Interaction of picrotoxin with GABA(A) receptor channel-lining residues probed in cysteine mutants

M. Xu, D. F. Covey, Myles Akabas

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Abstract

We used the substituted-cysteine-accessibility method to identify the channel-lining residues in a region (257-261) near the putative cytoplasmic end of the M2 membrane-spanning segment of the rat γ-aminobutyric acid type A (GABA(A)) receptor α1 subunit. The residues α1Val257 and α1Thr261 were accessible to charged, sulfhydryl-specific reagents applied extracellularly in both the open and closed states. The accessibility of α1V257C and α1T261C in the closed state implies that the gate must be at least as close to the cytoplasmic end of the channel as α1Val257. Also, the positively charged reagent methanethiosulfonate ethylammonium penetrated from the extracellular end of the channel to α1T261C, with which it reacted, indicating that the anion-selectivity filter is closer to the cytoplasmic end of the channel than this residue is. Co-application of picrotoxin prevented the sulfhydryl reagents from reacting with α1V257C but did not prevent reaction with the more extracellular residue α1T261C. Picrotoxin protection of α1V257C may be due to steric block by picrotoxin bound in the channel at the level of α1Val257; however, if this protection is allosteric, it is not due to the induction of the resting closed state in which α1V257C was accessible to sulfhydryl reagent.

Original languageEnglish (US)
Pages (from-to)1858-1867
Number of pages10
JournalBiophysical Journal
Volume69
Issue number5
StatePublished - 1995
Externally publishedYes

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Picrotoxin
Sulfhydryl Reagents
GABA-A Receptors
Cysteine
Aminobutyrates
Anions
Membranes

ASJC Scopus subject areas

  • Biophysics

Cite this

Interaction of picrotoxin with GABA(A) receptor channel-lining residues probed in cysteine mutants. / Xu, M.; Covey, D. F.; Akabas, Myles.

In: Biophysical Journal, Vol. 69, No. 5, 1995, p. 1858-1867.

Research output: Contribution to journalArticle

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