M. Xu et al., INTERACTION OF PICROTOXIN WITH GABA(A) RECEPTOR CHANNEL-LINING RESIDUES PROBED IN CYSTEINE MUTANTS, Biophysical journal, 69(5), 1995, pp. 1858-1867
We used the substituted-cysteine-accessibility method to identify the
channel-lining residues in a region (257-261) near the putative cytopl
asmic end of the M2 membrane-spanning segment of the rat gamma-aminobu
tyric acid type A (GABA(A)) receptor alpha(1) subunit. The residues al
pha(1)Val257 and alpha(1)Thr261 were accessible to charged, sulfhydryl
-specific reagents applied extracellularly in both the open and closed
states. The accessibility of alpha(1)V257C and alpha(1)T261C in the c
losed state implies that the gate must be at least as close to the cyt
oplasmic end of the channel as alpha(1)Val257. Also, the positively ch
arged reagent methanethiosulfonate ethylammonium penetrated from the e
xtracellular end of the channel to alpha(1)T261C, with which it reacte
d, indicating that the anion-selectivity filter is closer to the cytop
lasmic end of the channel than this residue is. Go-application of picr
otoxin prevented the sulfhydryl reagents from reacting with alpha(1)V2
57C but did not prevent reaction with the more extracellular residue a
lpha(1)T261C. Picrotoxin protection of alpha(1)V257C may be due to ste
ric block by picrotoxin bound in the channel at the level of alpha(1)V
al257; however, if this protection is allosteric, it is not due to the
induction of the resting closed state in which alpha(1)V257C was acce
ssible to sulfhydryl reagent.