13 research outputs found
Untersuchungen zur Spannungsabhängigkeit von Bacteriorhodopsin und Na<sup>+</sup>/K<sup>+</sup>ATPase
Voltage dependence of proton pumping by bacteriorhodopsin is regulated by the voltage-sensitive ratio of M1 to M2.
The voltage dependence of light-induced proton pumping was studied with bacteriorhodopsin (bR) from Halobacterium salinarum, expressed in the plasma membrane of oocytes from Xenopus laevis in the range -160 mV to +60 mV at different light intensities. Depending on the applied field, the quenching effect by blue light, which bypasses the normal photo and transport cycle, is drastically increased at inhibiting (negative) potentials, and is diminished at pump current increasing (positive) potentials. At any potential, two processes with different time constants for the M --> bR decay of approximately 5 ms (tau1) and approximately 20 ms (tau2) are obtained. At pump-inhibiting potentials, a third, long-lasting process with tau3 approximately 300 ms at neutral pH is observed. The fast processes (tau1, tau2) can be assigned to the decay of M2 in the normal pump cycle, i.e., to the reprotonation of the Schiff base via the cytoplasmic side, whereas tau3 is due to the decay of M1 without net pumping, i.e., the reprotonation of the Schiff base via the extracellular side. The results are supported by determination of photocurrents induced by bR on planar lipid films. The pH dependence of the slow decay of M1 is fully in agreement with the interpretation that the reprotonation of the Schiff base occurs from the extracellular side. The results give strong evidence that an externally applied electrical field changes the ratio of the M1 and the M2 intermediate. As a consequence, the transport cycle branches into a nontransporting cycle at negative potentials. This interpretation explains the current-voltage behavior of bR on a new basis, but agrees with the isomerisation, switch, transfer model for vectorial transport
An automatic electrophysiological assay for the neuronal glutamate transporter mEAAC1
A rapid and robust electrophysiological assay based on solid supported membranes (SSM) for the murine neuronal glutamate transporter mEAAC1 is presented. Measurements at different concentrations revealed the EAAC1 specific affinities for l-glutamate (Km = 24 μM), l-aspartate (Km = 5 μM) and Na+ (Km = 33 mM) and an inhibition constant Ki for dl-threo-β-benzyloxyaspartic acid (TBOA) of 1 μM. Inhibition by 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid (HIP-B) was not purely competitive with an IC50 of 13 μM. Experiments using SCN− concentration jumps yielded large transient currents in the presence of l-glutamate showing the characteristics of the glutamate-gated anion conductance of EAAC1. Thus, SSM-based electrophysiology allows the analysis of all relevant transport modes of the glutamate transporter on the same sample. K+ and Na+ gradients could be applied to the transporter. Experiments in the presence and absence of Na+ and K+ gradients demonstrated that the protein is still able to produce a charge translocation when no internal K+ is present. In this case, the signal amplitude is smaller and a lower apparent affinity for l-glutamate of 144 μM is found. Finally the assay was adapted to a commercial fully automatic system for SSM-based electrophysiology and was validated by determining the substrate affinities and inhibition constants as for the laboratory setup. The combination of automatic function and its ability to monitor all transport modes of EAAC1 make this system an universal tool for industrial drug discovery
Electrogenic Ion Pumps Investigated on a Solid Supported Membrane: Comparison of Current and Voltage Measurements
Current and voltage measurements were performed on Na,K-ATPase and sarcoplasmic reticulum (SR) Ca-ATPase. Measurements of current transients under short-circuit conditions and of voltage transients under open-circuit conditions were carried out by employing a solid supported membrane (SSM). Purified membrane fragments containing Na,K-ATPase or native SR vesicles were adsorbed on a SSM and were activated by performing substrate concentration jumps. Current and voltage transients were recorded in the external circuit, that are related to pump activity, and can be attributed to electrogenic events in the reaction cycles of the two enzymes. While current transients of very small amplitude are difficult to detect, the corresponding voltage transients can be measured with higher accuracy thanks to a much more favourable signal to noise ratio. Therefore, voltage measurements are preferable for the investigation of slow processes generating low current signals, e.g. for the analysis of low turnover transporters
Electrogenic Ion Pumps Investigated on a Solid Supported Membrane: Comparison of Current and Voltage Measurements
Transporter Assays Using Solid Supported Membranes: A Novel Screening Platform for Drug Discovery
Transporters are important targets in drug discovery. However, high throughput-capable assays for this class of membrane proteins are still missing. Here we present a novel drug discovery platform technology based on solid supported membranes. The functional principles of the technology are described, and a sample selection of transporter assays is discussed: the H(+)-dependent peptide transporter PepT1, the gastric proton pump, and the Na(+)/Ca(2+) exchanger. This technology promises to have an important impact on the drug discovery process
Transport Proteins on Solid-Supported Membranes: From Basic Research to Drug Discovery
Solid-supported membranes (SSM) can be used as capacitive electrodes for the investigation of electrogenic transport proteins (ion pumps). Membrane fragments or liposomes that contain the transport protein are adsorbed on the SSM. The proteins are activated by supplying a substrate via a rapid solution exchange at the SSM. The charge translocations during the reaction cycle of the transport protein can be measured via the capacitance of the SSM. The SSM and the adsorbed membrane fragments or proteoliposomes represent a rugged structure that can be used in basic research and drug discovery. On the basis of a few examples we demonstrate how this technique can be applied to the investigation of the transport mechanism of membrane proteins. In addition, we present the application of the system in drug discovery. A sensor based on the SSM technology represents a promising system for the rapid screening of pharmaceutically relevant compounds for transport proteins