Cysteine Modification 43
3.2 A Few Examples of SCAM in Other Channels
Extensive SCAM studies have been done in other ion channels, too many to de-
scribe in this chapter. A few highlight the versatility of the approach and empha-
size the unique types of information that can be obtained. In AMPA and NMDA
glutamate receptors SCAM studies identified channel-lining residues and dem-
onstrated that the accessibility of cysteines at aligned residues in different sub-
units was different (Kuner et al. 1996 ; Beck et al. 1999 ; Sobolevsky et al. 2002a,
b, 2005 ; Wollmuth and Sobolevsky 2004 ) This led to the hypothesis that the
subunits were not symmetrically arranged around the central channel axis (So-
bolevsky et al. 2002b). This prediction was borne out by the NMDA and AMPA
receptor crystal structures (Furukawa et al. 2005 ; Sobolevsky et al. 2009 ).
Finkelstein and colleagues have used SCAM and a variant of SCAM to identi-
fy the regions of bacterial channel-forming toxins that are translocated across the
lipid bilayer membrane during channel formation. These toxins, such as colicins
and diphtheria toxin, are synthesized as water soluble proteins and then insert
into membranes to form ion channels driven by either voltage or pH gradients
(Finkelstein 1994 , 2000 , 2009 ). To determine which parts of the proteins reached
the opposite side of the membrane, they biotinylated cysteines engineered into
the toxins and then added streptavidin to the opposite side of the planar bilayer
to see whether the biotinylated residue had crossed the membrane (Mindell et al.
1994 ; Qiu et al. 1994 , 1996 ; Slatin et al. 1994 ; Huynh et al. 1997 ; Senzel et al.
2000 ; Kienker et al. 2008 ). They have used this approach to establish the trans-
membrane topology of the resultant channels. Columbini and colleagues used a
similar approach to determine the transmembrane topology of the mitochondrial
outer membrane voltage dependent anion channel (VDAC) (Song et al. 1998a, b).
SCAM studies in bacterial channels such as those formed by anthrax toxin have
been used to establish the residues lining the β barrel pore formed by the toxin and
to demonstrate the translocation of lethal factor through the resultant β barrel chan-
nel (Nassi et al. 2002 ; Basilio et al. 2011 ). Using SCAM, Blount and coworkers
identified the residues critical for gating of the bacterial mechanosensitive MscL
channels (Levin and Blount 2004 ).
Deutsch and colleagues have used cysteine site-directed pegylation with sulf-
hydryl reactive pegylating reagents to determine the transmembrane topology of
potassium channels (Lu and Deutsch 2001 ). They have extended this approach to
their studies of nascent protein secondary structure in the ribosomal exit tunnel.
There, using reporter cysteines and pegylation mass tagging, they showed that
in the ribosomal exit tunnel the nascent potassium channels start to develop sec-
ondary structural elements (Gajewski et al. 2011 ; Lu and Deutsch 2005 ; Tu and
Deutsch 2010 ).