RESUMEN
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. Criteria to assess shifts in poise include (i) sensitivity to inhibition by inorganic orthovanadate to assess overall poise; (ii) K(+)-sensitivity of Na-ATPase measured at micromolar ATP to assess changes in the E(2)(K) + ATP --> E(1) x ATP + K(+) rate; (iii) K'(ATP) for low-affinity ATP binding at the latter step; (iv) overall catalytic turnover, and (v) the E(1)P --> E(2)P transition. The results of alanine replacements in H1 (31KKE) suggest that this site stabilizes E(2)P and to a lesser extent E(2). In H2, residues within 47HRK have a role in stabilizing E(2) but not E(2)P as revealed with double mutants 31KKE --> AAA/47H --> A and 31KKE --> AAA/47HRK --> AAA. Taken together, these observations suggest that sites 31KKE in H1 and 47HRK in H2 have distinct roles in modulating the enzyme's conformational transitions during the catalytic cycle of the enzyme.
Asunto(s)
Citoplasma/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/química , Alanina/química , Secuencia de Aminoácidos , Animales , Membrana Celular/metabolismo , Eliminación de Gen , Células HeLa , Humanos , Conformación Molecular , Datos de Secuencia Molecular , Mutagénesis , Potasio/química , Conformación Proteica , Ratas , Vanadatos/químicaRESUMEN
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. [Vanmolkot, K. R., et al. (2003) Ann. Neurol. 54, 360-366], 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 steady-state E(1) <==> E(2) poise of the enzyme is shifted toward E(1). 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 alpha2 pump isoform due to either decreased K(+) affinity (T345A) or catalytic turnover (R689Q and M731T), thus causing a delay in extracellular K(+) clearance and/or altered localized Ca(2+) handling/signaling secondary to reduced activity in colocalized Na(+)/Ca(2+) exchange.
Asunto(s)
Hemiplejía/genética , Hemiplejía/metabolismo , Trastornos Migrañosos/genética , Trastornos Migrañosos/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Adenosina Trifosfato/metabolismo , Alelos , Animales , Células HeLa , Humanos , Técnicas In Vitro , Cinética , Modelos Moleculares , Mutación Missense , Fenotipo , Potasio/metabolismo , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sodio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/química , TransfecciónRESUMEN
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. 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 alpha2 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 (approximately 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.
Asunto(s)
Hemiplejía/genética , Trastornos Migrañosos/genética , Mutación Missense , ATPasa Intercambiadora de Sodio-Potasio/genética , Sustitución de Aminoácidos , Animales , Línea Celular , Células HeLa , Humanos , Cinética , Ratas , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , TransfecciónRESUMEN
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 gamma 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 gamma mediates the Na+ affinity effect. Here we use a novel approach involving synthetic transmembrane mimetic peptides to demonstrate unequivocally that the TM domain of gamma effects the shift in apparent Na+ affinity. Specifically, we show that incubation of these peptides with membranes containing alphabeta pumps modulates Na+ affinity in a manner similar to transfected full-length gamma subunit. Using mutated gamma 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.
Asunto(s)
Péptidos/química , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Adenosina Trifosfato/química , Secuencia de Aminoácidos , Animales , Arginina/química , Biotinilación , Western Blotting , Línea Celular , Membrana Celular/metabolismo , Citoplasma/metabolismo , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Glicina/química , Células HeLa , Humanos , Riñón/metabolismo , Cinética , Magnesio/metabolismo , Datos de Secuencia Molecular , Mutación , Pruebas de Precipitina , Estructura Terciaria de Proteína , Ratas , Sodio/metabolismo , Sodio/farmacología , TransfecciónRESUMEN
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.
Asunto(s)
ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Secuencia de Aminoácidos , Animales , Células HeLa , Humanos , Cinética , Fragmentos de Péptidos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
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.
Asunto(s)
Proteínas de la Membrana/fisiología , ATPasa Intercambiadora de Sodio-Potasio/fisiología , Animales , Antibacterianos/farmacología , Colon/enzimología , Colon/metabolismo , Inhibidores Enzimáticos/farmacología , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Higromicina B/farmacología , Inmunohistoquímica , Riñón/enzimología , Riñón/metabolismo , Cinética , Ouabaína/farmacología , Pruebas de Precipitina , Ratas , Radioisótopos de Rubidio , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , TransfecciónRESUMEN
The two variants of the gamma subunit of the rat renal sodium pump, gamma(a) and gamma(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) <--> 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 gamma were expressed in rat alpha1-transfected HeLa cells. At the N terminus, the variant-distinct region was deleted (gammaNDelta7) or replaced by alanine residues (gammaN7A). At the C terminus, four (gamma(a)CDelta4) or ten (gamma(a)CDelta10) 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 (gammaNDelta7, gamma(a)CDelta4, and gamma(a)CDelta10) abolished the decrease in K'(ATP) seen with wild-type gamma(a) or gamma(b). It is concluded that different regions of the gamma chain mediate the distinct functional effects of gamma, 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 Mg(2+)-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 gamma but not alphabeta pumps to the cell surface and abrogates functional effects of gamma on alphabeta pumps. These findings underscore a potentially important role of gamma in affecting solute transport, in this instance Mg(2+) reabsorption, consequent to its primary effect on the sodium pump.