Identification of a Novel Residue within the Second Transmembrane Domain That Confers Use-facilitated Block by Picrotoxin in Glycine (cid:1) 1 Receptors*

The central nervous system convulsant picrotoxin (PTX) inhibits GABA A and glutamate-gated Cl (cid:2) channels in a use-facilitated fashion, whereas PTX inhibition of glycine and GABA C 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 (cid:1) 1 receptors from non-use-facilitated to use-facilitated. In wild type (cid:1) 1 receptors, PTX inhibited glycine-gated Cl (cid:2) 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 (cid:1) -serine to glutamine ( (cid:1) 1(S15 (cid:1) Q) receptors) converted the mechanism of PTX blockade from competitive to non-compet-itive. However, more notable was the fact that in (cid:1) 1(S15 (cid:1) Q) receptors, PTX had insignificant effects on peak current amplitude and dramatically enhanced current decay kinetics. Similar results were found in (cid:1) 1(S15 (cid:1) N) receptors. The reciprocal mutation in the (cid:3) 2 subunit of (cid:1) 1 (cid:3) 2 GABA A receptors ( (cid:1) 1 (cid:3) 2(N15 no use-facilitated block by picrotoxin. GABA A receptors incorporating the (cid:2) 2 subunit ( GABA (cid:2) 2 ) and glutamate-gated chloride channels or homomeric (cid:2) channels ( Glu-gated (cid:2) ) show use-facilitated inhibition by picrotoxin. These receptors express asparagine and glutamine, respectively, at the 15 (cid:3) position, which is the focus of this study. Residues at the 2 (cid:3) , 6 (cid:3) , and 19 (cid:3) positions are also known to affect picrotoxin sensitivity.

The central nervous system convulsant picrotoxin (PTX) inhibits GABA A and glutamate-gated Cl ؊ channels in a use-facilitated fashion, whereas PTX inhibition of glycine and GABA C 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 ␣1 receptors from non-use-facilitated to usefacilitated. In wild type ␣1 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 (␣1(S15Q) receptors) converted the mechanism of PTX blockade from competitive to non-competitive. However, more notable was the fact that in ␣1(S15Q) receptors, PTX had insignificant effects on peak current amplitude and dramatically enhanced current decay kinetics. Similar results were found in ␣1(S15N) receptors. The reciprocal mutation in the ␤2 subunit of ␣1␤2 GABA A receptors (␣1␤2(N15S) 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.
Glycine receptors belong to a superfamily of ligand-gated chloride channels that include GABA A 1 receptors, GABA C receptors, and glutamate-gated chloride channels (1). In native tissue, glycine receptors exist as either ␣ homomers or ␣␤ heteromers (1). They comprise five subunits (usually three ␣ subunits and two ␤ 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 (2). Glycine receptors are targets of therapeutics such as anesthet-ics as well as toxins like the central nervous system convulsant picrotoxin (1).
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 GABA C and glycine ␣1 receptors displays weak or no use-facilitation (13,21), picrotoxin inhibition of GABA A receptors and glutamate-gated Cl Ϫ channels is strongly use-facilitated (3,5,11,12). 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 Ϫ channels of the ligand-gated ion channel superfamily, we observed that GABA A and glutamate-gated channels have neutral acidic polar residues (Asn and Gln) at the 15Ј position; these receptors both demonstrate use-facilitated block by picrotoxin. Glycine ␣1 receptors, which do not display use-facilitated picrotoxin blockade, have a dissimilar residue (Ser) at the 15Ј position.
We demonstrate here that S15ЈQ and S15ЈN mutations in the TMII of glycine ␣1 receptors confer use-facilitated block by picrotoxin. In addition, the S15ЈQ mutation converted the picrotoxin block from competitive to non-competitive. This position also affects the mechanism of the picrotoxin blockade in GABA A receptors, because receptors expressing the reciprocal mutation (N15ЈS) in the ␤2 subunit of ␣1␤2 GABA A receptors displayed significantly less use-facilitated block than observed in wild type receptors. Our data demonstrate the involvement of the 15Ј position in the mechanism of picrotoxin block and suggest that PTX may be acting at two distinct sites in ligandgated anion channels.

EXPERIMENTAL PROCEDURES
Site-directed Mutagenesis and Transient Transfection-The wild type glycine receptor ␣1 cDNA was a generous gift from H. Betz. The mutant ␣1 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 (23). The mutation of the 15Ј-asparagine to serine in the human GABA A receptor ␤2 subunit was generated using QuikChange TM (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 (24). Transient expression of all glycine and GABA A receptors was obtained using the modified calcium phosphate precipitation method (25). 15-20 g of total DNA was used in the transfection step. Cells were analyzed electrophysiologically 48 -72 h after transfection.
Electrophysiology-Whole-cell patch recordings were made at room temperature (22-25°C). Cells were voltage-clamped at Ϫ60 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 (ϳ1.5 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 mM MgCl 2 , 3.0 mM CaCl 2 , 20 mM HEPES, 25 mM D-glucose, pH 7.3. Glycine/GABA-induced Cl Ϫ 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.
Experimental Protocol-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 m of the cell. With this system, the 10 -90% rise time of the junction potential at the open tip is 12-51 ms (26). Receptors were typically activated roughly with the EC 50 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.
Data Analysis-Glycine and GABA concentration-response profiles were generated for their respective receptors using the following 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 M, EC 50 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 where I is the steady-state current at a given concentration of picrotoxin, I max is the maximum current induced by agonist, IC 50 is the picrotoxin concentration that is half-maximally effective, and n is the Hill coefficient. Materials-Glycine, GABA, and picrotoxin were obtained from Sigma.

RESULTS
Previous work has shown that mutations at the 15Ј position affect glycine sensitivity to varying degrees (23). Thus, we first examined the glycine concentration-response curve for the wild type, the mutant ␣1(S15ЈQ), and mutant ␣1(S15ЈN) receptors (Fig. 2). The EC 50 and Hill coefficient values for glycine in the wild type receptors were 44 Ϯ 5.3 and 2.1 Ϯ 0.5 M, respectively. The substitution of the 15Ј-serine by glutamine caused a roughly 2-fold right shift in the glycine dose-response curve. The incorporation of Asn at position 15Ј caused a dramatic rightward shift, roughly 15-fold, in the concentration-response curve for glycine. The EC 50 and Hill coefficient values for all receptors are summarized in Table I. S15ЈQ Mutation Confers Use-facilitated Block by Picrotoxin-In wild type ␣1 receptors, picrotoxin lacks the use-facilitated feature that exists in GABA A and glutamate-gated Cl Ϫ channels (13). We observed a similar lack of use-facilitated block in the present studies. As shown in Fig. 3A, picrotoxin inhibited the peak as well as the steady-state current induced by 200 M 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 15ЈQ mutation had striking effects on the picrotoxin blockade of the glycine receptor. Co-application of variable concentrations of picrotoxin (1-1000 M) with 1 mM glycine in ␣1(S15ЈQ) receptors had little effect on the initial peak current but subsequently induced a time-dependent current decay to steady state (Fig. 3B). In addition, increasing the picrotoxin concentration enhanced the exponential decay rate of the glycine-induced current (Figs. 3B and 4). The application of increasing picrotoxin concentration caused a concentration-dependent decrease in the time constant (t) of the current decay from 11.5 Ϯ 1.0 s with 3 M PTX  Table I for Hill coefficients. All data points are from a minimum of 3-4 cells.
FIG. 1. TMII map of ligand-gated ion channels. Glycine ␣1 receptors (Gly ␣1), which express serine at the 15Ј position, display no use-facilitated block by picrotoxin. GABA A receptors incorporating the ␤2 subunit (GABA ␤2) and glutamate-gated chloride channels or homomeric ␤ channels (Glu-gated ␤) show use-facilitated inhibition by picrotoxin. These receptors express asparagine and glutamine, respectively, at the 15Ј position, which is the focus of this study. Residues at the 2Ј, 6Ј, and 19Ј positions are also known to affect picrotoxin sensitivity.
to 0.23 Ϯ 0.02 s with 1 mM PTX (Fig. 4B). 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 ␣1(S15ЈN) receptors. The conversion to use-facilitated block was evident in ␣1(S15ЈN) receptors as well (Fig. 3C). Thus, the incorporation of Asn or Gln changed the mechanism by which picrotoxin inhibits glycine receptors. The ␣1(S15ЈQ) mutation had an effect on picrotoxin inhibition similar to that observed with the ␣1(S15ЈN) mutation, and had a lesser effect on glycine sensitivity (Fig. 2). Hence, the remaining experiments were conducted using ␣1(S15ЈQ) receptors.
We next tested whether increasing the glycine concentration would increase the rate of the block by picrotoxin in ␣1(S15ЈQ) receptors. When the channel was gated by 100 M glycine, 100 M picrotoxin enhanced the glycine current decay rate to a time constant (t) of 4.7 Ϯ 0.3 s (Fig. 4A). When the ␣1(S15ЈQ) receptors were gated with a saturating glycine concentration (1 mM), 100 M picrotoxin caused a much greater enhancement of the decay of the glycine-activated current to a t of 0.87 Ϯ 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 neces-FIG. 3. Use-facilitated picrotoxin block conferred by the S15Q and S15N mutations. A, an example of response to picrotoxin in wild type ␣1 glycine receptors (a) and mean concentration-response profile for picrotoxin in the wild type receptors (b). Note that picrotoxin equally inhibited the peak and steady-state currents. B, a, response to picrotoxin in ␣1(S15ЈQ) receptors. Note insignificant effects on peak current and marked enhancement of current decay in this receptor, which are indicative of use-facilitated blockade. b, mean concentration-response profile for the effect of picrotoxin on initial peak and steady-state currents. Picrotoxin of up to 1 mM had minimal effect on peak current. C, similar use-facilitated blockade was observed in ␣1(S15ЈN) receptors (a), although the degree of use-facilitation was not as great as that induced with the S15ЈQ mutation (b). Calibration bar equals 360 pA in A, 285 pA in B, and 885 pA in C. See Table I   sarily 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 ␣1(S15ЈQ) receptors. The t 10 -90 was not different in the two receptors (85 Ϯ 17 ms (n ϭ 5) and 127 Ϯ 16 ms (n ϭ 7) in wild type and mutant receptors, respectively). Thus, a change in glycine activation kinetics is not responsible for the presence of usefacilitated block in glycine ␣1(S15ЈQ) receptors. S15ЈQ Mutation Converts Picrotoxin Antagonism from Competitive to Non-competitive-A TMII mutation has been shown to convert the mechanism of picrotoxin blockade in ␣1 glycine receptors from competitive to non-competitive (13). 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 ␣1S15ЈQ receptors. In this report, picrotoxin inhibited the current induced by the glycine EC 50 with an IC 50 of 39 Ϯ 6.0 M. Increasing the glycine concentration (4ϫ the glycine EC 50 ) shifted the picrotoxin IC 50 almost 10-fold to the right to 339 Ϯ 40 M (Fig. 5A), confirming the previous report of competitive inhibition in glycine ␣1 receptors (13). In ␣1S15ЈQ receptors, picrotoxin inhibited the current induced by the glycine EC 50 with an IC 50 of 54 Ϯ 14 M. 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 Ϯ 1.5 M (Fig. 5B). Thus, the (S15ЈQ) 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.
Picrotoxin Can Access Its Site in the S15ЈQ Mutant in the Absence of Channel Opening-The ability of picrotoxin to access its site is poor in the absence of the agonist in GABA A and glutamate-gated Cl Ϫ channels (3,5). However, picrotoxin can efficiently access its site without channel opening in glycine ␣1 receptors (13); we confirmed this finding in the present investigation (data not shown). We subsequently tested whether the S15ЈQ mutation affected the channel state dependence of picrotoxin access. Receptors were preincubated in 100 M picrotoxin for 3 min. At the 3-min time point, glycine (1 mM) and picrotoxin (100 M) 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 ϭ 4). A similar effect was found when using 10 M picrotoxin. Thus, whereas the S15ЈQ mutation clearly induced a use-facilitated picrotoxin effect, it did not prevent FIG. 5. S15Q mutation converts picrotoxin blockade from competitive to non-competitive inhibition. A, consistent with previous findings (13), picrotoxin inhibition of wild type glycine ␣1 receptors is competitive. B, in contrast, picrotoxin-induced blockade in ␣1(S15ЈQ) receptors was converted to non-competitive inhibition. See Table I for summarized information. FIG. 6. Picrotoxin can access its site in the absence of the glycine application in ␣1(S15Q) receptors. Following establishment of the control glycine current (1 mM) (solid line), receptors were incubated in 100 M picrotoxin for 3 min. Picrotoxin and glycine were subsequently co-applied to the cells. The dotted line represents the presence of picrotoxin in the bath solution. Dashed line represents a 5-s co-application of picrotoxin (100 M) and glycine (1 mM) separated by 3-s intervals. Similar results were obtained in four cells expressing the S15ЈQ mutation as well as wild type glycine ␣1 receptors (data not shown). Calibration bar equals 230 pA.

FIG. 7. N15S modifies picrotoxin inhibition of ␣1␤2 GABA A receptors.
A, picrotoxin inhibits wild type ␣1␤2 GABA A receptors in a non-competitive fashion. In these receptors, increasing the GABA-gating concentration from the GABA EC 50 (1.5 M) to 10ϫ the GABA EC 50 elicited a dramatic leftward shift in the picrotoxin inhibition curve. B, the mutation of the ␤2 subunit 15Ј asparagine to serine completely abolished the GABA concentration dependence of the ability of picrotoxin to block the channel. However, the mutation did not fully convert the mechanism of picrotoxin blockade to competitive inhibition. See Table I for IC 50 and Hill coefficient values under each condition.

Use-facilitated Block by Picrotoxin in Glycine Receptors
picrotoxin from accessing its site in the closed channel state.
The Reciprocal 15Ј-TMII Mutation in ␣1␤2 GABA A Receptors Alters Picrotoxin Blockade-The presence of Asn or Gln at the 15Ј position of TMII in glycine ␣1 receptors clearly converted the mechanism of picrotoxin blockade to use-facilitated noncompetitive inhibition. We next sought to evaluate the effects of the reciprocal mutation (N15ЈS) in GABA A receptors. The wild type GABA A receptor ␣1 subunit has a serine residue at the 15Ј position. Co-expression of the wild type ␣1 subunit with the ␤2(N15ЈS) subunit yields a ␣1␤2(N15ЈS) GABA A receptor with only serine residues at this position; thus, it is equivalent to the wild type glycine ␣1-homomeric receptor at the 15Ј position. The N15ЈS mutation caused a 3-fold shift in GABA sensitivity ( Table I). As expected, picrotoxin inhibition of wild type ␣1␤2 GABA A receptors showed strong use-facilitated non-competitive inhibition (Fig. 7A). In ␣1␤2(N15ЈS) receptors, blockade by picrotoxin was not completely shifted to that observed in wild type glycine ␣1 receptors. However, the magnitude of the usefacilitated block was greatly attenuated. In wild type receptors, increasing the GABA-gating concentration from the EC 50 to 10ϫ EC 50 decreased the picrotoxin IC 50 ϳ8-fold. In contrast, in ␣1␤2(N15ЈS) GABA A 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 GABA A receptors.

DISCUSSION
The Mechanism of Block by Picrotoxin-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 GABA A and glutamate-gated Cl Ϫ channels, it has been suggested that picrotoxin acts as an open channel blocker (3,5). However, single channel analysis has shown that picrotoxin lacks the flickery effect that is typical of a classical channel blocker (12). In addition, picrotoxin inhibition is voltage-independent in these channels (3,12,13). Furthermore, other reports have demonstrated little or no usefacilitated block by picrotoxin in glycine and GABA C receptors, respectively (13,21). Thus, picrotoxin might bind at an allosteric site to stabilize a closed or desensitized state of these channels (11,12,27). To resolve the complexity of the picrotoxin interaction with these channels, the existence of multiple binding sites for picrotoxin has been suggested (7,10). Upon analyzing the actions of picrotoxin and related compounds, Yoon et al. (7) suggested that picrotoxin might bind to both use-dependent and use-independent sites in GABA A receptors (7). Regardless of whether picrotoxin inhibition results from an interaction at one or two sites (discussed below), a molecular basis for the usefacilitated block has not been described.
In this study, the mutation of the wild type glycine ␣1 TMII 15Ј serine to glutamine or asparagine, which exists in glutamate-gated and GABA A -gated Cl Ϫ channels, respectively (3, 15), conferred use-facilitated block by picrotoxin. Use-facilitated block was concluded based on the following criteria: 1) peak glycine current was unaffected by picrotoxin, but it greatly enhanced current decay kinetics, 2) glycine current decay rate was picrotoxin concentration-dependent, 3) current decay rate in the presence of picrotoxin was dependent on glycine concentration, and 4) the magnitude of picrotoxin block was dependent on glycine concentration. None of these traits is present in wild type glycine ␣1 receptors, and all are observed in GABA A and glutamate-gated Cl Ϫ channels (3,7,11). Thus, the introduction of Asn or Gln at the 15Ј position of TMII appears to be sufficient to confer use-facilitated block by picrotoxin. At initial inspection, one aspect of our data appears to be inconsistent with this conclusion. Pretreatment studies showed that picrotoxin can still access its site in the absence of channel opening. In the strictest sense, "use-dependence" implies that the channel must be gated for the drug to access its site. There is a number of possibilities that could account for this somewhat unexpected finding. First, although picrotoxin is a highly oxygenated molecule, it is neutral at physiological pH and thus could access its site through both hydrophilic (the channel lumen) and hydrophobic (the lipid bilayer) pathways (5,28). Second, even in GABA A receptors, picrotoxin can gain access to its site in the closed channel state (11,12). However, the picrotoxin association rate is slowed ϳ100-fold when the channel is closed (11). Nevertheless, the inference is that channel opening is not an absolute requirement for picrotoxin to access its site. For this reason, we have chosen to refer to the phenomenon as use-facilitation rather than use dependence. Third, additional residues that are not yet identified may exist in the vicinity of the 15Ј position in both GABA A and glutamate-gated Cl Ϫ channels, but not in glycine receptors, that further interfere with picrotoxin access to its binding site in the closed state. Agonist binding in GABA A and glutamate-gated Cl Ϫ channels could induce a conformational change that relieves the hindrance of picrotoxin binding. This suggestion is speculative, and no structural data exist to support or refute it. Finally, we cannot definitively rule out the possibility that during our picrotoxin incubation experiments, some spontaneous channel openings may have occurred that allowed picrotoxin to access its site. Indeed, in a number of experiments, we did note a modest (30 -50 pA) decrease in holding current when cells were incubated in picrotoxin. This may account for some of the reduction of glycine current amplitude following picrotoxin incubation. It is worth noting that the picrotoxin-site ligand ␤-ethyl-␤-methyl-␥-butyrolactone shows a prominent use-dependent block in GABA A receptors and can also readily access its site in the absence of channel opening (7). Indeed, in many respects, the block of GABA A receptors by ␤-ethyl-␤-methyl-␥butyrolactone closely resembles the block of ␣1(S15ЈQ) receptors by picrotoxin described here. Yoon et al. (7) expressed similar thoughts pertaining to ␤-ethyl-␤-methyl-␥-butyrolactone access in the absence of channel opening.
Whereas the presence of asparagine or glutamine at the 15Ј position dramatically altered the mechanism of picrotoxin blockade in glycine receptors, the reciprocal mutation in GABA A receptors only partially altered picrotoxin pharmacology. There was a significant decrease in the ability of GABA to facilitate the block by picrotoxin in ␣1␤2(N15ЈS) GABA A receptors, although use-facilitation was not abolished. These results confirm the importance of this residue in the mechanism of block by picrotoxin and the cross-talk between the GABA and picrotoxin binding sites. However, because the mode of block was not completely converted in ␣1␤2(N15ЈS) GABA A receptors, the results suggest that other as yet unidentified residues also influence use-facilitated picrotoxin inhibition.
Location of the Picrotoxin Site(s)-Precisely where picrotoxin binds has not been determined conclusively. Several studies have implicated residues in the cytoplasmic aspect of TMII as the picrotoxin binding site (27, 29 -31). Mutations introduced at both the 2Ј and 6Ј positions of TMII confer PTX resistance (15, 16,27). Picrotoxin can protect a cysteine engineered into the 2Ј position from irreversible modification by reactive sulfhydryl reagents (29). When cysteine is mutated into the 6Ј position, picrotoxin cannot protect it from sulfhydryl modification. Based on these studies, it has been suggested that the picrotoxin binding site lies between the 2Ј and 6Ј positions of TMII (29,32). However, more recent work has shown that picrotoxin can also protect a cysteine engineered into the extracellular aspect of TMII (17Ј position) from irreversible modification by chemically reactive picrotoxin-related compounds (33). Although not definitive, these data are consistent with earlier suggestions that picrotoxin and related ligands may interact with multiple sites (7,10,34,35). The present data give additional credence to this possibility and indicate that the second site of picrotoxin interaction may exist in the 15Ј to 17Ј vicinity. In the GABA A receptor ␣1 subunit, the 15Ј residue appears to be directed away from the lumen of the channel (36). However, a cysteine residue at this position can be modified irreversibly by sulfhydryls, and it may form a water-filled crevice along with residues in the TMIII domain (36). Taken together, these studies indicate that this putative extracellular site and the more intracellular site near the 2Ј position could account for the use-dependent and use-independent picrotoxin sites described by Yoon et al. (7). A recent preliminary report based on kinetic analysis of picrotoxin binding and unbinding in varying states of the GABA A receptor also supports the existence of two sites of picrotoxin action (37). If two picrotoxin sites do exist, they must be of comparable affinity or of significantly different efficacy as Hill coefficients of near unity are routinely recorded in functional assays (3,9,11,13,16,29, but see Refs. 10 and 18).
Other interpretations of the data, however, must be considered. A number of recent studies has demonstrated the important role of the 15Ј residue in modulating the effects of other ligands including anesthetics, alcohol, barbiturates, and diazepam (23, 38 -41). The fact that the 15Ј position strongly influences such a structurally and functionally diverse class of drugs makes it difficult to argue that this residue might form a common binding site for all of these compounds. An alternative explanation is that it may be part of a pivotal signal transduction point through which the effects of many of these drugs converge (13). Indeed, conformational changes corresponding to different functional states are greatly influenced by mutations within TMII (13,31,(42)(43). In the present experiments, the substitution of glutamine or asparagine for the native serine may have influenced a state transition that altered picrotoxin inhibition of the channel, resulting in the use-facilitated blockade. Several studies have suggested that picrotoxin may inhibit GABA A receptors by stabilization of a desensitized state (11,12,31). We observed no evidence of this at the whole-cell level, but alterations in kinetic transitions can only be definitively observed with single channel recordings. It is notable that the wild type receptor showed more desensitization than the mutant receptors. However, because the alteration in the mechanism of picrotoxin block was independent of glycinegating concentration, it probably does not confound our interpretation (13).
One final interpretation of the present experiments must be considered. It is possible that mutations introduced at the 15Ј position may alter orientation of residues that are deeper in the channel. A change in orientation could influence the interaction of picrotoxin with these deeper channel residues and thus the mechanism by which it blocks channel activity. The idea of conformational change in the receptor structure induced by amino acid substitutions at quite distant locations is not without precedent. Jackson and colleagues (20) have recently postulated a similar mechanism to explain the observation that the presence of lysine, but not arginine, at the 19Ј position of TMII in Drosophila GABA receptors confers cation selectivity. Although there is no direct physical evidence to support a conformational change deep in the channel by alterations at the 15Ј position, we are aware of no evidence that eliminates this as a possible mechanism.
In conclusion, we have identified a residue at the extracellular aspect of TMII that confers use-facilitated (i.e. use-dependent) block by picrotoxin. It is possible that this 15Ј residue plays a role in a transduction step necessary for the expression of the inhibitory effect of picrotoxin. Alternatively, it may form part of a second binding site for picrotoxin, the existence to which has been alluded previously. Further studies should be conducted to explore the importance of this residue for picrotoxin action in other ligand-gated ion channels as well as the action of other convulsants that might interact with the picrotoxin site.