34 M. H. Akabas
their reaction rates increase at higher pH (Gorin et al. 1966 ). A disadvantage of the
maleimides is, however, that they also react with deprotonated primary amines so
that at pH > 8 they may react with the deprotonated ε-amino group of lysines as well
as cysteines.
2.4 “Silent” Reaction
In SCAM experiments, the interpretation that reaction took place when application
of a sulfhydryl reagent causes a change in protein function is straightforward if
modification causes an irreversible functional effect. In contrast, the interpretation
of results at positions where application of a sulfhydryl reagent has no functional ef-
fect are somewhat ambiguous; Either no reaction occurred or reaction has occurred
but is functionally silent. Although this may be rare, we have observed positions in
the GABAA receptor, such as α1A280C, where application of MTSES has a large
functional effect, but application of MTSET has no functional effect (Bera et al.
2002 ). In this case we can apply the reagents sequentially, first the apparently “non-
reactive” MTSET and then the MTSES that has a functional effect. If application
of MTSES still had an effect, then we would have inferred that the MTSET did
not react, but in this case, application of MTSES had no functional effect, thus, we
inferred that the MTSET reacted but has no functional effect except to modify the
cysteine and make it unreactive with MTSES. Thus, interpretation of apparently
non-reactive positions must be done with care.
One other potential cause for “non-reactive” cysteines is that they may react with
endogenous thiols that are present in the protein biosynthetic compartments (ER,
Golgi, etc.) or form a heavy metal binding site. In our initial SCAM study of the
M6 segment of the cystic fibrosis transmembrane conductance regulator (CFTR)
we found that a cysteine substituted for T338 was not reactive (Cheung and Akabas
1996 ). This was somewhat surprising because based on the pattern of reactivity
of the rest of the residues in the M6 transmembrane segment it should have been
reactive. Subsequent studies by Dawson and colleagues showed that its lack of re-
activity was due to binding or reaction with unknown substances: For some T338C
channels application of reducing agents such as DTT or 2-ME created MTS reactive
channels but for some T338C channels reduction had no effect on their lack of reac-
tion with MTS reagents (Liu et al. 2004 , 2006 ).
3 Main Body
3.1 Ligand-Gated Channels—Cys-Loop Receptors
The use of SCAM to study the structure and function of ion channels was pio-
neered in studies of members of the Cys-loop receptor superfamily of pentameric