Abstract
Chemical modification of ion channels using the substituted cysteine accessibility method has a rich and successful history in elucidating the structural basis of ion channel function. In this approach, cysteine residues are introduced in regions of interest into the protein and their accessibility to water soluble thiol-reactive reagents is determined by monitoring ion channel activity. Because a wide range of these reagents are available with differing size, charge, and membrane solubility, the physio-chemical environment of the introduced cysteine residue and therefore the protein domain of interest can be probed with great precision. The approach has been widely employed for determining the secondary structure of specific ion channel domains, the location and nature of the channel gate, and the conformational rearrangements in the channel pore that underlie the opening/closing of the pore. In this chapter, we describe the use of these and related approaches to probe the functional architecture and gating of store-operated Orai1 channels.