Ion flux assays

Pyrethroid insecticides (deltamethrin, NRDC 157, cismethrin), DDT analogs ( p,j> -DDT, (>,j> -DDT, methoxychlor, EDO), and a DDT-pyrethroid hybrid compound (GH401) enhanced veratridine-dependent sodium uptake by mouse brain synaptosomes The effectiveness of these compounds in the sodium uptake assay was in good agreement with their acute mammalian toxicities. , -DDT also enhanced veratridine-dependent sodium uptake by fish brain synaptosomes These findings demonstrate the utility of ion flux assays to study interactions of insecticides with sodium channels in the central nervous system and to explore species differences in insecticide target site sensitivity ... 

Second, the time-course of the assays are often of different dimensions depolarizations and ion fluxes are measured in fractions of minutes, while tumor promotion or receptor regulation may take tens of minutes to hours. 

In combination with methods such as fluorescence and flux assays, the new automated patch clamp electrophysiology systems have revolutionized the way in which ion channels are now screened. However, the future is not without major challenges, and important limitations of these technologies remain. 

Gill S, GiU R, Sen Lee S et al (2003) Flux assays in high throughput screening of ion channels in drug discovery. Assay Drug Dev Technol 1(5) 709-717... 

Our preliminary results with fish brain preparations suggest that ion flux techniques may be valuable in studies of target site differences between species. We have demonstrated veratridine-stimulated, tetrodotoxin-sensitive sodium uptake in a vesicular preparation from fish brain, thus confirming the presence of functional sodium channels in this preparation. Our results with , -DDT in this system also agree well with the action of DDT analogs and pyrethroids in mouse brain assays. Further studies wih both preparations should allow the exploration of target site differences between mammals and fish that have been inferred from whole animal toxicity studies. 

Table 6.6 lists voltage-gated sodium channelopathies (disease, mutations and phenotype), Table 6.7 describes the VGSC gene family and Table 6.8 describes ion-chatmel assay technologies, such as electrophysiology, membrane-potential measurement, flux assays, and binding assays. 

C-guanidine flux assay Scintillation counter 96-weII (20000), channel or delay Toxin to activate flux through inactivation hfeasurement of ion rather than relatively high-throughput, amenable to automation End-point assay, continuous functional recording... 

Developing an assay that measures the translocation or the flux of a substrate is more difficult the smaller the compartments. The difficulty peaks when it comes to detecting transport or ion channel function of a translocator in proUs. Some of the trouble results from the fact that with prolis the researcher does not know at which step the experiment failed. Was the translocator not incorporated into prolis in the correct conformation and orientation or was the flux protocol not suitable In addition, ion channels involve quick fluxes and rare membrane proteins (see Table 4.2). Often only few of the vesicles contain an ion channel. The experimenter has to distinguish the flux in this small volume from the nonspecific flux in the large remainder of the vesicle population. It helps a little bit to check whether other properties of the translocator were reconstituted (e.g., enzyme activity or binding sites). However, there is not necessarily a connection often (for example) a binding site of the translocator is reconstituted but not its translocation function. The question remains open whether the reconstitution failed or the flux assay was unsuitable. In the development of a flux assay, the following controls should be carried out independently of the type of compartment and translocator. 

Several methods are available today to test candidate ion channel active drugs (ICADs) electrophysiology (patch clamp), binding assays, radioactive flux assays, membrane potential-sensitive fluorescent dyes, ion-sensitive dyes, and voltage sensing based on fluorescence resonant energy transfer (FRET). 

Figure 15.2. Drosophila larval neuromuscular junction system. A wandering third-instar larva is dissected open to reveal the ventral neuromusculature (see Figure 15.1). The peripheral nerve is severed and stimulated with a glass suction electrode. The muscle is recorded from in two-electrode voltage-clamp (TEVC) configuration. The postsynaptic excitatory junctional current (EJC) is recorded to assay synaptic transmission bottom inset). Top inset) Basis of synaptic transmission event being evoked by nerve stimulation and recorded via ion flux through muscle glutamate receptors.

Fluorescence-based methods do not directly measure ionic current but, rather, measure either membrane-potential-dependent or ion-concentration-dependent changes of fluorescence signals (from fluorescent dyes loaded into the cytosol or cell membrane) as a result of ionic flux. Because fluorescence-based methods give robust and homogeneous cell population measurement, these assays are relatively easy to set up and achieve high throughput. 

Rubidium exhibits a very high flux through potassium ion channels. Upon depolarization, this flux can be quantified as a measure of channel integrity (Tang et al. 2001 Chang et al. 2002). Fluorometric assays have been developed that can detect drug-induced effects on hERG channels. 

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