Like the gamma-subunit of Na-K-ATPase, the corticosteroid hormone-induced factor (CHIF) is a member of the FXYD family of one-transmembrane-segment proteins. Both CHIF and two splice variants of gamma, gamma(a) and gamma(b), are expressed in the kidney. Immunolocalization experiments demonstrate mutually exclusive expression of CHIF and gamma in different nephron segments. Specific coimmunoprecipitation experiments demonstrate the existence in kidney membranes of the complexes alpha/beta/gamma(a), alpha/beta/gamma(b), and alpha/beta/CHIF and exclude mixed complexes such as alpha/beta/gamma(a)/gamma(b) and alpha/beta/gamma/CHIF. CHIF has been expressed in HeLa cells harboring the rat alpha(1)-subunit of Na-K-ATPase. (86)Rb flux experiments demonstrate that CHIF induces a two- to threefold increase in apparent affinity for cytoplasmic Na (K'(Na)) but does not affect affinity for extracellular K (Rb) ions (K'(K)) or V(max). Measurements of Na-K-ATPase using isolated membranes show similar but smaller effects of CHIF on K'(Na), whereas K'(K) and K'(ATP) are unaffected. The functional effects of CHIF differ from those of gamma. An implication of these findings is that other FXYD proteins could act as tissue-specific modulators of Na-K-ATPase.

The two variants of the ␥ subunit of the rat renal sodium pump, ␥ a and ␥ b , have similar effects on the Na,K-ATPase. Both increase the affinity for ATP due to a shift in the enzyme's E 1 7 E 2 conformational equilibrium toward E 1 . In addition, both increase K ؉ antagonism of cytoplasmic Na ؉ activation. To gain insight into the structural basis for these distinct effects, extramembranous N-terminal and C-terminal mutants of ␥ were expressed in rat ␣1-transfected HeLa cells. At the N terminus, the variant-distinct region was deleted (␥N⌬7) or replaced by alanine residues (␥N7A). At the C terminus, four (␥ a C⌬4) or ten (␥ a C⌬10) residues were deleted. None of these mutations abrogates the K ؉ /Na ؉ antagonism as evidenced in a similar increase in K Na seen at high (100 mM) K ؉ concentration. In contrast, the C-terminal as well as N-terminal deletions (␥N⌬7, ␥ a C⌬4, and

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 ...

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., Hamalainen, 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.Familial hemiplegic migraine (FHM) 1 is a rare autosomal dominant form of migraine with aura. This disorder is usually associated with hemiparesis and can be accompanied with clinical features ranging from ataxia to epileptic seizures. This genetically heterogeneous disease has been traced to at least two loci, FHM1 and FHM2. FHM1, which accounts for over 50% of all FHM families, has been traced to chromosome 19p13 and associated with missense mutations in the CACNA1A gene encoding the ␣1 subunit of the voltage-dependent neuronal (P/Q type) calcium channel. A recent breakthrough in migraine genetics is the discovery of missense mutations in the ATP1A2 gene on chromosome 1q23 that encodes the ␣2 isoform of the Na,K-ATPase. This finding gives strong support to the notion that FHM is caused by disruption of normal cation transport. The Na,K-ATPase is an integral membrane protein complex that comprises a large catalytic ␣ subunit of ϳ110 kDa as well as a smaller, highly glycosylated ␤ subunit that ensures the proper folding and mooring of ␣ in the plasma membrane. This P-type ion pump catalyzes the ATP-driven exchange of intrace...

Chimeras of the catalytic subunits of the gastric H,KATPase and Na,K-ATPase were constructed and expressed in LLC-PK 1 cells. The chimeras included the following: (i) a control, H85N (the first 85 residues comprising the cytoplasmic N terminus of Na,K-ATPase replaced by the analogous region of H,K-ATPase); (ii) H85N/H356 -519N (the N-terminal half of the cytoplasmic M4 -M5 loop also replaced); and (iii) H519N (the entire front half replaced). The latter two replacements confer a decrease in apparent affinity for extracellular K /K؉ exchange even at neutral pH. Overall, this study provides evidence for important roles in cation selectivity for both the N-terminal half of the M4 -M5 loop and the adjacent transmembrane helice(s).The gastric H,K-ATPase and ubiquitous Na,K-ATPase have the highest sequence similarity (62% amino acid sequence homology) of the phosphorylating class (P-type) of ion motive ATPases. The reaction sequence catalyzed by these enzymes involves ATP binding followed by cation-dependent phosphorylation and dephosphorylation of an aspartyl residue at the active site, as well as conformational and vectorial transitions of phospho-and dephosphoenzyme. These reactions transduce the chemical energy of ATP hydrolysis into cation binding at one side of the membrane followed by occlusion in an ionbinding pocket and then cation release at the opposite side of the membrane. Sequence similarity between these enzymes is greatest in the regions associated with ATP binding and phosphorylation (for review see Ref.

The enzymatic activity of the Na,K-ATPase, or sodium pump, is modulated by members of the so-called FXYD family of transmembrane proteins. The best characterized member, FXYD2, also referred to as the ␥ subunit, has been shown to decrease the apparent Na ؉ affinity and increase the apparent ATP affinity of the pump. The effect on ATP affinity had been ascribed to the cytoplasmic C-terminal end of the protein, whereas recent observations suggest that the transmembrane (TM) segment of ␥ mediates the Na ؉ affinity effect. Here we use a novel approach involving synthetic transmembrane mimetic peptides to demonstrate unequivocally that the TM domain of ␥ effects the shift in apparent Na ؉ affinity. Specifically, we show that incubation of these peptides with membranes containing ␣␤ pumps modulates Na ؉ affinity in a manner similar to transfected full-length ␥ subunit. Using mutated ␥ peptides and transfected proteins, we also show that a specific glycine residue, Gly-41, which is associated with a form of familial renal hypomagnesemia when mutated to Arg, is important for this kinetic effect, whereas Gly-35, located on an alternate face of the transmembrane helix, is not. The peptide approach allows for the analysis of mutants that fail to be expressed in a transfected system. The Na,K-ATPase or sodium pump is an integral membrane protein found in the cells of virtually all higher eukaryotes and is one of the most important systems involved in cellular energy transduction (1, 2). It catalyzes the electrogenic exchange of three intracellular Na ϩ for two extracellular K ϩ ions energized by the hydrolysis of one molecule of ATP. The transporter plays a major role in ion homeostasis, and, in epithelia, the sodium gradient created by the pump also plays an important role in secondary active transport mechanisms that are necessary for Na ϩ -dependent reabsorption of a variety of solutes including sugars and amino acids.There is an increasing body of evidence that members of a family of membrane proteins, the so-called FXYD family (3), associate with and modulate the kinetic behavior of the sodium pump (for a recent overview, see Ref 4). Members of this family of proteins are small, single transmembrane (TM) 1 proteins characterized by an N-terminal PFXYD motif that remains invariant in all mammals. There are at least seven known family members, of which several appear to modulate the kinetic behavior of the pump in a tissue-specific manner (5-8). To date, the ␥ "subunit" of the renal Na,K-ATPase is the best characterized member (reviewed in Refs. 9 and 10). Gamma, or FXYD2, exists as two main splice variants, ␥a and ␥b, with distinct as well as overlapping localization along the nephron (11). Mass spectrometry of ␥a and ␥b indicate that they differ only in the N terminus; in rat ␥a, TELSANH is replaced by Ac-MDRWYL in ␥b (12). Previous studies using membrane fragments isolated from ␥-transfected rat ␣1-HeLa cells have shown that ␥ serves at least two distinct functions in regulating the pump, and the effects are similar f...

After binding a sensitizing tumor antigen, human leukocytes undergo a series of changes that lead to a loss of their glass-adherent properties; a phenomenon called leukocyte adherence inhibition (LAI). After surgery or when patients have a large tumor burden, their test results become negative. This study shows that in vitro incubation of the leukocytes for 5 min with PGE2 converted to positive the negative test, in an immunologically specific manner. The effect was critically dose-dependent, too little or too much did not alter the result. The same effect was achieved with PGE2, PGI2, aminophylline or other drugs that raise intracellular nucleotides, including dibutyryl cyclic AMP and dibutyryl cyclic GMP. Dibutyryl cyclic AMP stimulated a stronger response and 100 times less was needed than of dibutyryl cyclic GMP. Prostaglandins did not mediate LAI since Indomethacin failed to inhibit a positive test. Nonetheless, arachidonate metabolites were critical for the LAI phenomenon since BPB and mepacrine, inhibitors of phospholipase A2, negated the LAI response. Moreover, ETYA, phenidone and NDGA, inhibitors of the lipoxygenase metabolic pathway, all negated the positive LAI response. The positive response was especially sensitive to nullification by ETYA. Although the last-named drugs inhibit other arachidonate metabolic pathways too, conclusive evidence that the metabolites of the lipoxygenase pathway, and leukotrienes in particular, mediate the LAI response was the fact that FPL 55712, a competitive antagonist of SRS, nullified a positive response at levels as low as 10(-13) M. The results imply that prostaglandins were able to modulate the expression of LAI by affecting intracellular nucleotides, but leukotrienes, it seems, were the metabolites that mediated leukocyte nonadherence after monocytes recognized and bound tumor antigen.

The Na,K-ATPase gamma subunit is present primarily in kidney as two splice variants, gammaa and gammab, which differ only at their extracellular N-termini. Two distinct effects of gamma are seen in biochemical Na,K-ATPase assays of mammalian (HeLa) cells transfected with gammaa or gammab, namely, (i) a decrease in K'(ATP) probably secondary to a shift in steady-state E(1) <--> E(2) poise in favor of E(1) and (ii) an increase in cytoplasmic K(+)/Na(+) antagonism seen as an increase in K'(Na) at high K(+) concentration. Mutagenesis experiments involving alterations in extramembranous regions of gamma indicate that different regions mediate the aforementioned distinct effects and that the effects appear to be long range. Studies of ouabain-sensitive fluxes with intact cells confirm the gamma effects seen with membranes and also suggest an additional effect (increase) in apparent affinity for extracellular K(+). Alteration in gamma function was also evidenced in the behavior of a G41 -->R mutation within the transmembrane domain of gamma. G41R is associated with autosomal dominant renal magnesium wasting. Our studies show that this mutation in the gammab variant retards trafficking of gamma, but not alphabeta pumps, to the cell surface and abolishes functional effects of gamma, consistent with the conclusion that the Mg(2+) transport defect is secondary to loss of gamma modulation of Na,K-ATPase function.