Cysteine Modification 37
tor (Cymes et al. 2005 ). Crystal structures have been solved for several Cys-loop
receptor homologues (Unwin 2005 ; Bocquet et al. 2009 ; Hilf and Dutzler 2008 ,
2009 ; Hibbs and Gouaux 2011 ; Unwin and Fujiyoshi 2012 ), although for most, it
is uncertain which conformational state has been crystallized (Akabas 2013 ). In
general, the residues aligned with those identified in the SCAM experiments are
the channel-lining residues in these crystal structures. Thus, the crystal structures
have validated the results of the SCAM studies.
SCAM studies have also raised questions about the crystal structures. For all
but the Torpedo nACh receptor, the crystals have been grown from detergent solu-
bilized protein. One issue that remains uncertain, is the extent to which detergent
solubilization alters the protein structure (Cross et al. 2011 ). Certainly, lipid com-
position affects ACh receptor function (Baenziger et al. 2008 ), so its absence in the
detergent solubilized proteins may affect structure in subtle ways (Labriola et al.
2013 ). An additional issue, is the extent to which cryopreservation of crystals alters
protein packing in crystal structures. Structures of cryopreserved crystals appear
more tightly packed than in structures performed on crystals of the same protein
at room temperature (Fraser et al. 2011 ). SCAM studies of the GLIC protein M2
segment suggest that it is more loosely packed than suggested by the x-ray crystal
structures (Parikh et al. 2011 ).
Localization of the Charge Selectivity Filter The Cys-loop receptor SCAM
experiments also provided insight into the location of the charge selectivity filter
that allows the channels to distinguish between cations and anions. It appears
to be distinct from the major determinants of single channel conductance. ACh,
5-HT 3 , GLIC and ELIC channels are cation-selective. In contrast, GABA, gly-
cine and GluCl channels are anion-selective. Early SCAM experiments showed
that sulfhydryl reagents with the opposite charge to the channel selectivity could
enter the channel from the extracellular side and react with residues between one-
third and two-thirds of the distance through the transmembrane channel (Akabas
et al. 1992 , 1994a; Xu and Akabas 1996 ; Reeves et al. 2001 ). This suggested
that the region responsible for the charge selectivity was located in or near the
cytoplasmic entrance to the transmembrane channel. Consistent with the charge
selectivity filter being near the cytoplasmic end of the channel, we and others
showed that extracellularly applied Zn2+ blocked anion-selective GABAA α1β1
receptors by interacting with channel-lining histidine residues at the M2 17’ posi-
tion, near the extracellular end of the channel (Wooltorton et al. 1997 ; Horenstein
and Akabas 1998 ). This suggested that residues lining the extracellular channel
vestibule did not have a major role in the anion vs cation selectivity of the chan-
nels. Experiments swapping residues between anion and cation selective family
members identified three residues that played a major role in charge selectivity.
Swapping these residues, two were near the cytoplasmic end of the channel, − 1’
and − 2’, and the other was at the 13’ position converted the charge selectivity
from cation to anion or visa versa (Galzi et al. 1992 ; Corringer et al. 1999 ; Gun-
thorpe and Lummis 2001 ; Keramidas et al. 2002 ; Sunesen et al. 2006 ). Much
attention has focused on the − 1’ position because it is generally a glutamate in