Measuring Channel Activity

Channel activity is best studied electrochemically as charged species cross a cell membrane or artificial lipid bilayer. There is a difference in electrical potential between the interior and exterior of a cell leading to the membrane itself having a resting potential between -50 and -100 mV. This can be determined by placing a microelectrode inside the cell and measuring the potential difference between it and a reference electrode placed in the extracellular solution. Subsequent changes in electrical current or capacitance are indicative of a transmembrane flux of ions. 

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Patch-clamp technique for measuring channel activity... 

The interaction of ACh with the Torpedo nAChR. The data shown compare the equilibrium binding parameters obtained from fluorescence studies using covalently attached fluorescent probes and those obtained from radiolabelled [ H]ACh binding studies or functional measurements of cation flux. These data support a model in which the Torpedo nAChR carries sites of different affinities for ACh. We have previously suggested that occupancy of the lower affinity sites leads to channel activation whereas the higher affinity sites may play a role in desensitization processes... 

Subsequently the ion channel activity was tested by single-channel current measurements using planar lipid bilayers. Single-channel conductances of ca. 55 in 500 mM NaCl and 65 pS in KCl were obtained. The weak ion selectivity was claimed to reflect a slightly larger mobility of K compared to that of Na ion in bulk solution. Therefore a large 7.5-A pore structure in lipid bilayers is assumed to resemble the bulk aqueous solution. 

Clear description of the electrophysiological methods used to measure the activity of single ion channels, by the Nobel Prize-winning developers of this technique. 

Fig. 7 Biosynthesis of NATs and TRP channel activation by NATs. (a) Evidence for a fatty acyl CoA taurine A-acyltransferase activity was detected in mouse tissue by incubating taurine and arachidonoyl-CoA with various tissue lysates, (b) arachidonyl NAT was tested as an activator of the TRPV1 (black line), TRPV4 (gray line), and TRPM8 (dashed line) ion channels. Channel activation was measured using a Fura-2-based calcium-imaging assay, where the ratio between the fluorescence at 340 and 380 nm is reflective of cellular calcium concentrations...

A medium throughput approach to evaluating sodium channel activity is the measurement of sodium flux across cell membranes [103]. In these experiments, a tracer that permeates the channel and is easily quantifiable is used to analyze sodium influx. Traditionally, radioactive tracers such as 22Na+ or [14C]guanidinium have been used. Alternatively, Li+ can be used as a tracer and analyzed by atomic absorption spectrometry. Sodium flux assays can be used to test approximately 105 compounds per year. They offer a robust readout of channel activity, but lack voltage control and temporal resolution. To examine sodium channel blockade by measuring sodium flux,... 

This technique essentially comprises two aqueous compartments containing electrolyte connected by a pinhole across which a bilayer of phospholipid molecules is spread. An electrical potential is applied across the bilayer and in the presence of an ionophore the resultant current flowing between the two chambers is measured as a function of time.12 The method is often used to assess the activity of synthetic peptides, and has been used to measure the channel activity of several non-peptide channels. Of particular significance is its use in allowing the measurement of the single channel properties of isolated channel-forming proteins that have been reintroduced back into planar bilayer membranes.13... 

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