RESUMO
A partially purified preparation of the lobster muscle Na(+)/Ca(2+) exchanger was reconstituted with, presumably, random orientation in liposomes. Ca(2+) efflux from (45)Ca-loaded vesicles was studied in exchanger molecules in which the transporter cytoplasmic surface was exposed to the extravesicular (ev) medium. Extravesicular Na(+) (Na(ev))-dependent Ca(2+) efflux depended directly upon the extravesicular Ca(2+) concentration ([Ca(2+)](ev)) with a half-maximal activation at [Ca(2+)](ev) = 0.6 microm. This suggests that the lobster muscle exchanger is catalytically upregulated by cytoplasmic Ca(2+), as in most other species. In contrast, at low [Na(+)](ev), the Ca(ev)-binding site (i.e., on the cytoplasmic surface) for Ca(2+) transported via Ca(2+)/Ca(2+) exchange was half-maximally activated by about 7.5 microm Ca(2+). Mild proteolysis of the Na(+)/Ca(2+) exchanger by alpha-chymotrypsin also upregulated the Na(ev)-dependent Ca(2+) efflux. Following proteolytic digestion in Ca-free medium, the exchanger was no longer regulated by nontransported ev Ca(2+). Proteolytic digestion in the presence of 1.9 microm free ev Ca(2+), however, induced only a 1. 6-fold augmentation of Ca(2+) efflux, whereas, after digestion in nominally Ca-free medium, a 2.3-fold augmentation was observed; Ca(2+) also inhibited proteolytic degradation of the Na(+)/Ca(2+) exchanger measured by immunoblotting. These data suggest that Ca(2+), bound to a high affinity binding site, protects against the activation of the Na(+)/Ca(2+) exchanger by alpha-chymotrypsin. Additionally, we observed a 6-fold increase in the Na(+)/Ca(2+) exchange rate, on average, when the intra- and extravesicular salt concentrations were increased from 160 to 450 mm, suggesting that the lobster muscle exchanger is optimized for transport at the high salt concentration present in lobster body fluids.
Assuntos
Músculo Esquelético/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Animais , Radioisótopos de Cálcio/metabolismo , Quimotripsina/metabolismo , Citoplasma/metabolismo , Nephropidae , ProteolipídeosRESUMO
The Na/Ca exchanger from lobster muscle crossreacts specifically with antibodies raised against the dog heart Na/Ca exchanger. Immunoblots of the lobster muscle and mammalian heart exchangers, following SDS-PAGE, indicate that the invertebrate and mammalian exchangers have similar molecular weights: about 120 kDa. The exchanger from lobster muscle was partially purified and functionally reconstituted into asolectin vesicles which were loaded with 160 mM NaCl. 45Ca uptake by these proteoliposomes was promoted by replacing 160 mM NaCl in the external medium with 160 mM KCl to produce an outwardly-directed Na+ concentration gradient. When the proteoliposomes were adsorbed onto black lipid membranes (BLM), and DM-Nitrophen-Ca2+ ("caged Ca2+") was added to the KCl medium, photolytically-evoked Ca2+ concentration jumps elicited transient electric currents. These currents corresponded to positive charge exiting from the proteoliposomes, and were consistent with the Na/Ca exchanger-mediated exit of 3 Na+ in exchange for 1 entering Ca2+. The current was dependent upon the Ca2+ concentration jump, the protein integrity, and the outwardly directed Na+ gradient. KCl-loaded proteoliposomes did not produce any current. Low external Na+ concentrations augmented the current, whereas Na+ concentrations > 25 mM reduced the current. The dependence of the current on free Ca2+ was Michaelis-Menten-like, with half-maximal activation (KM(Ca)) at < 10 microM Ca2+. Caged Sr2+ and Ba2+, but not Mg2+, also supported photolysis-evoked outward current, as did Ni2+, but not Mn2+. However, Mg2+ and Mn2+ augmented the Ca-dependent current, perhaps by facilitating the adsorption of proteoliposomes to the BLM. The Ca-dependent current was irreversibly blocked by La3+ (added as 200 microM DMN-La3+). The results indicate that the properties of the Na/Ca exchanger can be studied with these electro-physiological methods.
Assuntos
Acetatos/efeitos da radiação , Cálcio/metabolismo , Proteínas de Transporte/fisiologia , Etilenodiaminas/efeitos da radiação , Proteínas Musculares/fisiologia , Nephropidae/metabolismo , Fotólise , Proteolipídeos , Adsorção , Animais , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/isolamento & purificação , Cátions Bivalentes/farmacologia , Cinética , Lantânio/farmacologia , Bicamadas Lipídicas , Potenciais da Membrana/efeitos da radiação , Proteínas Musculares/isolamento & purificação , Fosfatidilcolinas , Fosfolipídeos , Cloreto de Potássio/metabolismo , Cloreto de Sódio/metabolismo , Trocador de Sódio e Cálcio , Raios UltravioletaAssuntos
Bacteriorodopsinas/fisiologia , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Canais Iônicos/fisiologia , Bicamadas Lipídicas , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Halobacterium salinarum/metabolismo , Halorrodopsinas , Cinética , Mamíferos , Matemática , Modelos BiológicosAssuntos
ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Rim/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Estômago/enzimologia , Animais , Condutividade Elétrica , Cinética , Bicamadas Lipídicas , Potenciais da Membrana , Modelos Biológicos , Potássio/farmacologia , Transdução de Sinais , SuínosAssuntos
Medula Renal/enzimologia , Potenciais da Membrana/fisiologia , ATPase Trocadora de Sódio-Potássio/fisiologia , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/fisiologia , Animais , Membrana Celular/química , Membrana Celular/enzimologia , Membrana Celular/fisiologia , Medula Renal/fisiologia , Medula Renal/ultraestrutura , Bicamadas Lipídicas/química , Sódio/metabolismo , SuínosRESUMO
Sarcoplasmic reticulum vesicles containing largely Ca2(+)-ATPase were incorporated into planar lipid membranes. The ATPase was activated by a UV flash-induced concentration jump of ATP from a photolabile caged ATP. Under these conditions stationary pump currents were observed. The dependence of these pump currents on applied voltages was investigated. The current-voltage curve of the Ca2(+)-ATPase shows monotonously increasing pump currents with increasing positive potentials of the ATP containing compartment. This indicates the existence of electrogenic steps in the direction of the transported Ca2+ ions. From the extrapolated reversal potentials of the curve is concluded that less than four positive net charges are transported per hydrolyzed ATP.