Identification of a Novel Residue within the Second Transmembrane Domain That Confers Use-facilitated Block by Picrotoxin in Glycine α1 Receptors
2002; Elsevier BV; Volume: 277; Issue: 11 Linguagem: Inglês
10.1074/jbc.m111356200
ISSN1083-351X
AutoresMohammed Dibas, Eric B. Gonzales, Paromita Das, Cathy L. Bell-Horner, Glenn H. Dillon,
Tópico(s)Epilepsy research and treatment
ResumoThe 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 lumen. Whereas picrotoxin block of GABAC and glycine α1 receptors displays weak or no use-facilitation (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 inhibition of GABAA receptors and glutamate-gated Cl− channels is strongly 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 molecular basis underlying this trait is unknown. Thus, we sought to determine the mechanism that confers use-facilitated blockade by picrotoxin. Because of its prominent role in picrotoxin action, we focused on the TMII domain as the probable region that would underlie the differing mechanisms of picrotoxin blockade. In our analysis of the TMII domain of Cl− channels of the ligand-gated ion channel superfamily, we observed that GABAA and glutamate-gated channels have neutral acidic polar residues (Asn and Gln) at the 15′ position; these receptors both demonstrate use-facilitated block by picrotoxin. Glycine α1 receptors, which do not display use-facilitated picrotoxin blockade, have a dissimilar residue (Ser) at the 15′ position. We demonstrate here that S15′Q and S15′N mutations in the TMII of glycine α1 receptors confer use-facilitated block by picrotoxin. In addition, the S15′Q mutation converted the picrotoxin block from competitive to non-competitive. This position also affects the mechanism of the picrotoxin blockade in GABAA receptors, because receptors expressing the reciprocal mutation (N15′S) in the β2 subunit of α1β2 GABAA receptors displayed significantly less use-facilitated block than observed in wild type receptors. Our data demonstrate the involvement of the 15′ position in the mechanism of picrotoxin block and suggest that PTX may be acting at two distinct sites in ligand-gated anion channels. The wild type glycine receptor α1 cDNA was a generous gift from H. Betz. The mutant α1 cDNAs were kindly provided by Qing Ye, N. L. Harrison, and R. A. Harris. All glycine cDNAs had been subcloned into the mammalian expression vector pCIS (23Ye Q. Koltchine V.V. Mihic S.J. Mascia M.P. Wick M.J. Finn S.E. Harrison N.L. Harris R.A. J. Biol. Chem. 1998; 273: 3314-3319Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). The mutation of the 15′-asparagine to serine in the human GABAA receptor β2 subunit was generated using QuikChangeTM (Stratagene, La Jolla, CA) and confirmed by DNA sequencing. Untransfected TSA-201 cells, which are transformed human embryonic kidney 293 cells, were plated onto 25-mm coverslips. These cells were cultured as described previously (24Steward L.J. Boess F.G. Steele J.A. Liu D. Wong N. Martin I.L. Mol. Pharmacol. 2000; 57: 1249-1255PubMed Google Scholar). Transient expression of all glycine and GABAA receptors was obtained using the modified calcium phosphate precipitation method (25Chen C. Okamaya H. Mol. Cell. Biol. 1987; 7: 2745-2750Crossref PubMed Scopus (5083) Google Scholar). 15–20 μg of total DNA was used in the transfection step. Cells were analyzed electrophysiologically 48–72 h after transfection. Whole-cell patch recordings were made at room temperature (22–25 °C). Cells were voltage-clamped at −60 mV. Patch pipettes of borosilicate glass (1B150F, World Precision Instruments, Inc., Sarasota, FL) were pulled (Flaming/Brown, P-87/PC, Sutter Instrument Co., Novato, CA) to a tip resistance of 1–2.5 megohms for whole-cell recordings. The pipette solution contained 140 mm CsCl, 10 mm EGTA, 10 mm HEPES, 4 mm Mg-ATP, pH 7.2. Coverslips containing cultured cells were placed in a small chamber (∼1.5 ml) on the stage of an inverted light microscope (Olympus IMT-2) and superfused continuously at 5–8 ml/min with the following external solution containing 125 mm NaCl, 5.5 mm KCl, 0.8 mmMgCl2, 3.0 mm CaCl2, 20 mm HEPES, 25 mm d-glucose, pH 7.3. Glycine/GABA-induced Cl− currents from the whole-cell configuration of the patch clamp technique were obtained using an Axoclamp 200A amplifier (Axon Instruments, Foster City, CA) equipped with a CV-4 headstage. Currents were low pass filtered at 5 kHz, monitored on an oscilloscope and a chart recorder (Gould TA240), and stored on a computer (pClamp 6.0, Axon Instruments) for subsequent analysis. To monitor the possibility that access resistance changed over time or during different experimental conditions, at the beginning of each recording we measured and stored on our digital oscilloscope the current response to a 5-mV voltage pulse. This stored trace was continuously referenced throughout the recording. If a change in access resistance was observed throughout the recording period, the patch was aborted, and the data were not included in the analysis. The agonist (glycine or GABA) with or without PTX was prepared in the extracellular solution and then was applied from independent reservoirs by gravity flow to cells using a Y-shaped tube positioned within 100 μm of the cell. With this system, the 10–90% rise time of the junction potential at the open tip is 12–51 ms (26Huang R.Q. Dillon G.H. J. Neurophysiol. 1999; 82: 1233-1243Crossref PubMed Scopus (53) Google Scholar). Receptors were typically activated roughly with the EC50 agonist concentration. Once a control agonist response was determined, the effect of PTX on the response was examined. Agonist applications were separated by at least 2-min intervals to ensure both adequate washout from the bath and recovery of receptors from desensitization if present. Glycine and GABA concentration-response profiles were generated for their respective receptors using the following Equation 1 I/Imax=1/(1+(EC50/[X]) n)Equation 1 where I and I max represent the normalized and maximal agonist-induced current at a given concentration, X represents the agonist (glycine or GABA) in μm, EC50 is the half-maximal effective agonist concentration, and n is the Hill coefficient. The antagonism profile of picrotoxin was analyzed by constructing concentration-inhibition relationships. The data were fitted to the Equation 2 I/Imax=1/(1+(IC50/[picrotoxin]) n)Equation 2 where I is the steady-state current at a given concentration of picrotoxin, I max is the maximum current induced by agonist, IC50 is the picrotoxin concentration that is half-maximally effective, and n is the Hill coefficient. Glycine, GABA, and picrotoxin were obtained from Sigma. Previous work has shown that mutations at the 15′ position affect glycine sensitivity to varying degrees (23Ye Q. Koltchine V.V. Mihic S.J. Mascia M.P. Wick M.J. Finn S.E. Harrison N.L. Harris R.A. J. Biol. Chem. 1998; 273: 3314-3319Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Thus, we first examined the glycine concentration-response curve for the wild type, the mutant α1(S15′Q), and mutant α1(S15′N) receptors (Fig.2). The EC50 and Hill coefficient values for glycine in the wild type receptors were 44 ± 5.3 and 2.1 ± 0.5 μm, respectively. The substitution of the 15′-serine by glutamine caused a roughly 2-fold right shift in the glycine dose-response curve. The incorporation of Asn at position 15′ caused a dramatic rightward shift, roughly 15-fold, in the concentration-response curve for glycine. The EC50and Hill coefficient values for all receptors are summarized in TableI.Table IAgonist sensitivity and picrotoxin inhibition in wild type and mutant glycine and GABAA receptorsReceptorEC50n HPTX IC50-APTXn H-APTX IC50-BPTXn H-BμmμmμmμmμmμmGlycineWT α144 ± 5.32.1 ± 0.539 ± 60.7 ± 0.05339 ± 401 ± 0.13α1 (S15′Q)102 ± 7.61.5 ± 0.254 ± 140.85 ± 0.259.3 ± 1.51 ± 0.17α1 (S15′N)670 ± 381.5 ± 0.15 ± 0.31.2 ± 0.07n.d.n.d.GABAAWT α1β21.5 ± 0.21.4 ± 0.125 ± 2.71.1 ± 0.13.4 ± 0.31 ± 0.1α1β2(N15′S)4.5 ± 0.51.5 ± 0.12.9 ± 0.40.7 ± 0.063.0 ± 0.70.6 ± 0.1Values for EC50 and picrotoxin IC50 are in μm. PTX IC50 is the value obtained when gated by the agonist EC50 (PTX IC50-A) or when gated by a saturating agonist concentration (PTX IC50-B). WT, wild type;n H, Hill coefficient; n.d., not determined. Open table in a new tab Values for EC50 and picrotoxin IC50 are in μm. PTX IC50 is the value obtained when gated by the agonist EC50 (PTX IC50-A) or when gated by a saturating agonist concentration (PTX IC50-B). WT, wild type;n H, Hill coefficient; n.d., not determined. In wild type α1 receptors, picrotoxin lacks the use-facilitated feature that exists 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 observed a similar lack of use-facilitated block in the present studies. As shown in Fig.3 A, picrotoxin inhibited the peak as well as the steady-state current induced by 200 μm glycine with equal potency in the wild type receptors. Picrotoxin did not increase the rate of current decay, even at a concentration of 3 mm. The introduction of the 15′Q mutation had striking effects on the picrotoxin blockade of the glycine receptor. Co-application of variable concentrations of picrotoxin (1–1000 μm) with 1 mm glycine in α1(S15′Q) receptors had little effect on the initial peak current but subsequently induced a time-dependent current decay to steady state (Fig. 3 B). In addition, increasing the picrotoxin concentration enhanced the exponential decay rate of the glycine-induced current (Figs. 3 B and4). The application of increasing picrotoxin concentration caused a concentration-dependent decrease in the time constant (t) of the current decay from 11.5 ± 1.0 s with 3 μm PTX to 0.23 ± 0.02 s with 1 mm PTX (Fig. 4 B). Thus, the time constant for the decay was inversely related to the concentration of picrotoxin. We also evaluated the effect of picrotoxin on the current induced by glycine in α1(S15′N) receptors. The conversion to use-facilitated block was evident in α1(S15′N) receptors as well (Fig. 3 C). Thus, the incorporation of Asn or Gln changed the mechanism by which picrotoxin inhibits glycine receptors. The α1(S15′Q) mutation had an effect on picrotoxin inhibition similar to that observed with the α1(S15′N) mutation, and had a lesser effect on glycine sensitivity (Fig. 2). Hence, the remaining experiments were conducted using α1(S15′Q) receptors.Figure 4Rate of block in α1(S15′Q) receptors is dependent on both glycine and picrotoxin concentrations. A, rate of picrotoxin-induced current decay is significantly slower when the channel is gated by 100 μm glycine (a) than when gated by 1000 μm glycine (b), a trait consistent with use-facilitated blockade. B, the time constant (t) for use-facilitated block picrotoxin is inversely proportional to picrotoxin concentration. Decaying currents were fitted with a single exponential function. Note also the summary data for the experiment in A, illustrating a 5-fold enhancement in current decay kinetics by 100 μmpicrotoxin when glycine is increased from 100 to 1000 μm. All data points are from a minimum of three cells. Calibration bar equals 175 pA in Aa and 375 pA in Ab.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next tested whether increasing the glycine concentration would increase the rate of the block by picrotoxin in α1(S15′Q) receptors. When the channel was gated by 100 μm glycine, 100 μm picrotoxin enhanced the glycine current decay rate to a time constant (t) of 4.7 ± 0.3 s (Fig.4 A). When the α1(S15′Q) receptors were gated with a saturating glycine concentration (1 mm), 100 μm picrotoxin caused a much greater enhancement of the decay of the glycine-activated current to a t of 0.87 ± 0.5 s. The enhancement of current decay with an increase in glycine concentration further demonstrates the use-facilitated nature of picrotoxin blockade in the mutant receptor. It might be argued that the mutation has affected the glycine activation kinetics by enhancing glycine binding and/or the gating transition. In this scenario, the data would not necessarily reflect a use-facilitated block but could instead be explained via an underlying alteration in the relative reaction rates for glycine and picrotoxin in the wild type compared with the mutant receptors. To test this possibility, we measured the 10–90% current rise time (t 10–90) in wild type and α1(S15′Q) receptors. The t 10–90 was not different in the two receptors (85 ± 17 ms (n = 5) and 127 ± 16 ms (n = 7) in wild type and mutant receptors, respectively). Thus, a change in glycine activation kinetics is not responsible for the presence of use-facilitated block in glycine α1(S15′Q) receptors. A TMII mutation has been shown to convert the mechanism of picrotoxin blockade in α1 glycine receptors from competitive to non-competitive (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 effect with increased concentrations of glycine as described above is suggestive of non-competitive inhibition. Thus, we assessed the nature of picrotoxin inhibition in both wild type and α1S15′Q receptors. In this report, picrotoxin inhibited the current induced by the glycine EC50 with an IC50 of 39 ± 6.0 μm. Increasing the glycine concentration (4× the glycine EC50) shifted the picrotoxin IC50 almost 10-fold to the right to 339 ± 40 μm (Fig.5 A), confirming the previous report 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 α1S15′Q receptors, picrotoxin inhibited the current induced by the glycine EC50 with an IC50 of 54 ± 14 μm. However, when the channel was gated with a saturating concentration of glycine (1 mm), the picrotoxin concentration-response curve was significantly shifted to the left to 9.3 ± 1.5 μm (Fig. 5 B). Thus, the (S15′Q) mutation converted the mechanism of picrotoxin block from competitive to non-competitive. A summary of the effects of picrotoxin in the different receptor configurations is presented in Table I. The ability of picrotoxin to access its site is poor in the absence of the agonist 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 can efficiently access its site without channel opening 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); we confirmed this finding in the present investigation (data not shown). We subsequently tested whether the S15′Q mutation affected the channel state dependence of picrotoxin access. Receptors were preincubated in 100 μm picrotoxin for 3 min. At the 3-min time point, glycine (1 mm) and picrotoxin (100 μm) were co-applied to the cell while still equilibrated in bath picrotoxin. As is evident from Fig.6, pretreatment with picrotoxin abolished the use-facilitated aspect of block (Fig. 6, n = 4). A similar effect was found when using 10 μm picrotoxin. Thus, whereas the S15′Q mutation clearly induced a use-facilitated picrotoxin effect, it did not prevent picrotoxin from accessing its site in the closed channel state. The presence of Asn or Gln at the 15′ position of TMII in glycine α1 receptors clearly converted the mechanism of picrotoxin blockade to use-facilitated non-competitive inhibition. We next sought to evaluate the effects of the reciprocal mutation (N15′S) in GABAA receptors. The wild type GABAA receptor α1 subunit has a serine residue at the 15′ position. Co-expression of the wild type α1 subunit with the β2(N15′S) subunit yields a α1β2(N15′S) GABAA receptor with only serine residues at this position; thus, it is equivalent to the wild type glycine α1-homomeric receptor at the 15′ position. The N15′S mutation caused a 3-fold shift in GABA sensitivity (Table I). As expected, picrotoxin inhibition of wild type α1β2 GABAAreceptors showed strong use-facilitated non-competitive inhibition (Fig. 7 A). In α1β2(N15′S) receptors, blockade by picrotoxin was not completely shifted to that observed in wild type glycine α1 receptors. However, the magnitude of the use-facilitated block was greatly attenuated. In wild type receptors, increasing the GABA-gating concentration from the EC50 to 10× EC50 decreased the picrotoxin IC50 ∼8-fold. In contrast, in α1β2(N15′S) GABAA receptors, increasing the GABA-gating concentration had no effect on picrotoxin sensitivity. The results confirm the involvement of this residue in the mechanism of picrotoxin blockade of both glycine and GABAA receptors. The inhibitory mechanism of picrotoxin in ligand-gated anion channels is a complex phenomenon. Based on the fact that the onset of picrotoxin block is facilitated in the presence of the agonist in GABAA and glutamate-gated Cl− channels, it has been suggested that picrotoxin acts as an open channel blocker (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, single channel analysis has shown that picrotoxin lacks the flickery effect that is typical of a classical channel blocker (12Newland C.F. Cull-Candy S.G. J. Physiol. (Lond.). 1992; 447: 191-213Crossref Scopus (177) Google Scholar). In addition, picrotoxin inhibition is voltage-independent 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). Furthermore, other reports have demonstrated 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). Thus, picrotoxin might bind at an allosteric site to stabilize a closed or desensitized state of these channels (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, 27Shan Q. Haddrill J.L. Lynch J.W. J. Neurochem. 2001; 76: 1109-1120Crossref PubMed Scopus (75) Google Scholar). To resolve the complexity of the picrotoxin interaction with these channels, the existence of multiple binding sites for picrotoxin has been suggested (7Yoon K.W. Covey D.F. Rothman S.M. J. Physiol. (Lond.). 1993; 464: 423-439Crossref Scopus (131) Google Scholar, 10Simmonds M.A. Eur. J. Pharmacol. 1980; 80: 347-358Crossref Scopus (87) Google Scholar). Upon analyzing the actions of picrotoxin and related compounds, Yoon et al.(7Yoon K.W. Covey D.F. Rothman S.M. J. Physiol. (Lond.). 1993; 464: 423-439Crossref Scopus (131) Google Scholar) suggested that picrotoxin might bind to both use-dependent and use-independent sites in GABAA receptors (7Yoon K.W. Covey D.F. Rothman S.M. J. Physiol. (Lond.). 1993; 464: 423-439Crossref Scopus (131) Google Scholar). Regardless of whether picrotoxin inhibition results from an interaction at one or two sites (discussed below), a molecular basis for the use-facilitated block has not been described. In this study, the mutation of the wild type glycine α1 TMII 15′ serine to glutamine or asparagine, which exists in glutamate-gated and GABAA-gated Cl− channels, respectively (3Etter A. Cully D.F. Liu K.K. Reiss B. Vassilatis D.B. Schaeffer J.M. Arena J.P. J. 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