Functional Consequences of the Loss of High Affinity Agonist Binding to γ-Aminobutyric Acid Type A Receptors
2002; Elsevier BV; Volume: 277; Issue: 24 Linguagem: Inglês
10.1074/jbc.m110312200
ISSN1083-351X
AutoresJ. Glen Newell, Susan M. J. Dunn,
Tópico(s)Neuroendocrine regulation and behavior
ResumoWe reported previously that tyrosine 62 of the β2 subunit of the γ-aminobutyric acid, type A (GABAA) receptor is an important determinant of high affinity agonist binding and that recombinant α1β2γ2Lreceptors carrying the Y62S mutation lack measurable high affinity sites for [3H]muscimol. We have now examined the effects of disrupting these sites on the macroscopic desensitization properties of receptors expressed in Xenopus oocytes. Desensitization was measured by the ability of low concentrations of bath-perfused agonist to reduce the current responses elicited by subsequent challenges with saturating concentrations of GABA. Wild-type receptors were desensitized by pre-perfused muscimol with an IC50∼0.7 μm, which correlates well with the lower affinity sites for this agonist that are measured in direct binding studies. Receptors carrying the β2 Y62S and Y62F mutations desensitized at slightly higher (2–7-fold) agonist concentrations. However, at low perfusate concentrations, the Y62S-containing receptor recovered from the desensitized state even in the continued presence of agonist. The characteristics of desensitization in the wild-type and mutant receptors lead us to suggest that the major role of the high affinity agonist-binding site(s) of the GABAA receptor is not to induce desensitization but rather to stabilize the desensitized state once it has been formed. We reported previously that tyrosine 62 of the β2 subunit of the γ-aminobutyric acid, type A (GABAA) receptor is an important determinant of high affinity agonist binding and that recombinant α1β2γ2Lreceptors carrying the Y62S mutation lack measurable high affinity sites for [3H]muscimol. We have now examined the effects of disrupting these sites on the macroscopic desensitization properties of receptors expressed in Xenopus oocytes. Desensitization was measured by the ability of low concentrations of bath-perfused agonist to reduce the current responses elicited by subsequent challenges with saturating concentrations of GABA. Wild-type receptors were desensitized by pre-perfused muscimol with an IC50∼0.7 μm, which correlates well with the lower affinity sites for this agonist that are measured in direct binding studies. Receptors carrying the β2 Y62S and Y62F mutations desensitized at slightly higher (2–7-fold) agonist concentrations. However, at low perfusate concentrations, the Y62S-containing receptor recovered from the desensitized state even in the continued presence of agonist. The characteristics of desensitization in the wild-type and mutant receptors lead us to suggest that the major role of the high affinity agonist-binding site(s) of the GABAA receptor is not to induce desensitization but rather to stabilize the desensitized state once it has been formed. γ-aminobutyric acid type A receptors γ-aminobutyric acid γ-aminobutyric acid, type A nicotinic acetylcholine receptors Desensitization, an intrinsic biophysical characteristic of ligand-gated ion channels, facilitates neurobiological adaptation to prolonged or repeated exposure to agonist. The molecular mechanisms by which this occurs have been subject to intense investigation but remain poorly understood (1Edmonds B. Gibb A.J. Colquhoun D. Annu. Rev. Physiol. 1995; 57: 495-519Crossref PubMed Scopus (133) Google Scholar). However, the general consensus is that agonist-induced conformational changes of the receptor protein induce the initial, if not all, phases of the process (2Ochoa E.R.M. Challopadhyay A. McNamee M.G. Cell. Mol. Neurobiol. 1989; 9: 141-177Crossref PubMed Scopus (179) Google Scholar, 3Lohse M.J. Biochim. Biophys. Acta. 1993; 1179: 171-188Crossref PubMed Scopus (402) Google Scholar). The γ-aminobutyric acid type A receptors (GABAARs)1 are members of a receptor gene family (see Ref. 4Barnard E.A. Skolnick P. Olsen R.W. Möhler H. Sieghart W. Biggio G. Braestrup C. Bateson A.N. Langer S.Z. Pharmacol. Rev. 1998; 50: 291-313PubMed Google Scholar) that includes the nicotinic acetylcholine receptors (nAChRs), glycine receptors, and the serotonin type 3 receptor. These receptors are structurally and functionally homologous and desensitize in the continued presence of agonist (5Schofield P.R. Darlison M.G. Fujita N. Burt D.R. Stephenson F.A. Rodriguez H. Rhee L.M. Ramachandran J. Reale V. Glencorse T.A. Seeburg P.H. Barnard E.A. Nature. 1987; 328: 221-227Crossref PubMed Scopus (1271) Google Scholar, 6Sieghart W. Pharmacol. Rev. 1995; 47: 181-234PubMed Google Scholar). GABAARs are large pentameric protein complexes composed of subunit isoforms from a number of classes (α1–6, β1–3, γ1–3, ρ1–3, δ, ε, and π). Although the precise subunit compositions of native receptors remain to be established, a major subtype in the brain appears to be a combination of α1, β2, and γ2 subunits in a likely stoichiometry of 2:2:1 (7Chang Y. Wang R. Barot S. Weiss D.S. J. Neurosci. 1996; 16: 5415-5424Crossref PubMed Google Scholar, 8Tretter V. Ehya N. Fuchs K. Sieghart W. J. Neurosci. 1997; 18: 2737-2738Google Scholar, 9Farrar S.J. Whiting P.J. Bonnert T.P. McKernan R.M. J. Biol. Chem. 1999; 274: 10100-10104Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). Radioligand binding studies have revealed the existence of (at least) two classes of agonist recognition sites on a single GABAAR (10Newell J.G. Davies M. Bateson A.N. Dunn S.M.J. J. Biol. Chem. 2000; 275: 14198-14204Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), which differ in affinity by more than 1 order of magnitude (see also Refs. 11Olsen R.W. Bergman M.O. Van Ness P.C. Lummis S.C. Watkins A.E. Napias C. Greenlee D.V. Mol. Pharmacol. 1981; 19: 217-227PubMed Google Scholar, 12Yang J.S.-J. Olsen R.W. Mol. Pharmacol. 1987; 32: 266-277PubMed Google Scholar, 13Agey M.W. Dunn S.M.J. Biochemistry. 1989; 28: 4200-4208Crossref PubMed Scopus (22) Google Scholar). By using the recombinant rat α1β2γ2 receptor expressed in tsA201 cells, we found two classes of binding sites for [3H]muscimol with Kd values of 8.1 and 430 nm (10Newell J.G. Davies M. Bateson A.N. Dunn S.M.J. J. Biol. Chem. 2000; 275: 14198-14204Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Because higher concentrations of muscimol are required to activate this receptor subtype (EC50 of 7.1 μm when expressed in Xenopus oocytes), sites of still lower affinity may be present (see below). Although the roles of these multiple sites in receptor function require clarification, it is generally accepted that the high affinity binding that is measured under equilibrium conditions reflects binding to a desensitized conformation(s) of the receptor (see Ref. 14Sigel E. Buhr A. Trends Pharmacol. Sci. 1997; 18: 425-429Abstract Full Text PDF PubMed Scopus (345) Google Scholar). Furthermore, it is assumed that agonist occupancy of these sites in the resting state of the protein induces the conformational changes that lead to this equilibrium desensitized state. One approach to delineate the role of individual sites in receptor function is to use site-directed mutagenesis to disrupt selectively binding domains and to examine the consequences on receptor function. By using this approach, we have recently identified an amino acid residue (Tyr-62) of the β2 subunit of the GABAAR that is important for high affinity agonist binding (10Newell J.G. Davies M. Bateson A.N. Dunn S.M.J. J. Biol. Chem. 2000; 275: 14198-14204Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Mutation of this residue to a phenylalanine (Y62F) caused a significant decrease in agonist affinity, and mutation to a serine (Y62S) led to a loss of measurable high affinity binding sites. In this study, we have expressed recombinant wild-type and mutant GABAA receptors in Xenopus oocytes and have developed methods to study aspects of the desensitization process. Receptors carrying the Tyr-62 mutations retained the ability to be desensitized upon prolonged exposure to agonists but with slightly altered concentration dependence compared with the wild type. At low GABA concentrations (1–10 μm), the Y62S mutant receptor displayed the unusual property of first becoming desensitized but then recovering from desensitization despite the continued presence of agonist. Thus, under these conditions, this receptor (which lacks measurable high affinity agonist-binding sites) seems to be unable to maintain the desensitized state. Therefore, we propose a new model in which the role of the high affinity sites is not to induce desensitization but rather to stabilize the desensitized state once it has been formed. GABAAR β2 subunit mutants were engineered using the Altered Sites II® in vitro Mutagenesis System (Promega, Madison, WI) as described previously (10Newell J.G. Davies M. Bateson A.N. Dunn S.M.J. J. Biol. Chem. 2000; 275: 14198-14204Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 15Newell J.G. Davies M. Bateson A.N. Mol. Biotech. 2000; 14: 25-45Crossref PubMed Google Scholar). Xenopusoocytes were prepared as described (16Belleli D. Callachan H. Hill-Venning C. Peters J.A. Lambert J.J. Br. J. Pharmacol. 1996; 118: 563-576Crossref PubMed Scopus (76) Google Scholar). Capped cRNA transcripts for GABAAR subunits were prepared from cDNA constructs that were generous gifts from Dr. David Weiss. Oocytes were injected with α1, β2 (or β2 mutants), and γ2L subunit cRNA (1 μg/μl total RNA) in a 1:1:1 ratio in a total volume of 50 nl. Individual oocytes were maintained in 96-well plates at 14 °C in modified Barth's medium (88 mm NaCl, 1 mm KCl, 0.5 mm CaCl2, 0.5 mmCa(NO3)2, 2.5 mm sodium pyruvate, 1 mm MgSO4, 2.4 mmNaHCO3, 15 mm HEPES, pH 7.4) that was supplemented with 100 μg/ml gentamicin. Electrophysiological experiments were performed 2–7 days following oocyte injection. Standard two-electrode voltage clamp techniques were carried out using a GeneClamp 500B Amplifier (Axon Instruments Inc.) at a holding potential of −60 mV. The microelectrodes were filled with 3m KCl and had resistances of 0.5–2.0 megohms in frog Ringer's medium (120 mm NaCl, 1.8 mmCaCl2, 2 mm KCl, and 5 mm HEPES, pH 7.4). In all experiments, oocytes were continuously bathed in frog Ringer's via a gravity perfusion system at a flow rate of ∼5 ml/min. GABA (Sigma) or muscimol (Sigma) was dissolved in the same medium. The current amplitude elicited by a 30-s application of a saturating concentration of GABA (1 mm) was first stabilized prior to experiments such that a maximum variation of 5% was observed over three successive applications. In desensitization experiments, changes in the current amplitudes elicited by challenge applications of GABA (1 mm) or muscimol (2 mm), i.e.concentrations that elicited a maximal response in all recombinant receptors, were measured. Bath pre-perfusion with lower concentrations of agonist was started after recovery of receptors from the desensitization induced by the challenge application itself. The relative current amplitude used in determination of IC50values for desensitization was defined by the steady state current (I) that was achieved in the presence of the indicated concentration of agonist in the pre-perfusate (see example in Fig. 1). Data were analyzed by iterative curve fitting using Equation 1 (GraphPad Prism Software) as follows: I=Imax/(1+(IC50/[A])n)(Eq. 1) where I is the peak amplitude of the current elicited by a given concentration of agonist ([A]); Imax is the maximum amplitude of the current; IC50 is the concentration required for half-maximal inhibition; and n is the Hill coefficient. The normalized data are presented as percent control to construct concentration-response curves. The assay protocols were optimized to ensure that any observed desensitization was due only to the presence of agonist in the bathing medium and not to other variables. In experiments to measure the effects of frequency of challenge and duration of pre-perfusion on current amplitude, desensitizing GABA concentrations equivalent to their IC50values (determined as above) were used in the pre-perfusion. Where noted in the text, pre-perfusion was started either after washout of the challenge concentration or immediately during the recovery phase from the agonist challenge. Both pre-perfusion protocols gave the same maximum depression of current amplitude under steady state conditions. The rate of recovery from desensitization was measured by first applying a desensitizing concentration of GABA (its Emax or EC50 concentration as noted) and then monitoring the magnitude of the peak current elicited by the same concentration of GABA administered at different time intervals (1–30 min) after the initial challenge. The recovery of the current amplitude as a function of time was fit by a single exponential model (GraphPad Prism, San Diego, CA; www.graphpad.com) to give estimates of the rate and extent of recovery. Similar experiments were carried out in the presence of bath-perfused GABA (3 μm) to assess the effects of pre-perfusion on the recovery from desensitization. Data were fitted to Equation 2, Recovery=Y0+YR(1−exp(−k*t))(Eq. 2) where YO represents the base-line response to GABA; YR is recovery; kis the rate of recovery; and t is the time. Data were analyzed by a one-way analysis of variance followed by either a post hoc Dunnett's test or Newman-Keuls to determine levels of significance. The data for GABA-mediated current shown in Figs. 2 and 3 were analyzed by a one-way repeated measures analysis of variance to compare the current before and after bath pre-perfusion of the indicated concentration of GABA.FIG. 3Experiments similar to those shown in Fig. 2 were carried out using recombinant GABAARs containing the Y62F (A) and Y62S (B) β2 subunits. Data represent the mean ± S.E. of 3 (Y62F) or 4 (Y62S) independent experiments. Following the third application (*) of GABA, the agonist was pre-perfused at concentrations of 5 and 25 μm,respectively (approximating their IC50 values for desensitization), to examine the effects of time (3 (▪), 6 (○), 9 (♦), and 12 min (■)) on functional recovery of the response from the challenge concentration. Inhibition of GABA-evoked current is significantly depressed during perfusion of agonist, and the Y62S mutant receptor shows accelerated recovery from desensitization induced by the challenge dose itself (see text for details).View Large Image Figure ViewerDownload (PPT) Fig. 1A shows representative two-electrode voltage clamp recordings from recombinant wild-type α1β2γ2L receptors expressed inXenopus oocytes. In this experiment, maximally effective concentrations of GABA (1 mm) elicited rapidly desensitizing currents that were reproducible in amplitude when applied at 12-min intervals (Fig. 1; see below). We have previously reported that the EC50 for GABA-activated currents in this receptor subtype is ∼33 μm (10Newell J.G. Davies M. Bateson A.N. Dunn S.M.J. J. Biol. Chem. 2000; 275: 14198-14204Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). After the challenge applications, the oocyte was washed with Ringer's solution for 12 min (to allow recovery from the desensitization induced by the challenge) and then continuously bath-perfused with 10 μm GABA, a concentration which itself evokes a significant current response (<15% of maximum). Subsequent challenges at 12-min intervals showed that the amplitudes of the evoked currents were reduced, declined to steady state levels, and were fully reversible upon washout of GABA from the perfusate. We interpret the observed reduction in current as receptor desensitization induced by GABA in the perfusion medium. Fig. 1B shows the effects of bath perfusion of a lower concentration of GABA (1 μm) which by itself induces only a minimal chloride conductance (<2% of maximum). In contrast to the results in Fig. 1A, there was no diminution of the response to the first GABA challenge; rather a small, but reproducible, increase in the current amplitude was observed. However, upon repeated challenges, the current again declined to a steady state level, and this was fully reversible upon removal of GABA from the perfusate. These data suggest that channel activation may be required before the receptor can desensitize and, furthermore, that the extent of desensitization is dependent on the number of activations (see also Ref. 17Bartrup J.T. Newberry N.R J. Physiol. (Lond.). 1996; 490: 679-690Crossref Scopus (24) Google Scholar). Comparison of the data in Fig. 1, A and B, demonstrates that the magnitude of steady state desensitization is also dependent upon the concentration of GABA in the perfusion medium (see below and Fig. 4). The experimental protocol used here to study GABAAreceptor desensitization was adapted from that of Bartrup and Newberry (17Bartrup J.T. Newberry N.R J. Physiol. (Lond.). 1996; 490: 679-690Crossref Scopus (24) Google Scholar) who used whole cell patch clamp techniques to study agonist-induced desensitization of the serotonin type 3 receptor in NG108-15 cells. Because GABAA receptor desensitization has not been quantified previously using the Xenopus oocyte system, we have optimized the experimental procedures to ensure that any desensitization observed was due to the presence of agonist in the perfusate and was not affected by other experimental variables. The data in Fig.2A illustrate the time interval between challenges required to avoid frequency-dependent effects in the wild-type receptor. As in Fig. 1, currents to a maximally effective GABA concentration (1 mm) were recorded at regular time intervals, but in these experiments, this interval was varied between 3 and 12 min as indicated. After the third control challenge, the oocyte was perfused with 3 μm GABA, a concentration that approximates its IC50 for desensitization (see below). Responses to subsequent regular challenges continued to be recorded prior to washout of GABA from the perfusate. In these experiments, perfusion with the lower concentration of GABA was started during the recovery phase from the challenge application, i.e. immediately after receptor activation. Under these conditions, there was an immediate reduction in the current amplitude to the next challenge (cf. Fig.1B), again demonstrating the activation dependence of this phenomenon (see below and also Ref. 17Bartrup J.T. Newberry N.R J. Physiol. (Lond.). 1996; 490: 679-690Crossref Scopus (24) Google Scholar). As noted under “Experimental Procedures,” both preperfusion protocols gave the same steady state levels of desensitization. By using a 9- or 12-min interval between challenges, the control responses were reproducible in amplitude, and there was no difference in the magnitude of the inhibition observed during agonist perfusion or in the recovery upon washout (Fig. 2A). A 3-min interval is clearly not suitable for measuring the concentration dependence of desensitization, because the receptor is unable to recover from the desensitization induced by the challenge itself. This is shown by the decrease in the amplitudes of successive control currents prior to inclusion of the lower concentration of GABA in the bath. By using a 6-min interval, there were apparent frequency-dependent effects on washout, i.e. the current did not immediately return to control levels. Similar control experiments have been carried out using muscimol (data not shown). In all cases, a 12-min interval between challenges gave rise to reproducible effects in which the apparent desensitization depends only on the presence of agonist in the perfusion medium and on the number (see e.g. Fig.1B) but not on the frequency of channel activations. The effects of the time of pre-perfusion were also investigated. Fig. 2B shows representative experiments using wild-type receptors. In these experiments, responses to 1 mm GABA were first stabilized; the oocytes were washed and then perfused with 3 μm GABA for different times prior to challenge. Increasing the pre-perfusion time from 1 to 12 min showed that the extent of current depression was dependent upon perfusion time, but there were no significant differences between 9 and 12 min, suggesting that desensitization reaches a steady state within 9 min. This also illustrates that unlike rapid application techniques (18Jones M.V. Westbrook G.L. Neuron. 1995; 15: 181-191Abstract Full Text PDF PubMed Scopus (541) Google Scholar), gravity perfusion requires longer periods of equilibration to achieve a stabilized desensitized state, which is in good agreement with theoretical considerations of Edelstein et al. (19Edelstein S.J. Schaad O. Henry E. Bertrand D. Changeux J.P. Biol. Cybern. 1996; 75: 361-379Crossref PubMed Scopus (113) Google Scholar). The data in Fig. 2 illustrate that a 12-min interval between challenges gives rise to reproducible measurements of desensitization induced by agonist in the pre-perfusate. Both mutant receptors desensitized upon bath perfusion with GABA as shown in Fig. 3. The data shown in this figure were obtained using perfusate GABA concentrations that approximated the IC50 value for desensitization for each receptor (see below). In receptors carrying the β2 subunit Y62F mutation, the desensitization characteristics measured in control experiments (Fig. 3A) were very similar to those of wild-type receptors. However, notable differences were observed using the Y62S mutant (Fig. 3B). For this receptor, few frequency-dependent effects were apparent using challenge intervals varying from 3 to 12 min. This indicates that this receptor recovers more quickly (within ∼3-min) from the desensitization induced by the challenge than does the wild-type receptor. Experiments similar to those illustrated in Fig. 1 were carried out to investigate the effects of agonist concentration on the steady state level of desensitization. Fig.4A shows the concentration dependence of both GABA- and muscimol-induced desensitization of the wild-type receptor, and curve fitting gave apparent IC50values of 3.3 and 0.7 μm, respectively (Table I). In direct binding studies using [3H]muscimol, we have reported that the wild-type receptor expressed in tsA201 cells has (at least) two classes of binding sites with affinities of about 8 nm and 0.43 μm (10Newell J.G. Davies M. Bateson A.N. Dunn S.M.J. J. Biol. Chem. 2000; 275: 14198-14204Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Although the latter value is subject to error due to rapid ligand dissociation from lower affinity sites during binding assays (see Ref. 13Agey M.W. Dunn S.M.J. Biochemistry. 1989; 28: 4200-4208Crossref PubMed Scopus (22) Google Scholar), the apparent KD value for this population correlates well with the IC50 value for muscimol-induced desensitization. Our preliminary interpretation is that desensitization may be induced by occupancy of the lower affinity binding sites measured in radioligand binding studies.Table IEffects of β2 subunit mutations on the concentration dependence of desensitization for GABA and muscimol using recombinant α1β2γ2 GABAAR expressed in Xenopus oocytesβ2 subunitIC50 (μm ± S.E.)nHMutant, wild-typeGABAWild-type (6)3.3 ± 1.0−0.99 ± 0.161.0Y62F (4)8.2 ± 1.11-ap < 0.05.−1.05 ± 0.072.5Y62S (3)24.7 ± 5.71-bp < 001.−1.03 ± 0.057.5MuscimolWild-type (3)0.7 ± 0.01−0.77 ± 0.081.0Y62F (3)2.1 ± 0.31-ap < 0.05.−0.86 ± 0.073.2Y62S (3)3.8 ± 0.51-bp < 001.−1.32 ± 0.345.71-a p < 0.05.1-b p < 001. Open table in a new tab The Y62F (Fig. 4B) and Y62S (Fig. 4C) mutations altered the concentration dependence for both GABA and muscimol-induced desensitization, the effects being greater in the case of the Y62S substitution (Table I). In both cases, muscimol was more potent than GABA in inducing desensitization. We have reported previously (10Newell J.G. Davies M. Bateson A.N. Dunn S.M.J. J. Biol. Chem. 2000; 275: 14198-14204Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) that muscimol is also more potent in activation of these mutant receptors. During the course of the above experiments, we observed several curious properties relating to the time dependence of desensitization of the Y62S mutant receptor. When the receptor was perfused with low concentrations of GABA (≤10 μm), i.e. below its IC50 for steady state desensitization, the receptor appeared to desensitize and then recover from this desensitization even in the continued presence of the perfused agonist. The representative traces in Fig. 5 illustrate this phenomenon. In these experiments, the wild-type (Fig. 5A) or Y62S (Fig. 5B) receptors were challenged successively with concentrations of GABA equivalent to their EC50 values for activation (see Table I). In control experiments, we have shown that the steady state level of desensitization is independent of whether the EC50 (used in Fig. 5) or Emax (as in FIG. 1, FIG. 2, FIG. 3, FIG. 4) concentration of agonist is used as the challenge application (data not shown). When the wild-type receptor was perfused with 3 μm GABA, the currents, as expected from the previous results, declined to a steady state level and returned to control values upon washout. In contrast, the Y62S mutant receptor showed desensitization during the pre-perfusion, but rather than reaching a steady state residual current, the currents began to grow again even in the continued presence of 3 μm GABA in the perfusate. Thus this receptor, which we have shown previously to lack measurable high affinity binding sites (10Newell J.G. Davies M. Bateson A.N. Dunn S.M.J. J. Biol. Chem. 2000; 275: 14198-14204Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), appears unable to maintain the desensitized state. Fig. 6 quantifies this recovery phenomenon when the GABA concentration in the perfusate was increased to 10 μm. In the wild type (Fig.6A), the receptor desensitized, and this state was maintained until washout of the GABA from the perfusate. In contrast, the Y62S mutant desensitized but then recovered to control values in the continued presence of perfused GABA (Fig. 6B). The recovery phenomenon in the mutant is concentration-dependent. Although it is clear at low perfusate concentrations, i.e. 3–10 μm (Figs. 5 and 6), it is less obvious at higher concentrations. This is discussed further below.FIG. 6Desensitization of wild-type (A) and Y62S (B) mutant receptors in response to bath-perfused GABA (10 μm). Experiments were carried out as in Fig. 5. After stabilization of the current to the control challenge, the magnitude of the current was measured after maximum desensitization (seen on challenge after the second 9-min application). This is referred to as an amplitude of 1.0, and the magnitude of all other currents is normalized to this current. The amplitude of the current was measured in response to challenges applied after additional perfusion times of 12, 15, and 20 min, and after a 20-min washout period with frog Ringer solution. In wild-type receptors, there is no recovery of IGABA during continuous agonist perfusion. However, receptors expressing Y62S recover to control levels as a function of time even in the presence of GABA. The asterisksdenote currents that are not significantly different from control values.View Large Image Figure ViewerDownload (PPT) To define further the recovery of GABAARs from desensitization, we have investigated the rate and extent of recovery using wild-type and mutant Y62S receptors. In these experiments, a desensitizing concentration of GABA was applied, and the amplitude of the peak current to a subsequent challenge administered at times varying from 1 to 30 min after the initial application was measured. Fig. 7 shows the complete recovery from desensitization of wild-type (Fig 7A) and mutant (Fig.7B) receptors from successive challenges at their respective EC50 concentrations. The Y62S mutant receptor recovers ∼2-fold faster than the wild type, and curve fitting by a single exponential model (Table II) gave half-times for recovery of 0.8 and 1.5 min, respectively. These results are in agreement with the data presented in Figs. 2 and 3, which show that the mutant receptor recovers more quickly from desensitization induced by the challenge application.Table IIRates of recovery for recombinant GABAAR receptors carrying either the wild-type or mutant β2 subunits when expressed in Xenopus oocytes[GABA challenge]k % min−1 ± S.E.Ymax % ± S.E.α1β2γ2LEC50(3)47.3 ± 4.796.2 ± 2.5EC50 + 3 μm GABA perfusion (3)63.6 ± 8.364.2 ± 1.72-ap < 0.001.α1β2(Y62S)γ2LEC50(3)84.1 ± 9.298.8 ± 1.9EC50 + 3 μm GABA perfusion (3)20.3 ± 2.42-bp < 0.01.92.9 ± 3.42-a p < 0.001.2-b p < 0.01. Open table in a new tab Similar experiments were carried out in the presence of 3 μm bath-perfused GABA, i.e. similar to the conditions used in Fig. 5. In the case of wild-type receptors, 3 μm GABA did not significantly alter the rate of recovery, but at longer time intervals, the current induced by the challenge application was reduced, reflecting the ability of the bath perfused GABA to “lock” a proportion of the receptors in a desensitized state. As expected from the previous results, the Y62S mutant receptor displayed different recovery properties (Fig. 7B). The receptor initially desensitized, but despite the continued presence of bath-perfused GABA (3 μm), the magnitude of the currents returned to control levels (Fig 7B) within about 20 min. This again demonstrates that low concentrations of GABA in the perfusate are able to induce desensitization but are unable to maintain the receptor in a desensitized state. First impressions of the data in Fig. 7B suggest that the rate of recovery may be slowed in the presence of GABA in the perfusate. However, this is unlikely to be due to a di
Referência(s)