30 M. H. Akabas
1996 ; Liu et al. 1996 ; Larsson et al. 1996 ; Yamagishi et al. 1997 ; Beck et al. 1999 ;
Boileau et al. 1999 ; Nassi et al. 2002 ; Panicker et al. 2002 ; Sobolevsky et al. 2002a;
Kronengold et al. 2003a, b; Dodier et al. 2004 ). Other sulfhydryl reagents that have
been used successfully include mercurials, particularly p-chloromercuribenzenesul-
fonate (pCMBS) (Xu and Akabas 1996 ; Williams and Akabas 1999 ; Filippova et al.
2004 ; Goren et al. 2004 ; Parikh et al. 2011 ), maleimides (Sahin-Toth and Kaback
1993 ; Slatin et al. 1994 ; Senzel et al. 2000 ; Lu and Deutsch 2001 ; Skerrett et al.
2001 ; Blaustein 2002 ) and even ions such as Ag+ and Cd2+ (Lu and Miller 1995 ;
Contreras et al. 2008 ). Strengths and weaknesses for some of these reagents are
discussed in the following paragraphs.
2.1 Methanethiosulfonates
MTS reagents have been used to probe endogenous cysteine residues (Wolf and
Pollock 1964 ; Lewis et al. 1976 ; Small 1976 ; Kenyon and Bruice 1977 ; Hersh
et al. 1979 ; Kluger and Tsui 1980 ; Berliner 1983 ). The first use of MTS reagents in
SCAM experiments was by Akabas et al. ( 1992 ). They used three charged reagents,
MTS-ethylammonium (MTSEA), MTS-ethyltrimethylammonium (MTSET) and
MTS-ethylsulfonate (MTSES). The original three reagents are small and will fit
into a right cylinder 0.6 nm in diameter and 1 nm in length (Akabas et al. 1992 ; Kar-
lin and Akabas 1998 ). Thus, they can hopefully enter the lumen of most ion chan-
nels. Subsequently, a large number of MTS reagents were synthesized by several
companies (Toronto Research Chemicals, Inc., http://www.trc-canada.com/index.
php; Biotium http://biotium.com/)..) The larger MTS derivatives that are now avail-
able can be used to assess steric factors related to access to an engineered cysteine
(Riegelhaupt et al. 2010 ).
The thiolate anion reacts by an SN2 nucleophilic attack on the MTS reagents
forming a mixed disulfide (Fig. 1a). As a result, MTS reagents react with the thio-
late anion 5 × 109 times faster than with the uncharged thiol (Roberts et al. 1986 ;
Stauffer and Karlin 1994 ; Karlin and Akabas 1998 ). Only thiols that are at least
transiently on the water accessible protein surface will ionize to any significant
extent. Thus, MTS reactive cysteine residues are likely to be, at least transiently,
on the water accessible protein surface. As a caveat, it should be noted that water
can penetrate into crevices and cavities within the transmembrane domain of an
integral membrane protein. Thus, MTS reagents may be able to react with cys-
teines substituted for residues that in a crystal structure appear to be located in
the protein interior (Williams and Akabas 1999 , 2000 , 2001 , 2002 ). Furthermore,
the local electrostatic environment can alter the ionization state of a substituted
cysteine, so this must be considered when comparing MTS reagent reaction rates
with different cysteine substitution mutants. For example, in the GABAA recep-
tor an Arg residue on the backside of the channel-lining M2 segment is in close
proximity to residues in the M1 membrane-spanning segment of the adjacent sub-
unit. The presence or absence of the positively charged residue at the M2-19’ Arg