38 M. H. Akabas
the cation-selective subunits and an alanine in the anion selective subunits. A
recent paper studying muscle-type nACh receptors pointed out the complicated
aspects of having four negatively charged residues in close proximity near the
cytoplasmic channel mouth (Cymes and Grosman 2012 ). They suggested that the
glutamates adopt alternative rotamer conformations so that only two are close
to the channel axis where they could influence selectivity and conductance. To
date, the Cys-loop crystal structures have not provided significant insight into
the mechanism by which the channels selectively allow either cations or anions
to permeate through the channel. This may be due in part to the fact that the
crystal structures are likely to represent either closed, desensitized or other non-
conducting channel states (Gonzalez-Gutierrez and Grosman 2010 ; Parikh et al.
2011 ; Akabas 2013 ).
Localization of the Picrotoxin Binding Site Picrotoxin is a plant derived toxin
that acts as an open channel blocker of Cys-loop receptors, particularly the anion-
selective family members. It is a roughly spherical, rigid compact molecule with a
diameter of about 8 Å. ffrench-Constant and coworkers had shown that a mutation
in the Drosophila GABAA receptor M2 segment 2’ position conferred resistance to
cyclodiene insecticides, such as dieldrin, and to picrotoxin (ffrench-Constant et al.
1993a, b; Zhang et al. 1994 ). Using SCAM, we showed that picrotoxin could pro-
tect a cysteine substituted at the GABAA receptor α1 M2-2’ position from modifi-
cation by extracellularly applied pCMBS but picrotoxin did not protect a cysteine
substituted at the 6’ position (Xu et al. 1995 ). Consistent with the SCAM results,
substitution of tryptophan residues at the 6’ position inhibited picrotoxin binding
(Gurley et al. 1995 ). We inferred that picrotoxin was an open channel blocker that
bound near the cytoplasmic end of the channel, below the 6’ level. This implies that
the channel diameter at the 2’ level should be about 8 Å (Xu et al. 1995 ). Based on
the size of permeant anions, the narrowest diameter in the GABAA receptor channel
was inferred to be 5.6 Å (Bormann et al. 1987 ). We subsequently showed that pic-
rotoxin could be trapped in the closed channel resulting in slowed reopening after
picrotoxin trapping (Bali and Akabas 2007 ). This allowed us to conclude that in the
closed state there is a constriction between the picrotoxin binding site and the extra-
cellular end of the channel that prevents release of bound picrotoxin in the closed
state (Bali and Akabas 2007 ). The GluCl channel was crystallized in the presence
of picrotoxin (Hibbs and Gouaux 2011 ). As inferred from SCAM and other experi-
ments, the picrotoxin binding site is formed by M2 segment residues from the − 2’
to the 2’ level (Fig. 4 ). Similar approaches have been used to identify ligand bind-
ing sites in channels and transporters (Javitch et al. 1995 ; Teissere and Czajkowski
2001 ; Bali and Akabas 2004 ).
Agonist Binding Site Residues Studies using affinity labeling reagents and pho-
toaffinity labeling had identified a subset of residues in the acetylcholine receptor
binding site (Kao et al. 1984 ; Kao and Karlin 1986 ; Dennis et al. 1988 ; Langen-
buch-Cachat et al. 1988 ; Galzi et al. 1990 ; Czajkowski and Karlin 1991 , 1993 ). Kar-
lin and coworkers used cysteines engineered into the ACh binding site to measure