A B S T R A C T Experiments were designed to characterize several partial reactions of the Na,K-ATPase and to demonstrate that a model can be defined that reproduces most of the transport features of the pump with a single set of kinetic parameters. We used the fluorescence label 5-iodoacetamidofluorescein, which is thought to be sensitive to conformational changes, and the styryl dye RH 421, which can be applied to detect ion-binding and -release reactions. In addition transient electric currents were measured, which are associated mainly with the E l ~ E2 conformational transition. Numerical simulations were performed on the basis of a reaction model, that has been developed from the Post-Albers cycle. Analysis of the experimental data allows the determination of several rate constants of the pump cycle. Our conclusions may be summarized as follows: (a) binding of one Na + ion at the cytoplasmic face is electrogenic. This Na + ion is specifically bound to a neutral binding site with an affinity of 8 mM in the presence of 10 mM Mg 2+. In the absence of divalent cations, the intrinsic binding affinity was found to be 0.7 mM. (b) The analysis of fluorescence experiments with the cardiotonic steroid strophanthidin indicates that the 5-iodoacetamidofluorescein label monitors the conformational transition (Na3)EI-P ---, P-Ez(Na2), which is accompanied by the release of one Na + ion. 5-IAF does not respond to the release of the subsequent two Na + ions, which can be monitored by the RH 421 dye. These experiments indicate further that the conformational transition E,P ~ P-E2 is the rate limiting process of the Na + translocation. The corresponding rate constant was determined to be 22 s -1 at 20~ From competition experiments with cardiotonic steroids, we estimated that the remaining 2 Na § ions are released subsequently with a rate constant of at least 5,000 s -1 from their negatively charged binding sites. (c) Comparing the fluorescence experiments with electric current transients, which were performed at various Na concentrations in the absence and presence of strophanthidin, we found that the transition (Na3)'EI-P --, P-E2"(Na2) is the major charge translocating step in the reaction sequence Na3'El ~ (Na3)'E1-P ~ P-E2"(Na~) ~ P-E 2. The subsequent release of 2 Na + ions contributed less than 25% to the total electric current transient. 198 THE JOURNAL OF GENERAL PHYSIOLOGY 9 VOLUME 104 9 1994 binding can be explained by a kinetic model. A quantitative description has been obtained under the assumption that these inhibitors bind only to the states P-E~(Na2) and P-E~ (K~). (e) Most of our experiments can be described by a modified Post-Albers scheme. A set of the kinetic parameters in this scheme has been determined by the experiments presented or by data from literature. Numerical simulations using this set are consistent with the presented data.

G protein-coupled receptors (GPCRs) constitute an abundant family of membrane receptors of high pharmacological interest. Cell-based assays are the predominant means of assessing GPCR activation, but are limited by their inherent complexity. Functional molecular assays that directly and specifically report G protein activation by receptors could offer substantial advantages. We present an approach to immobilize receptors stably and with defined orientation to substrates. By surface plasmon resonance (SPR), we were able to follow ligand binding, G protein activation, and receptor deactivation of a representative GPCR, bovine rhodopsin. Microcontact printing was used to produce micrometer-sized patterns with high contrast in receptor activity. These patterns can be used for local referencing to enhance the sensitivity of chip-based assays. The immobilized receptor was stable both for hours and during several activation cycles. A ligand dose-response curve with the photoactivatable agonist 11-cis-retinal showed a half-maximal signal at 120 nM. Our findings may be useful to develop novel assay formats for GPCRs based on receptor immobilization to solid supports, particularly to sensor surfaces.

Rhodopsin-transducin coupling was used as an assay to investigate a laterally patterned membrane reconstituted with a receptor and its G protein. It served as a model system to show the feasibility to immobilize G protein-coupled receptors on solid supports and investigate receptor activation and interaction with G proteins by one-dimensional imaging surface plasmon resonance. Supported membranes were formed by the self-assembly of lipids and rhodopsin from detergent solution onto functionalized gold surfaces. They formed micrometer-sized alternating regions of pure fluid phospholipid bilayers separated by bilayers composed of an outer phospholipid leaflet on a gold-attached inner thiolipid. Rhodopsin was found to incorporate preferentially into the phospholipid bilayer regions, whereas transducin was uniformly distributed over the entire outer surface of the supported patterned membrane. The influence of rhodopsin on the dark binding of transducin to lipid membranes was described quantitatively and compared with previously published data. Coupling reactions with transducin resembled closely the native system, indicating that the native functionality of rhodopsin was preserved in the supported membranes. The spatially varying properties of the membranes resulted in a pattern of rhodopsin activity on the surface. This combination of techniques is very promising for the investigation of the lateral diffusion of transducin, can be extended to include signalling proteins downstream of the G protein, and may be applied to functional screening of other G protein-coupled receptors. In the future, it may also serve as a basis for constructing biosensors based on receptor proteins.