Rhoda Blostein

Asymmetric interaction of inside-out and right-side-out erythrocyte membrane vesicles with ouabain

Abstract Inside-out and right-side-out human erythrocyte membrane vesicles were prepared as described (Steck, T. L., Weinstein, R. S., Strauss, J. H. and Wallach, D. F. H. (1970) Science 168, 255–257) and used to examine asymmetric properties of alkali cation-sensitive ATPase activity. 1. 1. The Na + - or (Na + -K + )-ATPase (ATP phosphohydrolase, EC 3.6.3.1) of inside-out vesicles was relatively insensitive to ouabain inhibition whereas the Na + - or (Na + -K + )-ATPase of right-side-out vesicles and ghosts was markedly ouabain sensitive. 2. 2. Ouabain added during vesiculation resulted in inside-out vesicles devoid of Na + - or (Na + -K + )-ATPase activity, whereas right-side-out vesicles retained ⩾ 70% of the activity observed without ouabain treatment. 3. 3. Specific [ 3 H]ouabain binding by inside-out vesicles was negligible, whereas erythrocyte ghosts and right-side-out vesicles bound approximately 1.1 and 0.7 pmoles/mg protein, respectively. A direct relation between the degree of ouabain binding to the membranes and the degree of ouabain inhibition of (Na + -K + )-ATPase was observed. 4. 4. These results provide further direct evidence that (i) ouabain binding sites are located on the outer membrane surface and (ii) ‘inside-out’ vesicles are indeed inside-out with respect to the sidedness of the ouabain-binding sites.

Kinetic Alterations due to a Missense Mutation in the Na,K-ATPase α2 Subunit Cause Familial Hemiplegic Migraine Type 2

A number of missense mutations in the ATP1A2 gene, which encodes the Na,K-ATPase α2 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 α2 enzyme does not alter cell growth or catalytic turnover but causes a substantial decrease in apparent K+ affinity (2-fold increase in K0.5(K+)). In view of the location of Thr-345 in the cytoplasmic stalk domain adjacent to transmembrane segment 4, the 2-fold increase in K0.5(K+) is probably due to T345A replacement altering K+ occlusion/deocclusion. Faster K+ deocclusion of the mutant via the E2(K) + ATP → E1·ATP + 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 KATP, and (iii) only a modest increase (∼3-fold) in I50 for vanadate, which was used as a probe of the steady state E1/E2 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.

Distinct Regulatory Effects of the Na,K-ATPase γ Subunit

The two variants of the γ subunit of the rat renal sodium pump, γa and γb, have similar effects on the Na,K-ATPase. Both increase the affinity for ATP due to a shift in the enzymes E1 ↔ E2 conformational equilibrium toward E1. 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 γ were expressed in rat α1-transfected HeLa cells. At the N terminus, the variant-distinct region was deleted (γNΔ7) or replaced by alanine residues (γN7A). At the C terminus, four (γaCΔ4) or ten (γaCΔ10) 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 (γNΔ7, γaCΔ4, and γaCΔ10) abolished the decrease in K′ATP seen with wild-type γa or γb. It is concluded that different regions of the γ chain mediate the distinct functional effects of γ, and the effects can be long-range. In the transmembrane region, the impact of G41R replacement was analyzed since this mutation is associated with autosomal dominant renal Mg2+-wasting in man (Meij, I. C., Koenderink, J. B., van Bokhoven, H., Assink, K. F. H., Groenestege, W. T., de Pont, J. J. H. H. M., Bindels, R. J. M., Monnens, L. A. H., Van den Heuvel, L. P. W. J., and Knoers, N. V. A. M. (2000) Nat. Genet. 26, 265–266). The results show that Gly-41 → Arg prevents trafficking of γ but not αβ pumps to the cell surface and abrogates functional effects of γ on αβ pumps. These findings underscore a potentially important role of γ in affecting solute transport, in this instance Mg2+ reabsorption, consequent to its primary effect on the sodium pump.

Molecular and Functional Studies of the Gamma Subunit of the Sodium Pump

This article reviews our studies of the γ subunit of the sodium pump. γ is a member of the FXYD family of small, single transmembrane proteins and is expressed predominantly in the kidney tubule. There are two major variants of γ which function similarly to bring about two distinct effects, one on K′ATP and the other, on KK, the affinity of the pump for K+ acting as a competitor of cytoplasmic Na+. In this way, γ is believed to provide a self-regulatory mechanism for maintaining the steady-state activity of the pump in the kidney. Our studies also suggest that K+ antagonism of cytoplasmic Na+ activation of the pump is relevant not only to the presence of γ in the kidney, but probably some hitherto undefined factor(s) in other tissues, most notably heart. The interesting possibility that not only γ but other members of the FXYD family regulate ion transport in a tissue-specific manner is discussed.

Modulation of Na,K-ATPase by the γ Subunit STUDIES WITH TRANSFECTED CELLS AND TRANSMEMBRANE MIMETIC PEPTIDES

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 γ 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 γ mediates the Na+ affinity effect. Here we use a novel approach involving synthetic transmembrane mimetic peptides to demonstrate unequivocally that the TM domain of γ effects the shift in apparent Na+ affinity. Specifically, we show that incubation of these peptides with membranes containing αβ pumps modulates Na+ affinity in a manner similar to transfected full-length γ subunit. Using mutated γ 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.

Asymmetric iodination of the human erythrocyte membrane

Abstract Exposure of erythrocyte membrane protein components on the inner and outer cell surfaces was probed using lactoperoxidase-catalyzed iodination. Resealed ghosts derived from I 125 -labelled red cells were reiodinated with I 131 a) on the outside by adding lactoperoxidase after the resealing process or b) on the inside by resealing with lactoperoxidase inside the ghost. SDS-polyacrylamide gel electrophoresis revealed two major radioactive components. One, ∼ 90,000 daltons, was labelled on both surfaces; the other, a glycoprotein, was labelled only on the outer surface. Labelling of large molecular weight protein (> 200,000 daltons) was also observed, to a greater extent inside than outside.

K+/Na+antagonism at cytoplasmic sites of Na+-K+-ATPase: a tissue-specific mechanism of sodium pump regulation

Tissue-distinct interactions of the Na+-K+-ATPase with Na+ and K+, independent of isoform-specific properties, were reported previously (A. G. Therien, N. B. Nestor, W. J. Ball, and R. Blostein. J. Biol. Chem. 271: 7104-7112, 1996). In this paper, we describe a detailed analysis of tissue-specific kinetics particularly relevant to regulation of pump activity by intracellular K+, namely K+ inhibition at cytoplasmic Na+ sites. Our results show that the order of susceptibilities of α1 pumps of various rat tissues to K+/Na+antagonism, represented by the ratio of the apparent affinity for Na+ binding at cytoplasmic activation sites in the absence of K+ to the affinity constant for K+ as a competitive inhibitor of Na+ binding at cytoplasmic sites, is red blood cell < axolemma ≈ rat α1-transfected HeLa cells < small intestine < kidney < heart. In addition, we have carried out an extensive analysis of the kinetics of K+ binding and occlusion to the cytoplasmic cation binding site and find that, for most tissues, there is a relationship between the rate of K+ binding/occlusion and the apparent affinity for K+ as a competitive inhibitor of Na+activation, the order for both parameters being heart ≥ kidney > small intestine ≈ rat α1-transfected HeLa cells. The notion that modulations in cytoplasmic K+/Na+antagonism are a potential mode of pump regulation is underscored by evidence of its reversibility. Thus the relatively high K+/Na+antagonism characteristic of kidney pumps was reduced when rat kidney microsomal membranes were fused into the dog red blood cell.

Characteristics of Na+-ATPase of low-K+ sheep red cell membranes stimulated by blood group L antiserum

1. 1. The effect of isoimmune (anti-L) serum on the Na+-activated ATPase activity of low-K+ (LK) type red cell membranes was studied at low ATP concentration. 2. 2. The L-antigen-antibody reaction results in a marked increase in the velocity of Na+-dependent ATP hydrolysis. 3. 3. The response of anti-L-stimulated Na+-ATPase in homozygous LL (low-K+) membranes to K+ resembles that of low-K+ rather than high-K+ (HK) membranes.

Structure/Function Studies of the Gamma Subunit of the Na,K‐ATPase

Abstract: The Na,K‐ATPase γ subunit is present primarily in kidney as two splice variants, γa and γb, 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 γa or γb, namely, (i) a decrease in K′ATP probably secondary to a shift in steady‐state E1↔ E2 poise in favor of E1 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 γ 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 γ 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 γb variant retards trafficking of γ, but not αβ pumps, to the cell surface and abolishes functional effects of γ, consistent with the conclusion that the Mg2+ transport defect is secondary to loss of γ modulation of Na,K‐ATPase function.

Functional consequences of a posttransfection mutation in the H2-H3 cytoplasmic loop of the alpha subunit of Na,K-ATPase.

During kinetic studies of mutant rat Na,K-ATPases, we identified a spontaneous mutation in the first cytoplasmic loop between transmembrane helices 2 and 3 (H2-H3 loop) which results in a functional enzyme with distinct Na,K-ATPase kinetics. The mutant cDNA contained a single G950 to A substitution, which resulted in the replacement of glutamate at 233 with a lysine (E233K). E233K and α1 cDNAs were transfected into HeLa cells and their kinetic behavior was compared. Transport studies carried out under physiological conditions with intact cells indicate that the E233K mutant and α1 have similar apparent affinities for cytoplasmic Na+ and extracellular K+. In contrast, distinct kinetic properties are observed when ATPase activity is assayed under conditions (low ATP concentration) in which the K+ deocclusion pathway of the reaction is rate-limiting. At 1 μM ATP K+ inhibits Na+-ATPase of α1, but activates Na+-ATPase of E233K. This distinctive behavior of E233K is due to its faster rate of formation of dephosphoenzyme (E1) from K+-occluded enzyme (E2(K)), as well as 6-fold higher affinity for ATP at the low affinity ATP binding site. A lower ratio of Vmax to maximal level of phosphoenzyme indicates that E233K has a lower catalytic turnover than α1. These distinct kinetics of E233K suggest a shift in its E1/E2 conformational equilibrium toward E1. Furthermore, the importance of the H2-H3 loop in coupling conformational changes to ATP hydrolysis is underscored by a marked (2 orders of magnitude) reduction in vanadate sensitivity effected by this Glu233 → Lys mutation.