28 M. H. Akabas
2000 ; Riegelhaupt et al. 2010 ) and receptors (Javitch et al. 1995 ), in their native
lipid membrane environment. SCAM has provided a wealth of information on the
structure and conformational changes in ion channels and will be the focus of the
remainder of this chapter.
2 Approach/Methods
The Substituted Cysteine Accessibility Method (SCAM) assesses the chemical re-
activity of engineered cysteine residues with sulfhydryl reactive reagents (Akabas
et al. 1992 , 1994a, b; Xu and Akabas 1993 ; Xu and Akabas 1996 ; Karlin and Aka-
bas 1998 ). SCAM experiments require that either a functional or chemical assay
exist to assess the extent of reaction of the sulfhydryl reactive reagents with each
engineered cysteine. For ion channels, the assays have usually been electrophysi-
ological, using either macroscopic or single channel recording techniques. The spe-
cific assay does not matter as much as its ability to detect reproducibly the effects
of covalent modification of the engineered cysteine residues. Because of the labor
intensive nature of single channel analysis and the large number of cysteine mu-
tants investigated in typical SCAM experiments, most investigators have opted to
use macroscopic current recordings in their SCAM assays. While one can perform
SCAM experiments on isolated engineered cysteine residues, much of the power of
the approach comes from systematically investigating the residues in one region of
a protein, such as a membrane-spanning segment or those residues forming a ligand
binding site. Another powerful aspect of the approach is that one can probe the re-
activity of the substituted cysteine residues in different functional states, such as for
an ion channel; closed, open or desensitized/inactivated states.
An essential starting point for SCAM experiments is that the protein background
in which the cysteine mutants will be made, must not have sulfhydryl reactive cyste-
ines. This means that application of the sulfhydryl reagents to be used in the SCAM
experiments must not have a functional effect on the wild type background. If the
wild type protein is functionally effected by application of the sulfhydryl reagents
then one must first identify the reactive cysteine(s) (Javitch et al. 1994 ; Gkountelias
et al. 2010 ). Although a cysteine-free construct may seem desirable as a starting
point, if generating it requires multiple mutations that may create additional prob-
lems for protein function.
Performing a typical SCAM experiment involves several steps: (1) Generation
of cysteine substitution mutants, (2) heterologous expression of the mutants, (3)
functional characterization of the mutants, and (4) determination of their reactivity
with sulfhydryl reactive reagents. Interpretation of the data requires several assump-
tions. It is important to recognize that the choice of sulfhydryl reactive reagent(s)
can have a significant impact on the interpretation of the data, as will be discussed
below. The only comment I would make on the first two steps is that one should
choose a heterologous expression system that facilitates the process of expression