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Magnitude of a Conformational Change in the Glycine Receptor β1-β2 Loop Is Correlated with Agonist Efficacy

2009; Elsevier BV; Volume: 284; Issue: 40 Linguagem: Inglês

10.1074/jbc.m109.048405

ISSN

1083-351X

Autores

Stephan A. Pless, Joseph W. Lynch,

Tópico(s)

Synthesis and Biological Evaluation

Resumo

The efficacy of agonists at Cys-loop ion channel receptors is determined by the rate they isomerize receptors to a pre-open flip state. Once the flip state is reached, the shut-open reaction is similar for low and high efficacy agonists. The present study sought to identify a conformational change associated with the closed-flip transition in the α1-glycine receptor. We employed voltage-clamp fluorometry to compare ligand-binding domain conformational changes induced by the following agonists, listed from highest to lowest affinity and efficacy: glycine > β-alanine > taurine. Voltage-clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. Agonist affinity and efficacy correlated inversely with maximum fluorescence magnitudes at labeled residues in ligand-binding domain loops D and E, suggesting that large conformational changes in this region preclude efficacious gating. However, agonist affinity and efficacy correlated directly with maximum fluorescence magnitudes from a label attached to A52C in loop 2, near the transmembrane domain interface. Because glycine experiences the largest affinity increase between closed and flip states, we propose that the magnitude of this fluorescence signal is directly proportional to the agonist affinity increase. In contrast, labeled residues in loops C, F, and the pre-M1 domain yielded agonist-independent fluorescence responses. Our results support the conclusion that a closed-flip conformation change, with a magnitude proportional to the agonist affinity increase from closed to flip states, occurs in the microenvironment of Ala-52. The efficacy of agonists at Cys-loop ion channel receptors is determined by the rate they isomerize receptors to a pre-open flip state. Once the flip state is reached, the shut-open reaction is similar for low and high efficacy agonists. The present study sought to identify a conformational change associated with the closed-flip transition in the α1-glycine receptor. We employed voltage-clamp fluorometry to compare ligand-binding domain conformational changes induced by the following agonists, listed from highest to lowest affinity and efficacy: glycine > β-alanine > taurine. Voltage-clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. Agonist affinity and efficacy correlated inversely with maximum fluorescence magnitudes at labeled residues in ligand-binding domain loops D and E, suggesting that large conformational changes in this region preclude efficacious gating. However, agonist affinity and efficacy correlated directly with maximum fluorescence magnitudes from a label attached to A52C in loop 2, near the transmembrane domain interface. Because glycine experiences the largest affinity increase between closed and flip states, we propose that the magnitude of this fluorescence signal is directly proportional to the agonist affinity increase. In contrast, labeled residues in loops C, F, and the pre-M1 domain yielded agonist-independent fluorescence responses. Our results support the conclusion that a closed-flip conformation change, with a magnitude proportional to the agonist affinity increase from closed to flip states, occurs in the microenvironment of Ala-52. Glycine receptors (GlyRs) 3The abbreviations used are: GlyRglycine receptor5-HT3Rserotonin type-3 receptorΔFchange in fluorescenceΔIchange in currentAChBPacetylcholine-binding proteinAF546Alexa Fluor 546ECextracellularGABAA/CRGABA type-A/type-C receptorLBDligand-binding domainMTSRmethanethiosulfonate-rhodamineMTS-TAMRA2-((5(6)-tetramethylrhodamine)carboxylamino)ethyl methanethiosulfonatenAChRnicotinic acetylcholine receptorpre-M1the domain preceding the first transmembrane segmentTMDtransmembrane domainTMRMtetramethylrhodamine-maleimideVCFvoltage-clamp fluorometryWTwild type. are pentameric chloride-selective ion channels that mediate fast inhibitory neurotransmission (1Lynch J.W. Physiol. Rev. 2004; 84: 1051-1095Crossref PubMed Scopus (615) Google Scholar). They are members of the Cys-loop receptor family that includes the prototypical nicotinic acetylcholine receptor (nAChR), the γ-aminobutyric acid type-A receptors (GABAARs), and serotonin type-3 receptors (5-HT3Rs). Recent structural studies have provided a wealth of information on the structure and function of this receptor family (2Bocquet N. Nury H. Baaden M. Le Poupon C. Changeux J.P. Delarue M. Corringer P.J. Nature. 2008; 457: 111-114Crossref PubMed Scopus (591) Google Scholar, 3Brejc K. van Dijk W.J. Klaassen R.V. Schuurmans M. van Der Oost J. Smit A.B. Sixma T.K. Nature. 2001; 411: 269-276Crossref PubMed Scopus (1579) Google Scholar, 4Hilf R.J. Dutzler R. Nature. 2008; 452: 375-379Crossref PubMed Scopus (579) Google Scholar, 5Hilf R.J. Dutzler R. Nature. 2008; 457: 115-118Crossref PubMed Scopus (470) Google Scholar, 6Miyazawa A. Fujiyoshi Y. Unwin N. Nature. 2003; 423: 949-955Crossref PubMed Scopus (1081) Google Scholar). In Cys-loop receptors, the ligand-binding domain (LBD) preceding the four transmembrane helices consists of two twisted β-sheets. The inner (vestibule facing) β-sheet comprises seven β-strands, while the outer β-sheet is formed by three β-strands (3Brejc K. van Dijk W.J. Klaassen R.V. Schuurmans M. van Der Oost J. Smit A.B. Sixma T.K. Nature. 2001; 411: 269-276Crossref PubMed Scopus (1579) Google Scholar). The ligand binding site is located at the interface of adjacent subunits and is lined by six domains: three loops from the principal and the complementary sides, termed A-C and D-F, respectively (3Brejc K. van Dijk W.J. Klaassen R.V. Schuurmans M. van Der Oost J. Smit A.B. Sixma T.K. Nature. 2001; 411: 269-276Crossref PubMed Scopus (1579) Google Scholar). glycine receptor serotonin type-3 receptor change in fluorescence change in current acetylcholine-binding protein Alexa Fluor 546 extracellular GABA type-A/type-C receptor ligand-binding domain methanethiosulfonate-rhodamine 2-((5(6)-tetramethylrhodamine)carboxylamino)ethyl methanethiosulfonate nicotinic acetylcholine receptor the domain preceding the first transmembrane segment transmembrane domain tetramethylrhodamine-maleimide voltage-clamp fluorometry wild type. GlyRs are activated by endogenous amino acid agonists in the following order of efficacy: glycine > β-alanine > taurine (7Lewis T.M. Schofield P.R. McClellan A.M. J. Physiol. 2003; 549: 361-374Crossref PubMed Scopus (39) Google Scholar, 8Schmieden V. Kuhse J. Betz H. EMBO J. 1992; 11: 2025-2032Crossref PubMed Scopus (149) Google Scholar). As these amino acids share considerable structural similarity (Fig. 1A), they are likely to compete for the same binding site (9De Saint Jan D. David-Watine B. Korn H. Bregestovski P. J. Physiol. 2001; 535: 741-755Crossref PubMed Scopus (72) Google Scholar, 10Schmieden V. Betz H. Mol. Pharmacol. 1995; 48: 919-927PubMed Google Scholar, 11Grudzinska J. Schemm R. Haeger S. Nicke A. Schmalzing G. Betz H. Laube B. Neuron. 2005; 45: 727-739Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). A recent ground-breaking study on an intermediate pre-open state, the so-called "flip" state (12Burzomato V. Beato M. Groot-Kormelink P.J. Colquhoun D. Sivilotti L.G. J. Neurosci. 2004; 24: 10924-10940Crossref PubMed Scopus (157) Google Scholar), has provided new insights into the mechanism of partial agonism in Cys-loop receptors (13Lape R. Colquhoun D. Sivilotti L.G. Nature. 2008; 454: 722-727Crossref PubMed Scopus (286) Google Scholar). This study suggested that agonist efficacy depends on the ability of the agonist to convert the inert agonist-bound receptor to the pre-open flip state. Once the flip state is reached, the shut-open reaction is similar for high and low efficacy agonists. To date there is, however, very little information concerning the structural basis for the lower efficacies of partial agonists. To address this, the present study employed the voltage-clamp fluorometry (VCF) technique (14Pless S.A. Lynch J.W. Clin. Exp. Pharmacol. Physiol. 2008; 35: 1137-1142Crossref PubMed Scopus (31) Google Scholar) to compare the conformational changes induced by glycine, β-alanine, and taurine at various positions in the GlyR LBD. VCF involves tethering of an environmentally sensitive fluorophore to a cysteine engineered into a domain of interest. If ligand-binding and/or channel opening leads to a changed dielectric environment surrounding the fluorophore, a change in quantum yield or emission spectrum can be detected. VCF was first employed on voltage-gated potassium channels (15Mannuzzu L.M. Moronne M.M. Isacoff E.Y. Science. 1996; 271: 213-216Crossref PubMed Scopus (400) Google Scholar) and has since provided a wealth of information on Cys-loop receptor structure and function (16Chang Y. Weiss D.S. Nature Neurosci. 2002; 5: 1163-1168Crossref PubMed Scopus (86) Google Scholar, 17Dahan D.S. Dibas M.I. Petersson E.J. Auyeung V.C. Chanda B. Bezanilla F. Dougherty D.A. Lester H.A. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 10195-10200Crossref PubMed Scopus (77) Google Scholar, 18Mourot A. Bamberg E. Rettinger J. J. Neurochem. 2008; 105: 413-424Crossref PubMed Scopus (17) Google Scholar, 19Muroi Y. Czajkowski C. Jackson M.B. Biochemistry. 2006; 45: 7013-7022Crossref PubMed Scopus (43) Google Scholar, 20Pless S.A. Dibas M.I. Lester H.A. Lynch J.W. J. Biol. Chem. 2007; 282: 36057-36067Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 21Khatri A. Sedelnikova A. Weiss D.S. Biophys. J. 2009; 96: 45-55Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 22Muroi Y. Theusch C.M. Czajkowski C. Jackson M.B. Biophys. J. 2009; 96: 499-509Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 23Pless S.A. Lynch J.W. J. Neurochem. 2009; 108: 1585-1594Crossref PubMed Scopus (12) Google Scholar). Here we employ VCF to identify an agonist-specific conformational change that may control or reflect the rate at which the GlyR isomerizes to the flip state. Glycine, taurine, and β-alanine (all Sigma Aldrich) were dissolved in water and stored at 4 °C. Strychnine (Sigma Aldrich), was dissolved in DMSO (Sigma Aldrich) and stored at −20 °C. Sulforhodamine methanethiosulfonate (MTSR) and 2-((5(6Miyazawa A. Fujiyoshi Y. Unwin N. Nature. 2003; 423: 949-955Crossref PubMed Scopus (1081) Google Scholar)-tetramethylrhodamine)carboxylamino)ethyl methanethiosulfonate (MTS-TAMRA) (Toronto Research Chemicals, North York, ON, Canada) were dissolved in DMSO and stored at −20 °C. Alexa Fluor 546 C5 maleimide (AF546, Invitrogen Corp.) was dissolved in water on the day of the experiment. The human GlyR α1 cDNA was subcloned into the pGEMHE vector. All constructs contained the functionally silent C41A mutation to remove the only uncross-linked cysteine in the LBD. Site-directed mutagenesis was carried out with the QuikChange mutagenesis kit (Stratagene, La Jolla, CA) and incorporation of mutations was confirmed by automated sequencing. The mMessage mMachine Kit (Ambion, Austin, TX) was used to generate capped mRNA. Oocytes from female Xenopus laevis frogs (Xenopus Express) were prepared as described (20Pless S.A. Dibas M.I. Lester H.A. Lynch J.W. J. Biol. Chem. 2007; 282: 36057-36067Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) and injected with 10 ng of capped mRNA. Oocytes were incubated for 3–5 days at 18 °C in a solution containing 96 mm NaCl, 2 mm KCl, 1 mm MgCl2, 1.8 mm CaCl2, 5 mm HEPES, 0.6 mm theophylline, 2.5 mm pyruvic acid, 50 μg/ml gentamycin (Cambrex Corporation, East Rutherford, NJ), pH 7.4. Prior to recording oocytes were transferred into ND96 (96 mm NaCl, 2 mm KCl, 1 mm MgCl2, 1.8 mm CaCl2, 5 mm HEPES, pH 7.4) containing 5–20 μm dye for 30 s (for MTS-linked dyes) or 45 min (for AF546). Oocytes were washed in ND96 and stored up to 4 h before recording. The GlyR LBD model in Fig. 1 shows the positions of the labeled residues employed in this study. Wild type (WT) GlyRs never displayed detectable ΔF or ΔI changes after incubation with MTS-TAMRA (Table 1) or any of the other fluorophores employed here (n = 3 each, data not shown). We thus rule out nonspecific effects of the fluorophores.TABLE 1Summary of results for glycine, β-alanine, and taurine-evoked current and fluorescence recordingsConstructGlycineβ-AlanineTaurineEC50nHImax/ΔFmaxEC50nHRI/RFEC50nHRI/RFμmμA/%μmμmWTWT ΔI no label15.5 ± 0.32.6 ± 0.18.3 ± 0.422.2 ± 0.1†2.4 ± 0.10.97 ± 0.0364.4 ± 1.0†/‡2.2 ± 0.10.94 ± 0.02WT ΔI label115.6 ± 0.42.7 ± 0.17.9 ± 0.220.8 ± 0.2†2.5 ± 0.10.97 ± 0.0260.4 ± 1.5†/‡2.5 ± 0.10.98 ± 0.02Loop EL127C ΔI14950 ± 1802.4 ± 0.27.7 ± 0.86690 ± 140†2.7 ± 0.11.04 ± 0.1018400 ± 100†/‡2.8 ± 0.11.00 ± 0.12L127C ΔF6070 ± 2401.8 ± 0.146.7 ± 7.147100 ± 33001.2 ± 0.12.67 ± 0.35*66100 ± 46001.2 ± 0.13.19 ± 0.41*Loop DQ67C ΔI163.1 ± 0.62.9 ± 0.17.2 ± 0.5240 ± 6†2.4 ± 0.11.11 ± 0.19665 ± 10†/‡2.3 ± 0.10.92 ± 0.04Q67C ΔF1180 ± 501.7 ± 0.118.0 ± 0.62440 ± 1501.2 ± 0.17.20 ± 0.78*3000 ± 901.2 ± 0.111.38 ± 1.50*/#Loop 2A52C ΔI117.4 ± 0.13.0 ± 0.17.7 ± 0.240.8 ± 0.3†2.6 ± 0.10.85 ± 0.13105 ± 1†/‡2.1 ± 0.10.99 ± 0.15A52C ΔF201 ± 101.4 ± 0.1−8.6 ± 1.1380 ± 380.9 ± 0.10.42 ± 0.02*730 ± 761.1 ± 0.10.23 ± 0.01*/#Loop FV178C ΔI340.7 ± 0.12.6 ± 0.110.3 ± 2.397.8 ± 9.6†1.8 ± 0.30.85 ± 0.22400 ± 18†/‡1.7 ± 0.11.11 ± 0.22V178C ΔF313 ± 201.3 ± 0.112.6 ± 0.6131 ± 11.4 ± 0.10.95 ± 0.15283 ± 191.3 ± 0.10.84 ± 0.13G181C ΔI239.8 ± 1.31.9 ± 0.19.9 ± 2.1121 ± 4†2.3 ± 0.20.95 ± 0.10351 ± 16†/‡1.7 ± 0.10.92 ± 0.08G181C ΔF503 ± 651.3 ± 0.211.6 ± 2.7553 ± 201.4 ± 0.10.92 ± 0.241400 ± 1701.2 ± 0.10.88 ± 0.19Loop CH201C ΔI116.3 ± 0.62.9 ± 0.29.3 ± 0.862.3 ± 0.1†4.6 ± 0.11.26 ± 0.07117 ± 3†/‡2.6 ± 0.20.82 ± 0.07H201C ΔF126 ± 32.1 ± 0.110.2 ± 1.0209 ± 391.3 ± 0.10.95 ± 0.12365 ± 341.2 ± 0.10.78 ± 0.12N203C ΔI145.7 ± 1.23.0 ± 0.26.9 ± 0.256.1 ± 1.9†3.3 ± 0.20.93 ± 0.0490.8 ± 4.2†/‡2.6 ± 0.40.89 ± 0.06N203C ΔF526 ± 251.2 ± 0.144.3 ± 5.9228 ± 140.8 ± 0.10.92 ± 0.07286 ± 150.8 ± 0.10.90 ± 0.14Pre-M1Q219C ΔI29.2 ± 0.22.6 ± 0.28.4 ± 0.412.3 ± 1.32.1 ± 0.40.86 ± 0.0922.3 ± 0.4†/‡2.6 ± 0.10.84 ± 0.07Q219C ΔF98.6 ± 9.91.5 ± 0.2−13.3 ± 0.869.3 ± 5.21.1 ± 0.10.90 ± 0.1390.8 ± 5.01.3 ± 0.10.81 ± 0.13M227C ΔI313.1 ± 0.12.4 ± 0.16.5 ± 0.528.8 ± 0.7†2.1 ± 0.10.79 ± 0.0281.5 ± 1.9†/‡2.0 ± 0.10.75 ± 0.12M227C ΔF226 ± 241.0 ± 0.1−3.5 ± 0.398.5 ± 4.61.1 ± 0.11.10 ± 0.06336 ± 361.1 ± 0.11.13 ± 0.09 Open table in a new tab The VCF set up has previously been described in detail (20Pless S.A. Dibas M.I. Lester H.A. Lynch J.W. J. Biol. Chem. 2007; 282: 36057-36067Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). In brief, an inverted microscope (Nikon Instruments, Kawasaki, Japan) was fitted with a high-Q tetramethylrhodamine isothiocyanate filter set (Chroma Technology, Rockingham, VT), a Plan Fluor 40× objective (Nikon Instruments, Kawasaki, Japan), a PhotoMax 200 photodiode (Dagan Corporation, Minneapolis, MN), a xenon arc lamp (Sutter Instruments, Novato, CA) and an automated perfusion system (AutoMate Scientific, San Francisco, CA). A detailed description of the recording chamber can be found in Ref. 17Dahan D.S. Dibas M.I. Petersson E.J. Auyeung V.C. Chanda B. Bezanilla F. Dougherty D.A. Lester H.A. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 10195-10200Crossref PubMed Scopus (77) Google Scholar. Electrodes for two-electrode voltage clamp recordings were operated by automated micromanipulators (Sutter Instruments, Novarto, CA). All cells were voltage-clamped at −40 mV and current recordings were made with a Gene Clamp 500B amplifier (Axon Instruments, Union City, CA). Fluorescence and current traces were acquired at 200 Hz with a Digidata 1322A interface and digitally filtered at 2 Hz with an eight-pole Bessel filter for analysis and display (Axon Instruments, Union City, CA). Fluorescence baselines were corrected for bleaching where necessary. Values for half-maximal concentrations (EC50) and Hill coefficients (nH) for ligand-induced activation of current and fluorescence signals were obtained with the Hill equation, fitted with a non-linear least squares algorithm (SigmaPlot 9.0, Systat Software, Point Richmond, CA). All results expressed as means ± S.E. of at least three independent experiments. The molecular model was generated with PyMOL 0.99 (DeLano Scientific LLC, San Francisco, CA). GlyRs exhibit the agonist efficacy (and affinity) sequence: glycine > β-alanine > taurine (7Lewis T.M. Schofield P.R. McClellan A.M. J. Physiol. 2003; 549: 361-374Crossref PubMed Scopus (39) Google Scholar, 8Schmieden V. Kuhse J. Betz H. EMBO J. 1992; 11: 2025-2032Crossref PubMed Scopus (149) Google Scholar). When α1-GlyRs are expressed in mammalian HEK293 cells, the efficacies of all three agonists are relatively high so that taurine and β-alanine act as full or near-full agonists (7Lewis T.M. Schofield P.R. McClellan A.M. J. Physiol. 2003; 549: 361-374Crossref PubMed Scopus (39) Google Scholar, 9De Saint Jan D. David-Watine B. Korn H. Bregestovski P. J. Physiol. 2001; 535: 741-755Crossref PubMed Scopus (72) Google Scholar, 24Rajendra S. Lynch J.W. Pierce K.D. French C.R. Barry P.H. Schofield P.R. Neuron. 1995; 14: 169-175Abstract Full Text PDF PubMed Scopus (142) Google Scholar). However, when expressed in Xenopus oocytes, the efficacies of all three agonists are reduced, which results in an increase in their EC50 values and a conversion of β-alanine and taurine into weak partial agonists relative to glycine (8Schmieden V. Kuhse J. Betz H. EMBO J. 1992; 11: 2025-2032Crossref PubMed Scopus (149) Google Scholar). Because the reduced efficacies seen in oocyte-expressed GlyRs can be reversed by expressing receptors at high densities (9De Saint Jan D. David-Watine B. Korn H. Bregestovski P. J. Physiol. 2001; 535: 741-755Crossref PubMed Scopus (72) Google Scholar, 25Taleb O. Betz H. EMBO J. 1994; 13: 1318-1324Crossref PubMed Scopus (68) Google Scholar), we injected oocytes with a high amount (10 ng) of mRNA to ensure that β-alanine and taurine evoked maximal currents that were comparable in magnitude to those elicited by glycine. To facilitate comparison of agonist-induced responses, the maximum currents (ΔImax) and maximum fluorescence changes (ΔFmax) elicited by β-alanine and taurine at all labeled residues investigated here were normalized to those elicited by glycine via the relations, RI = (ΔImax(β-Ala or tau))/(ΔImax(Gly)) and RF = (ΔFmax(β-Ala or tau))/(ΔFmax(Gly)), respectively. In this nomenclature, R refers to ratio and subscripts I and F refer to current and fluorescence, respectively. These values are summarized in Table 1 for the WT and each mutant GlyR investigated here. We investigated labeled residues in three domains of the LBD inner β-sheet: loop D, loop E, and loop 2 (Fig. 1B). Loop D contributes to the complementary (−) side of the GlyR-binding pocket (11Grudzinska J. Schemm R. Haeger S. Nicke A. Schmalzing G. Betz H. Laube B. Neuron. 2005; 45: 727-739Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 26Pless S.A. Millen K.S. Hanek A.P. Lynch J.W. Lester H.A. Lummis S.C. Dougherty D.A. J. Neurosci. 2008; 28: 10937-10942Crossref PubMed Scopus (63) Google Scholar). As shown in Fig. 1B, Loop D forms part of β-strand 2 that in turn forms part of the inner β-sheet. We recently demonstrated that the MTS-TAMRA-labeled Q67C residue in loop D reports a much smaller ΔFmax in response to saturating glycine (Fig. 2A) than to strychnine (27Pless S.A. Lynch J.W. J. Biol. Chem. 2009; 284: 15847-15856Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Here we quantitated the ΔF and ΔI dose-response relationships for both β-alanine and taurine (Table 1 and Fig. 2, B and C, supplemental Fig. S1). Importantly, the mean ΔImax values for glycine, β-alanine, and taurine at this mutant GlyR were indistinguishable in magnitude (Fig. 2D). While the ΔI EC50 values for all three agonists were increased in the labeled Q67C GlyR relative to their WT GlyR values, their rank order was unchanged implying that the mutation did not affect the agonist affinity or efficacy sequence (Table 1). However, taurine activated the largest ΔFmax signals, which were only marginally smaller than those induced by the antagonist, strychnine (RF(strychnine) = 11.8 ± 2.1 versus RF(taurine) = 11.1 ± 0.2, both n = 4). The agonist-activated ΔFmax values decreased significantly from taurine to β-alanine and from β-alanine to glycine (Fig. 2D). The strong linear correlation between ΔI EC50 and ΔFmax values for the three tested agonists (R2 = 0.89), demonstrates that the ΔFmax reported by the labeled Q67C is inversely related to both agonist efficacy and affinity. Although the ΔI EC50 values for activation by glycine, β-alanine, and taurine were significantly lower than the corresponding ΔF EC50 values (Table 1 and supplemental Fig. S1), both current and fluorescence responses were apparent at low glycine concentrations (Fig. 2, A–C). Furthermore, for each of the three agonists, the nH values for the ΔF dose response were significantly lower than their values for the ΔI dose-response (Table 1), indicating substantially lower cooperativity for ΔF signals than for ΔI signals. Indeed, this phenomenon applied to all mutants investigated in this study. The reasons for this will be considered in the "Discussion." We next investigated MTS-TAMRA-labeled L127C in loop E. This domain forms part of β-strand 6 on the inner β-sheet (3Brejc K. van Dijk W.J. Klaassen R.V. Schuurmans M. van Der Oost J. Smit A.B. Sixma T.K. Nature. 2001; 411: 269-276Crossref PubMed Scopus (1579) Google Scholar, 11Grudzinska J. Schemm R. Haeger S. Nicke A. Schmalzing G. Betz H. Laube B. Neuron. 2005; 45: 727-739Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). We recently demonstrated that while glycine and strychnine both evoke large ΔFmax responses at this labeled site, those activated by strychnine were around four times larger than those activated by glycine (27Pless S.A. Lynch J.W. J. Biol. Chem. 2009; 284: 15847-15856Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). This prompted us to conclude that the two ligands induce distinct local conformational rearrangements at this site. In the present study, we found that β-alanine and taurine both induce large ΔI and ΔF responses (Table 1). The ΔImax values were virtually identical for glycine, β-alanine, and taurine, while the ΔFmax signals for β-alanine and taurine were significantly (∼3 times) larger than those elicited by glycine (Table 1). The pattern of ΔI and ΔF responses observed at the labeled L127C residue was therefore similar to that observed at Q67C in the adjacent β-strand, where ΔFmax responses were also inversely related to agonist efficacy. We also found that for both β-alanine and taurine the ΔI EC50 was significantly lower than that for ΔF (Table 1 and supplemental Fig. S1). Additionally, for all three agonists the nH for ΔF was significantly lower that that for ΔI (Table 1). It should be noted, however, that both ΔI and ΔF responses exhibited similar thresholds at low β-alanine and taurine concentrations. As loop 2 is also part of the inner β-sheet and is linked to loop D via β-strand 2 (Fig. 1), we investigated the possibility that a label attached to this domain might also report agonist-specific movements. As shown previously (27Pless S.A. Lynch J.W. J. Biol. Chem. 2009; 284: 15847-15856Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), the MTS-TAMRA-labeled A52C mutant GlyR yielded reliable ΔF decreases in response to glycine application. A sample ΔI and ΔF dose-response for glycine is shown in Fig. 3A, with averaged results presented in Table 1 and supplemental Fig. S1. β-Alanine and taurine application also evoked large ΔI responses but smaller ΔF responses (Fig. 3, B and C and Table 1). Although all three agonists displayed virtually identical ΔImax values and WT-like ΔI EC50 values (Fig. 3D and Table 1), the averaged ΔFmax values decreased significantly from glycine to β-alanine and from β-alanine to taurine (Fig. 3D and Table 1). This trend of ΔFmax being directly proportional to agonist efficacy is consistent with our previous finding that the competitive antagonist strychnine induces no measureable ΔF at the labeled A52C GlyR (27Pless S.A. Lynch J.W. J. Biol. Chem. 2009; 284: 15847-15856Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Indeed, there was a strong linear correlation between ΔI EC50 and ΔFmax values for the three tested agonists (R2 = 0.97), indicating that the local environmental change sensed by the label attached to A52S is directly proportional to agonist affinity and efficacy. The EC50 values of both the glycine- and β-alanine-induced ΔF responses were significantly higher than those of the corresponding ΔI responses (Table 1 and supplemental Fig. S1). In addition, as with the previously described mutants, the nH values were significantly higher for ΔI compared with ΔF (Table 1). Loop C, loop F, and the pre-M1 domain all flank the outer β-sheet of the Cys-loop receptor LBD (Fig. 1B). In the present study, we investigated the ligand sensitivity of ΔI and ΔF responses at the following labeled residues: V178C and G181C in loop F, H201C and N203C in loop C, and Q219C and M227C in the pre-M1 domain. The locations of G181C, N203C, and Q219C are shown in Fig. 1B. We have previously demonstrated that labels attached to these sites yield robust ΔF values in response to glycine activation (27Pless S.A. Lynch J.W. J. Biol. Chem. 2009; 284: 15847-15856Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The fluorescent labels that were successfully attached to each of these sites are indicated in Table 1. In our previous study, we showed that ΔFmax values at most labeled residues in loops F and C were indistinguishable for glycine and strychnine, suggesting these domains mainly undergo local ligand-independent conformational changes (27Pless S.A. Lynch J.W. J. Biol. Chem. 2009; 284: 15847-15856Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). In the present study, we found that all six labeled residues also responded with large ΔI and ΔF signals in response to β-alanine and taurine application (supplemental Fig. S1 and Table 1). Indeed, glycine, β-alanine, and taurine induced virtually identical ΔImax and ΔFmax values at all six labeled residues (Table 1 and Fig. 4), indicating that agonist-induced movements in loop F, loop C, and in the pre-M1 domain do not discriminate between different agonists. This is an important finding, as we previously showed that H201C in loop C and Q219C and M227C in the pre-M1 domain did discriminate between glycine and strychnine (27Pless S.A. Lynch J.W. J. Biol. Chem. 2009; 284: 15847-15856Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). ΔF values can be produced by either fluorophore environmental changes or by direct quench/dequench interactions between ligand and fluorophore. A direct ligand-fluorophore interaction cannot explain results from labeled residues in loop 2 or the pre-M1 domain as they lie well away from the ligand-binding site. Direct interactions between fluorophore and ligand are difficult to categorically rule out for the remaining labeled residues in binding loops C, D, E, and F. However, many of the sites investigated here have been studied previously in VCF studies on Cys-loop receptors and in each case a variety of criteria has been used to rule out direct ligand-induced quenching or dequenching events (16Chang Y. Weiss D.S. Nature Neurosci. 2002; 5: 1163-1168Crossref PubMed Scopus (86) Google Scholar, 19Muroi Y. Czajkowski C. Jackson M.B. Biochemistry. 2006; 45: 7013-7022Crossref PubMed Scopus (43) Google Scholar, 21Khatri A. Sedelnikova A. Weiss D.S. Biophys. J. 2009; 96: 45-55Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 22Muroi Y. Theusch C.M. Czajkowski C. Jackson M.B. Biophys. J. 2009; 96: 499-509Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 27Pless S.A. Lynch J.W. J. Biol. Chem. 2009; 284: 15847-15856Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). For almost all labeled residues examined here, the ΔF dose-response relationship was right-shifted with a shallower slope relative to the corresponding ΔI dose-response relationship. We propose that this is because the binding of three glycine molecules is sufficient for a maximal ΔI (7Lewis T.M. Schofield P.R. McClellan A.M. J. Physiol. 2003; 549: 361-374Crossref PubMed Scopus (39) Google Scholar, 28Beato M. Groot-Kormelink P.J. Colquhoun D. Sivilotti L.G. J. Gen. Physiol. 2002; 119: 443-466Crossref PubMed Scopus (57) Google Scholar, 29Beato M. Groot-Kormelink P.J. Colquhoun D. Sivilotti L.G. J. Neurosci. 2004; 24: 895-906Crossref PubMed Scopus (78) Google Scholar), whereas ΔF, which responds to local conformational changes or individual binding events, does not reach a maximum until five glycine molecules are bound. Importantly, we assume that significantly different ΔFmax values for labeled loop 2 or pre-M1 residues indicate distinct local conformational rearrangements. All three agonists plus strychnine induced large ΔF increases at the labeled Q67C and L127C residues. These residues are located adjacent to each other in loops D and E of the inner β-sheet. Because glycine, β-alanine, and taurine elicit identical single channel conductances (7Lewis T.M. Schofield P.R. McClellan A.M. J. Physiol. 2003; 549: 361-374Crossref PubMed Scopus (39) Google Scholar) and activated identical ΔImax values at all labeled mutant receptors examined here (Table 1), we conclude that the mean number of receptors activated by the different agonists remains con

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