Cysteine Modification 29
and screening of a large number of mutants and functional characterization of the
mutants. We have found that with ion channels, the Xenopus laevis expression sys-
tem is very robust for SCAM experiments and the simplicity of doing two electrode
voltage clamp recording makes it especially attractive for ion channel studies. Its
major limitations are time resolution for solutions changes, ~ 0.5–1 s, and the lack
of access to the cytoplasmic regions of the channel. Other investigators have suc-
cessfully used transient expression in cell culture systems and patch clamp record-
ing. This allows faster solution exchange times and with inside-out patches, access
to residues from the cytoplasmic side of the channel. For bacterial channel-forming
toxins the planar lipid bilayer system has been extensively used (Jakes et al. 1990 ;
Kienker et al. 2008 ; Mindell et al. 1994 ; Nassi et al. 2002 ; Slatin et al. 1994 ).
In general, cysteine substitution has been well tolerated at numerous positions in
countless membrane proteins, including channels, transporters and receptors. While
cysteine substitution rarely causes total disruption of protein folding/expression, it
may cause alterations in channel function that depend on the specific site of the sub-
stitution. One fundamental assumption for SCAM experiments is that a substituted
cysteine acts as a good reporter for the accessibility of the endogenous amino acid
that it is replacing. How strong this assumption is depends, in part, on the extent to
which the mutation perturbs the protein structure. In the absence of an x-ray crystal
structure of the wild type and cysteine substitution mutant proteins, one can try to
infer the extent to which the mutation perturbs the structure from its effects on chan-
nel function. However, it is often difficult to correlate the magnitudes of functional
and structural changes (Brown et al. 1993 ). For example, in the nicotinic acetylcho-
line receptor (nAChR) α subunit M2 segment, the apparent affinity for acetylcho-
line (ACh) measured in 20 consecutive cysteine substitution mutants, from α1E241
(− 1’) to α1E262 (20’), ranged from 22-fold less to 50-fold greater than wild type
(Akabas et al. 1994a). Functional changes could arise from structural changes on
the order of angstroms or changes in the energetics of side chain interactions with
minimal perturbation of the local or global protein structure (Brown et al. 1993 ).
In fact, because all of the nAChR mutants responded to ACh, we inferred that their
overall structure was similar to wild type receptors. An additional consideration is
that channels have multiple functional states, closed/open/desensitized. The reac-
tivity of a cysteine could be different in different functional states. Mutations may
have a greater impact on the relative energy/stability of one or more states. This
could change the probability that under a given set of experimental conditions, e.g.,
a given agonist concentration, the relative occupancy of the functional states is the
same for a set of cysteine substitution mutants. Thus, knowledge of the functional
effects of the individual cysteine substitutions is important in the design of the ac-
tual experimental protocols for determining sulfhydryl reagent reaction rates and
for interpretation of the experimental data.
A major consideration in conducting SCAM experiments is the choice of sulfhy-
dryl reactive reagents to be used. Many SCAM experiments have used the methane-
thiosulfonates (MTS) derivatives that were used in the original experiments (Aka-
bas et al. 1992 , 1994a, b; Xu and Akabas 1993 ; Mindell et al. 1994 ; Kuner et al.