Establishment of cell-free electrophysiology for ion transporters: application for pharmacological profiling.

Ion transporters are emerging targets of increasing importance to the pharmaceutical industry because of their relevance to a wide range of numerous indications of cardiovascular, metabolic, and inflammatory diseases. However, traditional ion transporter assay technologies using radioactive or fluorescent ligands and substrates or manual patch clamping suffer from several problems: limited sensitivity and robustness, significant numbers of false positives and false negatives, and cost. The authors describe a novel method for the measurement of ion transporters using cell-free electrophysiology based on the SURFE (2) R (surface electrogenic event reader) technology platform. The main advantages of the method described here are high sensitivity and simple handling. Material for assays is mainly a simple membrane preparation, which can be stored over weeks and months. Thus, the application of the method does not depend on a permanently running cell-culture lab. The application of the technology itself uses a bench-top system and chips loaded with membrane fragments. The SURFE (2) R technology was used to establish an Na+/Ca2+-exchanger assay. The assay performance, as judged by the Z' value of 0.73 and the signal-to-background ratio of 7.6, suggests that this is a reliable and robust assay. The authors compared the technology with patch-clamp experiments: The measurement of activity of 17 different inhibitors and the determination of an IC (50)value indicated a good correlation between SURFE (2) R technology and patch clamp results. Using the SURFE (2) R technology, results were obtained with 20 times higher throughput and required less-qualified personnel compared with manual patch clamping.

Light signaling in scotopic conditions in the rabbit, mouse and rat retina: a physiological and anatomical study.

In the dark, light signals are conventionally routed through the following circuit: rods synapse onto rod bipolar (RB) cells, which in turn contact AII amacrine cells. AII cells segregate the light signal into the on and off pathways by making electrical synapses with on cone bipolar (CB) cells and glycinergic inhibitory chemical synapses with off CB cells. These bipolar cells synapse onto their respective ganglion cells, which transfer on and off signals to the visual centers of the brain. Two alternative pathways have recently been postulated for the signal transfer in scotopic conditions: 1) electrical coupling between rods and cones, and 2) a circuit independent of cone photoreceptors, implying direct contacts between rods and off CB cells. Anatomical evidence supports the existence of both these circuits. To investigate the contribution of these alternative pathways to scotopic vision in the mammalian retina, we have performed patch-clamp recordings from ganglion cells in the dark-adapted retina of the rabbit, mouse, and rat. Approximately one-half of the ganglion cells in the rabbit retina received off signals through a circuit that was independent of RB cells. This was shown by their persistence in the presence of the glutamate agonist 2-amino-4-phosphonobutyric acid (APB), which blocks rod-->RB cell signaling. Consistent with this result, strychnine, a glycine receptor antagonist, was unable to abolish these off responses. In addition, we were able to show that some off cone bipolar dendrites terminate at rod spherules and make potential contacts. In the mouse retina, however, there seems to be a very low proportion of off signals carried by an APB-resistant pathway. No ganglion cells in the rat retina displayed APB- and strychnine-resistant responses. Our data support signaling through flat contacts between rods and off CB cells as the alternative route, but suggest that the significance of this pathway differs between species.