The central nervous system convulsant picrotoxin (PTX) inhibits GABAA and glutamate-gated Cl− channels in a use-facilitated fashion, whereas PTX inhibition of glycine and GABAC receptors displays little or no use-facilitated block. We have identified a residue in the extracellular aspect of the second transmembrane domain that converted picrotoxin inhibition of glycine α1 receptors from non-use-facilitated to use-facilitated. In wild type α1 receptors, PTX inhibited glycine-gated Cl− current in a competitive manner and had equivalent effects on peak and steady-state currents, confirming a lack of use-facilitated block. Mutation of the second transmembrane domain 15′-serine to glutamine (α1(S15′Q) receptors) converted the mechanism of PTX blockade from competitive to non-competitive. However, more notable was the fact that in α1(S15′Q) receptors, PTX had insignificant effects on peak current amplitude and dramatically enhanced current decay kinetics. Similar results were found in α1(S15′N) receptors. The reciprocal mutation in the β2 subunit of α1β2 GABAA receptors (α1β2(N15′S) receptors) decreased the magnitude of use-facilitated PTX inhibition. Our results implicate a specific amino acid at the extracellular aspect of the ion channel in determining use-facilitated characteristics of picrotoxin blockade. Moreover, the data are consistent with the suggestion that picrotoxin may interact with two domains in ligand-gated anion channels. The central nervous system convulsant picrotoxin (PTX) inhibits GABAA and glutamate-gated Cl− channels in a use-facilitated fashion, whereas PTX inhibition of glycine and GABAC receptors displays little or no use-facilitated block. We have identified a residue in the extracellular aspect of the second transmembrane domain that converted picrotoxin inhibition of glycine α1 receptors from non-use-facilitated to use-facilitated. In wild type α1 receptors, PTX inhibited glycine-gated Cl− current in a competitive manner and had equivalent effects on peak and steady-state currents, confirming a lack of use-facilitated block. Mutation of the second transmembrane domain 15′-serine to glutamine (α1(S15′Q) receptors) converted the mechanism of PTX blockade from competitive to non-competitive. However, more notable was the fact that in α1(S15′Q) receptors, PTX had insignificant effects on peak current amplitude and dramatically enhanced current decay kinetics. Similar results were found in α1(S15′N) receptors. The reciprocal mutation in the β2 subunit of α1β2 GABAA receptors (α1β2(N15′S) receptors) decreased the magnitude of use-facilitated PTX inhibition. Our results implicate a specific amino acid at the extracellular aspect of the ion channel in determining use-facilitated characteristics of picrotoxin blockade. Moreover, the data are consistent with the suggestion that picrotoxin may interact with two domains in ligand-gated anion channels. γ-aminobutyric acid γ-aminobutyric acid, types A and C picrotoxin transmembrane domain picoAmperes Glycine receptors belong to a superfamily of ligand-gated chloride channels that include GABAA1 receptors, GABAC receptors, and glutamate-gated chloride channels (1Rajendra S. Lynch J.W. Schofield P.R. Pharmacol. Ther. 1997; 73: 121-146Crossref PubMed Scopus (266) Google Scholar). In native tissue, glycine receptors exist as either α homomers or αβ heteromers (1Rajendra S. Lynch J.W. Schofield P.R. Pharmacol. Ther. 1997; 73: 121-146Crossref PubMed Scopus (266) Google Scholar). They comprise five subunits (usually three α subunits and two β subunits) arranged asymmetrically around the ion pore. Each subunit is made up of a large extracellular N-terminal region, four transmembrane domains (TM), and a large cytoplasmic domain; TMII forms the channel lumen (2Langosch D. Thomas L. Betz H. Proc. Natl. Acad. Sci. U. S. A. 1988; 185: 7394-7398Crossref Scopus (324) Google Scholar). Glycine receptors are targets of therapeutics such as anesthetics as well as toxins like the central nervous system convulsant picrotoxin (1Rajendra S. Lynch J.W. Schofield P.R. Pharmacol. Ther. 1997; 73: 121-146Crossref PubMed Scopus (266) Google Scholar). Picrotoxin inhibits all known anionic ligand-gated Cl−channels (3Etter A. Cully D.F. Liu K.K. Reiss B. Vassilatis D.B. Schaeffer J.M. Arena J.P. J. Neurochem. 1999; 72: 318-326Crossref PubMed Scopus (91) Google Scholar, 4ffrench-Constant R.H. Mortlock D.P. Shaffer C.S. Macintyre R.J. Roush R.T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7209-7213Crossref PubMed Scopus (264) Google Scholar, 5Inoue M. Akaike N. Neurosci. Res. 1988; 5: 380-394Crossref PubMed Scopus (65) Google Scholar). The mechanism of action and the exact location of picrotoxin binding are still unknown (6Pribilla I. Takagi T. Langosch D. Bormann J. Betz H. EMBO J. 1992; 11: 4305-4311Crossref PubMed Scopus (314) Google Scholar, 7Yoon K.W. Covey D.F. Rothman S.M. J. Physiol. (Lond.). 1993; 464: 423-439Crossref Scopus (131) Google Scholar, 8Akaike N. Hattori K. Oomura Y. Carpenter D.O. Experientia (Basel). 1985; 41: 70-71Crossref PubMed Scopus (117) Google Scholar, 9Krishek B.J. Moss S.J. Smart T.G. Neuropharmacology. 1996; 35: 1289-1298Crossref PubMed Scopus (81) Google Scholar, 10Simmonds M.A. Eur. J. Pharmacol. 1980; 80: 347-358Crossref Scopus (87) Google Scholar, 11Dillon G.H. Im W.B. Carter D.B. McKinley D.D. Br. J. Pharmacol. 1995; 115: 539-545Crossref PubMed Scopus (36) Google Scholar, 12Newland C.F. Cull-Candy S.G. J. Physiol. (Lond.). 1992; 447: 191-213Crossref Scopus (177) Google Scholar). However, several studies have indicated that TMII is the probable site for picrotoxin action (6Pribilla I. Takagi T. Langosch D. Bormann J. Betz H. EMBO J. 1992; 11: 4305-4311Crossref PubMed Scopus (314) Google Scholar, 13Lynch J.W. Rajendra S. Bany P.H. Schofield P.R. J. Biol. Chem. 1995; 270: 13799-13806Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 14Enz R. Bormann J. Neuroreport. 1995; 6: 1569-1572Crossref PubMed Scopus (27) Google Scholar, 15ffrench-Constant R.H. Rocheleau T.A. Steichen J.C. Chalmers A.E. Nature. 1993; 363: 449-451Crossref PubMed Scopus (472) Google Scholar, 16Gurley D. Amin J. Ross P. Weiss D.S. White G. Receptor Channels. 1995; 3: 13-20PubMed Google Scholar, 17Reddy G.L. lwamoto T. Tomich J.M. Montal M. J. Biol. Chem. 1993; 268: 14608-14615Abstract Full Text PDF PubMed Google Scholar, 18Wang T.-L. Hackam A.S. Guggino W.S. Cutting G.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11751-11755Crossref PubMed Scopus (89) Google Scholar, 19Wang T.L. Guggino W.B. Cutting G.R. J. Neurosci. 1994; 14: 6524-6531Crossref PubMed Google Scholar, 20Wang C.-T. Zhang H.-G. Rocheleau T.A. ffrench-Constant R.H. Jackson M.B. Biophys. J. 1999; 77: 691-700Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 21Dong C. Werblin F. Vision Res. 1996; 36: 3997-4005Crossref PubMed Scopus (22) Google Scholar, 22Cully D.F. Vassilatis D.K. Liu K.K. Paress P.S. Van der Ploeg L.H.T. Schaeffer J.S. Arena J.P. Nature. 1994; 371: 707-711Crossref PubMed Scopus (597) Google Scholar) (Fig. 1). For example, the TMII of the glycine β subunit was found to be responsible for conferring resistance to picrotoxin in heteromeric glycine αnβ receptors (n = 1–3) (6Pribilla I. Takagi T. Langosch D. Bormann J. Betz H. EMBO J. 1992; 11: 4305-4311Crossref PubMed Scopus (314) Google Scholar). Subsequent work has defined the existence of a phenylalanine residue at the 6′ position of the TMII glycine β subunit in conferring insensitivity to picrotoxin (16Gurley D. Amin J. Ross P. Weiss D.S. White G. Receptor Channels. 1995; 3: 13-20PubMed Google Scholar). In addition, other TMII residues (2′ and 19′) have also been implicated directly or indirectly in the mechanism by which picrotoxin inhibits these channels (13Lynch J.W. Rajendra S. Bany P.H. Schofield P.R. J. Biol. Chem. 1995; 270: 13799-13806Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 15ffrench-Constant R.H. Rocheleau T.A. Steichen J.C. Chalmers A.E. Nature. 1993; 363: 449-451Crossref PubMed Scopus (472) Google Scholar, 16Gurley D. Amin J. Ross P. Weiss D.S. White G. Receptor Channels. 1995; 3: 13-20PubMed Google Scholar). The mutations at positions 2′ and 19′ have been shown to affect the type of the inhibition (competitive versus non-competitive) by picrotoxin in GABAC and glycine α1 receptors, respectively (13Lynch J.W. Rajendra S. Bany P.H. Schofield P.R. J. Biol. Chem. 1995; 270: 13799-13806Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 18Wang T.-L. Hackam A.S. Guggino W.S. Cutting G.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11751-11755Crossref PubMed Scopus (89) Google Scholar). The ability of some antagonists to block channel activity is enhanced when the channel is open. This trait is generally referred to as a use-dependent or use-facilitated block and suggests that the site of action of the antagonist may be in the channel picrotoxin block of GABAC and glycine α1 receptors displays or no (13Lynch J.W. Rajendra S. Bany P.H. Schofield P.R. J. Biol. Chem. 1995; 270: 13799-13806Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 21Dong C. Werblin F. Vision Res. 1996; 36: 3997-4005Crossref PubMed Scopus (22) Google picrotoxin inhibition of GABAA receptors and glutamate-gated Cl− channels is use-facilitated (3Etter A. Cully D.F. Liu K.K. Reiss B. Vassilatis D.B. Schaeffer J.M. Arena J.P. J. Neurochem. 1999; 72: 318-326Crossref PubMed Scopus (91) Google Scholar, 5Inoue M. Akaike N. Neurosci. Res. 1988; 5: 380-394Crossref PubMed Scopus (65) Google Scholar, 11Dillon G.H. Im W.B. Carter D.B. McKinley D.D. Br. J. Pharmacol. 1995; 115: 539-545Crossref PubMed Scopus (36) Google Scholar, 12Newland C.F. Cull-Candy S.G. J. Physiol. (Lond.). 1992; 447: 191-213Crossref Scopus (177) Google Scholar). The trait is to the mechanism that use-facilitated blockade by of in picrotoxin on the TMII domain as the probable that the of picrotoxin blockade. In of the TMII domain of Cl− channels of the ligand-gated ion channel that GABAA and glutamate-gated channels have residues and at the these receptors use-facilitated block by Glycine α1 receptors, which use-facilitated picrotoxin have a residue at the We that and mutations in the TMII of glycine α1 receptors use-facilitated block by In addition, the mutation converted the picrotoxin block from competitive to non-competitive. This position also the mechanism of the picrotoxin blockade in GABAA receptors, receptors the reciprocal mutation in the β2 subunit of α1β2 GABAA receptors use-facilitated block in wild type receptors. Our data the of the position in the mechanism of picrotoxin block and that PTX may be at two in ligand-gated anion channels. The wild type glycine α1 was a from H. The α1 were by N. L. and R. A. glycine had been the S.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The mutation of the to in the GABAA β2 subunit was and by which are were were as Liu D. N. Pharmacol. Google Scholar). of all glycine and GABAA receptors was the C. H. Biol. PubMed Scopus Google Scholar). of was in the were were made at were at of were to a resistance of for The were in a on the of and at with the Cl− from the of the were with a were at on and a and on a for the that resistance or at the of and on the current to a This was the a in resistance was the the was and the data were in the The or with or PTX was in the extracellular and was from by to a of the the of the at the is G.H. J. 1999; PubMed Scopus Google Scholar). were with the a was the of PTX on the was were by at to from the and of receptors from Glycine and were for receptors the and the and current at a the or in is the and is the The of picrotoxin was by The data were to the is the steady-state current at a of is the current by is the picrotoxin that is and is the and picrotoxin were from work has shown that mutations at the position affect glycine to S.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the glycine for the wild the and α1(S15′N) receptors The and for glycine in the wild type receptors were and The of the 15′-serine by glutamine a in the glycine The of at position a in the for The for all receptors are in and picrotoxin inhibition in wild type and glycine and GABAA for and picrotoxin are in PTX is the when by the or when by a wild in a for and picrotoxin are in PTX is the when by the or when by a wild In wild type α1 receptors, picrotoxin the use-facilitated that in GABAA and glutamate-gated Cl−channels (13Lynch J.W. Rajendra S. Bany P.H. Schofield P.R. J. Biol. Chem. 1995; 270: 13799-13806Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). We a lack of use-facilitated block in the shown in picrotoxin inhibited the peak as well as the steady-state current by glycine with in the wild type receptors. Picrotoxin the of current at a of The of the mutation had effects on the picrotoxin blockade of the glycine of of picrotoxin with glycine in α1(S15′Q) receptors had little on the peak current a current decay to (Fig. In addition, the picrotoxin enhanced the decay of the current The of picrotoxin a in the of the current decay from with PTX to with PTX (Fig. the for the decay was to the of We also the of picrotoxin on the current by glycine in α1(S15′N) receptors. The to use-facilitated block was in α1(S15′N) receptors as well (Fig. the of or the mechanism by which picrotoxin inhibits glycine receptors. The α1(S15′Q) mutation had on picrotoxin inhibition to that with the α1(S15′N) and had a on glycine (Fig. the were α1(S15′Q) of block in α1(S15′Q) receptors is on glycine and picrotoxin of current decay is when the channel is by glycine when by glycine a trait consistent with use-facilitated blockade. the for use-facilitated block picrotoxin is to picrotoxin were with a also the data for the in a in current decay by when glycine is from to data are from a of three in and in We the glycine the of the block by picrotoxin in α1(S15′Q) receptors. the channel was by picrotoxin enhanced the glycine current decay to a of the α1(S15′Q) receptors were with a glycine picrotoxin a of the decay of the current to a of The of current decay with in glycine the use-facilitated of picrotoxin blockade in the be that the mutation has the glycine by glycine binding the In the data a use-facilitated block be in the for glycine and picrotoxin in the wild type with the receptors. the current in wild type and α1(S15′Q) receptors. The was in the two receptors (n = and (n = in wild type and receptors, a in glycine is responsible for the of use-facilitated block in glycine α1(S15′Q) receptors. A TMII mutation has been shown to the mechanism of picrotoxin blockade in α1 glycine receptors from competitive to (13Lynch J.W. Rajendra S. Bany P.H. Schofield P.R. J. Biol. Chem. 1995; 270: 13799-13806Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). The enhanced picrotoxin with of glycine as is of inhibition. the of picrotoxin inhibition in wild type and receptors. In picrotoxin inhibited the current by the glycine with of the glycine the glycine the picrotoxin to the to confirming the of competitive inhibition in glycine α1 receptors (13Lynch J.W. Rajendra S. Bany P.H. Schofield P.R. J. Biol. Chem. 1995; 270: 13799-13806Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). In receptors, picrotoxin inhibited the current by the glycine with of However, when the channel was with a of glycine the picrotoxin was to the to (Fig. the mutation converted the mechanism of picrotoxin block from competitive to non-competitive. A of the effects of picrotoxin in the is in I. The ability of picrotoxin to site is in the of the in GABAA and glutamate-gated Cl− channels (3Etter A. Cully D.F. Liu K.K. Reiss B. Vassilatis D.B. Schaeffer J.M. Arena J.P. J. Neurochem. 1999; 72: 318-326Crossref PubMed Scopus (91) Google Scholar, 5Inoue M. Akaike N. Neurosci. Res. 1988; 5: 380-394Crossref PubMed Scopus (65) Google Scholar). However, picrotoxin site channel in glycine α1 receptors (13Lynch J.W. Rajendra S. Bany P.H. Schofield P.R. J. Biol. Chem. 1995; 270: 13799-13806Abstract Full Text Full Text PDF PubMed Scopus (107) Google in the We the mutation the channel of picrotoxin were in picrotoxin for the glycine and picrotoxin were to the still in is from with picrotoxin the use-facilitated aspect of block (Fig. = A was found when whereas the mutation a use-facilitated picrotoxin picrotoxin from site in the channel The of or at the position of TMII in glycine α1 receptors converted the mechanism of picrotoxin blockade to use-facilitated inhibition. We to the effects of the reciprocal mutation in GABAA receptors. The wild type GABAA α1 subunit has a residue at the of the wild type α1 subunit with the subunit a GABAA with residues at is equivalent to the wild type glycine at the The mutation a in picrotoxin inhibition of wild type α1β2 use-facilitated inhibition (Fig. In receptors, blockade by picrotoxin was to that in wild type glycine α1 receptors. However, the magnitude of the use-facilitated block was In wild type receptors, the from the to decreased the picrotoxin In in GABAA receptors, the had no on picrotoxin The results the of residue in the mechanism of picrotoxin blockade of glycine and GABAA receptors. The mechanism of picrotoxin in ligand-gated anion channels is a on the fact that the of picrotoxin block is in the of the in GABAA and glutamate-gated Cl− has been that picrotoxin as channel (3Etter A. Cully D.F. Liu K.K. Reiss B. Vassilatis D.B. Schaeffer J.M. Arena J.P. J. Neurochem. 1999; 72: 318-326Crossref PubMed Scopus (91) Google Scholar, 5Inoue M. Akaike N. Neurosci. Res. 1988; 5: 380-394Crossref PubMed Scopus (65) Google Scholar). However, channel has shown that picrotoxin the that is of a channel C.F. Cull-Candy S.G. J. Physiol. (Lond.). 1992; 447: 191-213Crossref Scopus (177) Google Scholar). In addition, picrotoxin inhibition is in these channels (3Etter A. Cully D.F. Liu K.K. Reiss B. Vassilatis D.B. Schaeffer J.M. Arena J.P. J. Neurochem. 1999; 72: 318-326Crossref PubMed Scopus (91) Google Scholar, 12Newland C.F. Cull-Candy S.G. J. Physiol. (Lond.). 1992; 447: 191-213Crossref Scopus (177) Google Scholar, 13Lynch J.W. Rajendra S. Bany P.H. Schofield P.R. J. Biol. Chem. 1995; 270: 13799-13806Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). other have little or no use-facilitated block by picrotoxin in glycine and GABAC receptors, respectively (13Lynch J.W. Rajendra S. Bany P.H. Schofield P.R. J. Biol. Chem. 1995; 270: 13799-13806Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 21Dong C. Werblin F. Vision Res. 1996; 36: 3997-4005Crossref PubMed Scopus (22) Google Scholar). picrotoxin at site to a or of these channels G.H. Im W.B. Carter D.B. McKinley D.D. Br. J. Pharmacol. 1995; 115: 539-545Crossref PubMed Scopus (36) Google Scholar, 12Newland C.F. Cull-Candy S.G. J. Physiol. 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Neurochem. 1999; 72: 318-326Crossref PubMed Scopus (91) Google R.H. Rocheleau T.A. Steichen J.C. Chalmers A.E. Nature. 1993; 363: 449-451Crossref PubMed Scopus (472) Google use-facilitated block by block was on the peak glycine current was by enhanced current decay glycine current decay was picrotoxin current decay in the of picrotoxin was on glycine and the magnitude of picrotoxin block was on glycine of these is in wild type glycine α1 receptors, and all are in GABAA and glutamate-gated Cl− channels (3Etter A. Cully D.F. Liu K.K. Reiss B. Vassilatis D.B. Schaeffer J.M. Arena J.P. J. Neurochem. 1999; 72: 318-326Crossref PubMed Scopus (91) Google Scholar, 7Yoon K.W. Covey D.F. Rothman S.M. J. Physiol. (Lond.). 1993; 464: 423-439Crossref Scopus (131) Google Scholar, 11Dillon G.H. Im W.B. Carter D.B. McKinley D.D. Br. J. 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