RESUMEN
Interactions of rat FXYD4 (corticosteroid hormone-induced factor (CHIF)), FXYD2 (gamma), or FXYD1 (phospholemman (PLM)) proteins with rat alpha1 subunits of Na(+),K(+)-ATPase have been analyzed by co-immunoprecipitation and covalent cross-linking. In detergent-solubilized membranes from HeLa cells expressing both gamma and CHIF or CHIF and hemagglutinin A-tagged CHIF, mixed complexes of CHIF and gamma or CHIF and hemagglutinin A-tagged CHIF with alpha/beta subunits are undetectable. This implies that the alpha/beta/FXYD protomer is the major species in detergent solution. A lipid-soluble cysteine-cysteine bifunctional reagent, dibromobimane, cross-links CHIF to alpha in colonic membranes but not gamma or PLM to alpha in kidney or heart membranes, respectively. Sequence comparisons of the FXYD proteins suggested that Cys-49 in the trans-membrane segment of CHIF could be involved. In detergent-solubilized HeLa cell membranes, dibromobimane cross-links wild-type CHIF to alpha but not the C49F mutant, and also the corresponding F36C mutant but not wild-type gammab, and F48C but not wild-type PLM. C140S, C338A, C804A, and C966S mutants of the alpha subunit have been expressed. Only the C140S mutant prevents cross-linking with CHIF. The data demonstrated the proximity of trans-membrane segments of CHIF, gamma, and PLM to M2 of alpha. Molecular modeling is consistent with location of the trans-membrane segment of all FXYD proteins between M2, M6, and M9 and the proximity of Cys-49 of CHIF or Phe-36 of gamma with Cys-140 of M2. Cross-linking also demonstrated CHIF-alpha and CHIF-beta proximities in extra-membrane regions, similar to the evidence for gamma-alpha and gamma-beta cross-links.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Canales de Potasio/metabolismo , Conformación Proteica , Subunidades de Proteína/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Secuencia de Aminoácidos , Animales , Membrana Celular/metabolismo , Reactivos de Enlaces Cruzados/metabolismo , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Modelos Moleculares , Datos de Secuencia Molecular , Fosfoproteínas/química , Fosfoproteínas/genética , Canales de Potasio/química , Canales de Potasio/genética , Subunidades de Proteína/química , Subunidades de Proteína/genética , Ratas , Alineación de Secuencia , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
This study describes specific intramolecular covalent cross-linking of the gamma to alpha and gamma to beta subunits of pig kidney Na,K-ATPase and rat gamma to alpha co-expressed in HeLa cells. For this purpose pig gammaa and gammab sequences were determined by cloning and mass spectrometry. Three bifunctional reagents were used: N-hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA), disuccinimidyl tartrate (DST), and 1-ethyl-3-[3dimethylaminopropyl]carbodiimide (EDC). NHS-ASA induced alpha-gamma, DST induced alpha-gamma and beta-gamma, and EDC induced primarily beta-gamma cross-links. Specific proteolytic and Fe(2+)-catalyzed cleavages located NHS-ASA- and DST-induced alpha-gamma cross-links on the cytoplasmic surface of the alpha subunit, downstream of His(283) and upstream of Val(440). Additional considerations indicated that the DST-induced and NHS-ASA-induced cross-links involve either Lys(347) or Lys(352) in the S4 stalk segment. Mutational analysis of the rat gamma subunit expressed in HeLa cells showed that the DST-induced cross-link involves Lys(55) and Lys(56) in the cytoplasmic segment. DST and EDC induced two beta-gamma cross-links, a major one at the extracellular surface within the segment Gly(143)-Ser(302) of the beta subunit and another within Ala(1)-Arg(142). Based on the cross-linking and other data on alpha-gamma proximities, we modeled interactions of the transmembrane alpha-helix and an unstructured cytoplasmic segment SKRLRCGGKKHR of gamma with a homology model of the pig alpha1 subunit. According to the model, the transmembrane segment fits in a groove between M2, M6, and M9, and the cytoplasmic segment interacts with loops L6/7 and L8/9 and stalk S5.
Asunto(s)
Reactivos de Enlaces Cruzados/química , Mapeo de Interacción de Proteínas/métodos , Subunidades de Proteína/química , ATPasa Intercambiadora de Sodio-Potasio/química , Secuencia de Aminoácidos , Animales , Análisis Mutacional de ADN , Células HeLa , Humanos , Datos de Secuencia Molecular , Subunidades de Proteína/metabolismo , Ratas , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , PorcinosRESUMEN
CHIF (corticosteroid hormone-induced factor) is a member of the FXYD family that shares approximately 50% homology with the gamma subunit of Na,K-ATPase. It is expressed in renal collecting duct and distal colon, and is upregulated by Na(+) deprivation and high K(+) diet. Both CHIF and gamma are coimmunoprecipitated by an anti-alpha subunit antibody, and alpha is immunoprecipitated by anti-gamma and anti-CHIF antibodies. (86)Rb(+) flux experiments in CHIF-transfected HeLa cells demonstrate that CHIF increases the affinity for cytoplasmic Na(+), but does not affect the affinity for extracellular K(Rb). A physiological role of CHIF in kidney function is further elucidated by the phenotypic analysis of CHIF knockout mice. Taken together with data by others, it appears that FXYD proteins are tissue-specific subunits or regulators of the Na,K-ATPase whose function is to adjust the pump kinetics to particular physiological needs.
Asunto(s)
Proteínas de la Membrana/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Secuencia de Aminoácidos , Células HeLa , Homeostasis , Humanos , Bombas Iónicas/metabolismo , Proteínas de la Membrana/química , Subunidades de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Rubidio/metabolismo , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
The recently defined FXYD protein family contains seven members that are small, single-span membrane proteins characterized by a signature sequence containing an FXYD motif and three other conserved amino acid residues. Until recently, the functional role of FXYD proteins was largely unknown, with the exception of the gamma subunit of Na,K-ATPase, which was shown to be a specific regulator of renal alpha1-beta1 isozymes. We have investigated whether other members of the FXYD family may have a similar role as the gamma subunit and have found that CHIF (corticosteroid hormone-induced factor, FXYD4), FXYD7, as well as phospholemman (FXYD1) specifically associate with Na,K-ATPase and preferentially with alpha1-beta isozymes in native tissues, and produce distinct effects on the transport properties of Na,K-ATPase that are adapted to the physiological demands of the tissues in which they are expressed. These results provide evidence for a unique and novel mode of regulation of Na,K-ATPase by FXYD proteins that involves a tissue-specific expression of an auxiliary subunit of distinct Na,K-ATPase isozymes.
Asunto(s)
Riñón/enzimología , Proteínas de la Membrana/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Secuencia de Aminoácidos , Animales , Colon , Secuencia Conservada , Mucosa Intestinal/enzimología , Isoenzimas/química , Isoenzimas/metabolismo , Lisosomas/enzimología , Neuronas/enzimología , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/químicaAsunto(s)
Proteínas de Unión al Calcio/metabolismo , Proteínas de la Membrana/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Sitios de Unión , Proteínas de Unión al Calcio/química , Bovinos , Membrana Celular/enzimología , Colon , Mucosa Intestinal/enzimología , Médula Renal/enzimología , Proteínas de la Membrana/química , Ratas , Sarcolema/enzimologíaAsunto(s)
Proteínas de la Membrana/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Sitios de Unión , Células HeLa , Humanos , Proteínas de la Membrana/química , Subunidades de Proteína , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/química , 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
Corticosteroid hormone-induced factor (CHIF) is a short epithelial-specific protein that is independently induced by aldosterone and a high-K(+) diet. It is a member of the FXYD family of single-span transmembrane proteins that include phospholemman, Mat-8, and the gamma-subunit of Na(+)-K(+)-ATPase. A number of studies have suggested that these proteins are involved in the regulation of ion transport and, in particular, functionally interact with the Na(+)-K(+)-ATPase. The present study describes the characterization, targeted disruption, and phenotypic analysis of the mouse CHIF gene. The CHIF knockout mice are viable and not distinguishable from wild-type littermates under normal conditions. Under K(+) loading, they have a twofold higher urine volume and an increased glomerular filtration rate. Similar but smaller effects are observed in mice fed a low-Na(+) diet. Treating K(+)-loaded mice for 10 days with furosemide resulted in lethality in the knockout mice (17 of 39) but not in the wild-type group (1 of 39). The data are consistent with an effect of CHIF on the Na(+)-K(+)-ATPase that is specific to the outer and inner medullary duct, its major expression site.
Asunto(s)
Proteínas de la Membrana/fisiología , Fenotipo , Secuencia de Aminoácidos , Animales , Sangre , Southern Blotting , Enzimas de Restricción del ADN/metabolismo , Dieta , Dieta Hiposódica , Embrión de Mamíferos/citología , Exones , Furosemida/farmacología , Tasa de Filtración Glomerular , Intrones , Médula Renal/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Concentración Osmolar , Potasio/administración & dosificación , Cloruro de Potasio/administración & dosificación , ARN Mensajero/análisis , ARN Mensajero/química , Análisis de Secuencia de ADN , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Células Madre/metabolismo , Transfección , OrinaRESUMEN
A family of small, single-span membrane proteins (the FXYD family) has recently been defined based on their sequence and structural homology. Some members of this family have already been identified as tissue-specific regulators of Na,K-ATPase (NKA). In the present study, we demonstrate that phospholemman (PLM) (FXYD1), so far considered to be a heart- and muscle-specific channel or channel-regulating protein, associates specifically and stably with six different alpha-beta isozymes of NKA after coexpression in Xenopus oocytes, and with alpha1-beta, and less efficiently with alpha2-beta isozymes, in native cardiac and skeletal muscles. Stoichiometric association of PLM with NKA occurs posttranslationally either in the Golgi or the plasma membrane. Interaction of PLM with NKA induces a small decrease in the external K+ affinity of alpha1-beta1 and alpha2-beta1 isozymes and a nearly 2-fold decrease in the internal Na+ affinity. In conclusion, this study demonstrates that PLM is a tissue-specific regulator of NKA that may play an essential role in muscle contractility.