familial hemiplegic migraine type 2

Abstract

A number of missense mutations in the Na,K-ATPase ␣2 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,KATPase kinetics. This study describes two additional mutants, R689Q and M731T, originally identified by Vanmolkot et al. , which we show here to also be functional and kinetically altered. Both mutants have reduced catalytic turnover and increased apparent affinity for extracellular K ؉ . For both R689Q and M731T, sensitivity to vanadate inhibition is decreased, suggesting that the steadystate E 1 7 E2 poise of the enzyme is shifted toward E1. Whereas the K ATP is not affected by the R689Q replacement, the M731T mutant has an increase in apparent affinity for ATP. Analysis of the structural changes effected by T345A, R689Q, and M731T mutations, based on homologous replacements in the known crystal structure of the sarcoplasmic reticulum Ca-ATPase, provides insights into the molecular bases for the kinetic alterations. It is suggested that the disease phenotype is the consequence of lowered molecular activity of the ␣2 pump isoform due to either decreased K ؉ affinity (T345A) or catalytic turnover (R689Q and M731T), thus causing a delay in extracellular K ؉ clearance and͞or altered localized Ca 2؉ handling͞signaling secondary to reduced activity in colocalized Na ؉ ͞Ca 2؉ exchange.sodium pump kinetics ͉ missense mutations ͉ ATP1A2 gene T he Na,K-ATPase catalyzes the ATP-driven exchange of three intracellular Na ϩ ions for two extracellular K ϩ ions across the plasma membrane of virtually all animal cells and is essential to the maintenance of the electrochemical alkali cation gradients that are dissipated by ion channels in the propagation of action potentials (for recent reviews, see refs. 1 and 2). The catalytic cycle of this P type ion pump involves phosphorylation and dephosphorylation of a conserved aspartate residue in its catalytic ␣ subunit and conformational transitions of phosphoand dephosphoenzyme, commonly referred to as E 1 P 7 E 2 P and E 1 7 E 2 conformational transitions, respectively (see Scheme 1). Four isoforms of ␣ and three isoforms of ␤ have been described thus far. All are distributed in a tissue-and developmentally dependent manner. In adult mammals, ␣2 is located principally in skeletal muscle and brain, in particular in glial cells, and, to a lesser extent, in heart, adipocytes, and the eye (see refs. 3-6).The discovery of the association of missense mutations in the ATP1A2 gene on chromosome 1q23 that encodes the Na,KATPase ␣2 isoform with familial hemiplegic migraine (FHM) has been an important breakthrough in that it reflects a disease caused by a kinetically altered sodium pump and is therefore an important lead in migraine pathophysiology. Although a migraine is a common polygenic disorder, FHM is a rare autosomal dominant form of migraine with aura and is usually additionally associated with hemiparesis and other clinical features ...

CaV2.1 voltage activated calcium channels and synaptic transmission in familial hemiplegic migraine pathogenesis

“…When this mechanism fails, it leads to elevated extracellular levels of glutamate and potassium, and accordingly, to an increased susceptibility to CSD (Pietrobon 2007, Koenderink et al 2005. All FHM2 mutations studied in heterologous expression systems result in a "loss-of-function" or a kinetically altered Na + , K + pump (Pietrobon 2007, Tavraz et al 2008, Vanmolkot et al 2006, Segall et al 2005). …”

Familial Hemiplegic Migraine

“…2) have been investigated in HeLa cells expressing recombinant (human or rat) ␣ 2 Na ϩ ,K ϩ -ATPase subunits containing mutations that make the pump insensitive to ouabain, which allows one to distinguish the activity of the exogenous pumps from the endogenous pumps of the cell line. 10,68,69,84,85 Three FHM2 mutations (L764P, W887R, G615R, and also the AHC mutation) produced severe or complete loss-offunction of pump activity, because HeLa cells transfected with the FHM2 mutants showed rapid mortality in the presence of ouabain. 10,69,71 Complete loss of pump activity (as measured by lack of pump current) with unaltered expression in the plasma membrane has been shown for the L764P and W887R mutants expressed in Xenopus ocytes.…”

“…86 In contrast, the other five FHM2 mutants analyzed so far conferred sufficient Na ϩ /K ϩ pump activity to allow survival of HeLa cells although at a reduced rate in the case of the R593W and V628M mutants; survival was similar to that conferred by the WT pump in the case of the T345A, M731T, and R689Q mutants. 68,84,85 However, the T345A mutant had a lower affinity for extracellular K ϩ , which would slow removal of K ϩ from the extracellular space and slow the recovery from neuronal excitation. 84 The M731T and R689Q mutants had a decreased catalytic turnover and increased affinity for extracellular Kϩ, which would also slow extracellular Kϩ clearance in the plausible hypothesis that the lower catalytic turnover overrides the effect of increased Kϩ affinity, at least at high extracellular Kϩ concentrations.…”

“…84 The M731T and R689Q mutants had a decreased catalytic turnover and increased affinity for extracellular Kϩ, which would also slow extracellular Kϩ clearance in the plausible hypothesis that the lower catalytic turnover overrides the effect of increased Kϩ affinity, at least at high extracellular Kϩ concentrations. 85 Impaired clearance of K ϩ and glutamate by astrocytes during cortical neuronal activity, consequent to a decreased number of functional Na ϩ ,K ϩ -ATPases, or a decreased affinity for K ϩ or a decreased catalytic turnover of the ␣ 2 Na ϩ ,K ϩ -ATPase pumps, would depolarize neurons and enhance glutamate concentration in the synaptic cleft, thus impairing recovery from neuronal excitation. One may predict enhanced susceptibility to CSD in FHM2 considering that relatively weak depolarizing stimuli, that would be without consequences in healthy individuals whose efficient glial uptake mechanisms keep the concentration of K ϩ within the physiological range, may be able to produce a local buildup of K ϩ concentration above the critical value that ignites CSD in FHM2 patients with impaired K ϩ and glutamate reuptake mechanisms.…”

Novel Mutation Confirms Seizure Locus SCN1A is Also Familial Hemiplegic Migraine Locus FHM3

“…CACNA1A, encoding the α1 subunit of the neuronal P/Q Ca ++ channel, and ATP1A2, encoding the α2 isoform of the major subunit of the Na,K-ATPase, give rise to FHM1 [OMIM #141500] and FHM2 [OMIM #602481], respectively, and together account for most FHM patients [2,4,5]. The FHM2 mutations produce hypomorphic kinetic alterations in Na/K pump function [6,7], but it remains controversial whether FHM1 mutations are hypermorphic [8] or hypomorphic [9].…”