40 M. H. Akabas
Conformational Change During Gating and Gate Location Another powerful
use of SCAM is to detect channel regions that undergo conformational changes
during activation or desensitization. This is best done by measurement of the sulf-
hydryl reagent reaction rates in the absence and presence of agonist. One important
assumption is that in the absence of agonist the channels are predominantly in the
closed/resting state. It is important to recognize that several factors that may affect
this assumption. First, the cysteine substitution mutations may alter the channel’s
open probability. In the nACh receptor α1 M2 segment, we found that at some posi-
tions cysteine substitutions altered the apparent affinity for ACh by several orders
of magnitude (Akabas et al. 1992 , 1994a). We inferred to be due to a change in
the probability of channel opening, the spontaneous open probability, rather than a
change in ACh binding affinity. Thus, for some of the nACh receptor M2 segment
mutants, the spontaneous open probability was significant even in the absence of
agonist. Thus, MTS reagent reaction was not necessarily occurring in the closed
state even though no agonist was present. A second issue that must be considered
is that the sulfhydryl reagents themselves might act as agonists. Even if they act as
weak agonists, reaction that occurs in the ostensible absence of agonist may actu-
ally be occurring in the open state due to channel opening by the sulfhydryl reagents
interacting with the agonist binding site. The extent of opening may be small, but
if the reaction rate is high in the open state then reagents may appear to react in the
closed state even though the channels are only open for a small percentage of the
time. For example, many quaternary ammonium compounds act as nACh receptor
agonists (Karlin 1989 ). MTSET is a quaternary ammonium compound. It activates
measurable currents with some nACh receptor α1 M2 segment cysteine mutants.
Thus, attempts to measure its closed state reaction rate is complicated at some posi-
tions by the reagent’s ability to open the channels (Akabas et al. 1994a). These two
issues may have complicated attempts to use SCAM to determine the location of the
channel gate as will be discussed below.
Determining the state(s) in which cysteine modification occurs in the presence of
agonist is complicated because in the presence of agonist, channels undergo rapid
transitions between open, desensitized and closed states. Thus, for reaction rates
measured in the presence of agonist it is difficult to know whether reaction is taking
place in a specific state or in multiple states. Only if the rates are significantly dif-
ferent would it be potentially feasible to detect that in the measured rates, i.e. rates
that require multi-exponential fits. To be safe, one can refer to rates measured in the
presence of agonist as activated state rates, with the understanding that they poten-
tially constitute an average of rates in multiple states weighted by the proportion of
time the channel spends in each state and the relative reactivity of the cysteine in
each state.
Investigators have long sought to identify the location of the closed channel
gate in Cys-loop receptors. SCAM studies in the nACh and GABAA receptors
showed that channel-lining residues were accessible to charged sulfhydryl re-
agents in both the absence and presence of agonist (Akabas et al. 1994a; Xu and
Akabas 1996 ). Because the sulfhydryl reagents were applied from the extracellu-