Voltage-dependent pump currents of the sarcoplasmic reticulum Ca²⁺-ATPase in planar lipid membranes

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

Voltage dependence of the Na⁺K⁺-ATPase incorporated into planar lipid membranes

The aim of the work presented here was to investigate the electrogenic properties and the current-voltage behavior of a highly purified preparation of pig kidney Na,K-ATPase reconstituted into artificial planar bilayer membranes

Charge transport of ion pumps on lipid bilayer membranes

Ion pumps create ion gradients across cell membranes while consuming light energy or chemical energy. The ion gradients are used by the corresponding cell types for passive-ion transport via ion channels or carriers or for accumulation of nutrients like sugar or amino acids via cotransport systems or antiporter

Regulatory Processes on the Cytosplasmic Surface of the Na⁺/Ca²⁺ Exchanger from Lobster Exoskeletal Muscle

A partially purified preparation of the lobster muscle Na+/Ca2+ exchanger was reconstituted with, presumably, random orientation in liposomes. Ca2+ efflux from 45Ca-loaded vesicles was studied in exchanger molecules in which the transporter cytoplasmic surface was exposed to the extravesicular (ev) medium. Extravesicular Na+ (Naev)-dependent Ca2+ efflux depended directly upon the extravesicular Ca2+ concentration ([Ca2+]ev) with a half-maximal activation at [Ca2+] ev = 0.6 μm. This suggests that the lobster muscle exchanger is catalytically upregulated by cytoplasmic Ca2+, as in most other species. In contrast, at low [Na+]ev , the Ca ev -binding site (i.e., on the cytoplasmic surface) for Ca2+ transported via Ca2+/Ca2+ exchange was half-maximally activated by about 7.5 μm Ca2+. Mild proteolysis of the Na+/Ca2+ exchanger by α-chymotrypsin also upregulated the Naev -dependent Ca2+ efflux. Following proteolytic digestion in Ca-free medium, the exchanger was no longer regulated by nontransported ev Ca2+. Proteolytic digestion in the presence of 1.9 μm free ev Ca2+, however, induced only a 1.6-fold augmentation of Ca2+ efflux, whereas, after digestion in nominally Ca-free medium, a 2.3-fold augmentation was observed; Ca2+ also inhibited proteolytic degradation of the Na+/Ca2+ exchanger measured by immunoblotting. These data suggest that Ca2+, bound to a high affinity binding site, protects against the activation of the Na+/Ca2+ exchanger by α-chymotrypsin. Additionally, we observed a 6-fold increase in the Na+/Ca2+ 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

Computer Assisted Instruction (CAI): The Bottom Line

The stated purpose of this report was to summarize the effectiveness and effects of CAI and CMI and the conditions under which they work best. It reviews the positive effects demonstrated by CAI evaluations, including four large-scale studies done by Educational Testing Service (ETS). Another section looks at the characteristics of quality courseware

ANWR: The Musical

This film is a lengthy rock opera sung by sock puppets about the long-running controversy over whether to drill for oil in the Arctic National Wildlife Refug

Electrical currents generated by a partially purified Na/Ca exchanger from lobster muscle reconstituted into liposomes and adsorbed on black lipid membranes: Activation by photolysis of caged Ca²⁺

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 DMNitrophen-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 halfmaximal activation (KM(Ca)) at 2+. Caged Sr2+ and Ba2+, but not Mg2+, also supported photolysisevoked outward current, as did Ni2+, but not Mn2+. However, Mg2+ and Mn2+ augmented the Cadependent current, perhaps by facilitating the adsorption of proteoliposomes to the BLM. The Ca-dependent current was irreversibly blocked by La3+ (added as 200 μm DMN-La3+). The results indicate that the properties of the Na/Ca exchanger can be studied with these electrophysiological methods