Understanding ion channels: The Cysteine Scanning Accessibility Method for eejits - Part The Third (whereupon I finally arrive at my point)

The Cysteine Scanning Accessibility Method involves the use of site-directed mutagenesis to substitute individual amino acids within a protein for Cys and then probing whether or not the introduced Cys side chain is subsequently accessible to water-soluble thiol-reactive agents. Conventionally, this involves generating a series of mutant ion channel subunits bearing single Cys point mutations at different amino acid positions along a stretch of the polypeptide. Each of these mutant subunits is then individually expressed into a model cell line (e.g. HEK293, or CHO) to form the functional mutant ion channels, and the accessibility of the introduced Cys to water-soluble thiol-reactive assessed.  Usually, assessment focuses on whether the function of the ion channel is altered by exposure to water-soluble thiol-reactive agents, and the principal method for studying channel function is patch clamp electrophysiology. Assuming that a manufactured Cys mutant receptor retains the ability to open and close, one can test whether the transmembrane currents evoked by gating the ion channel are modified by the addition of thiol-reactive chemicals, such as the methanethiosulfonates. Generally, one is simply looking to see whether the application of a thiol-reactive agent to a channel bearing an introduced Cys side chain will change its current profile in someway. For example, addition of the thiol-reactive chemical may cause a decrease in the current ampl...
Source: Across the Bilayer - Category: Medical Scientists Source Type: blogs