Open-channel Conformation Research Articles

Thermal stability of purified and reconstituted CFTR in a locked open channel conformation

CFTR is unique among ABC transporters as the only one functioning as an ion channel and from a human health perspective because mutations in its gene cause cystic fibrosis. Although considerable advances have been made towards understanding CFTR’s mechanism of action and the impact of mutations, the lack of a high-resolution 3D structure has hindered progress. The large multi-domain membrane glycoprotein is normally present at low copy number and when over expressed at high levels it aggregates strongly, limiting the production of stable mono-disperse preparations. While the reasons for the strong self-association are not fully understood, its relatively low thermal stability seems likely to be one. The major CF causing mutation, ΔF508, renders the protein very thermally unstable and therefore a great deal of attention has been paid to this property of CFTR. Multiple second site mutations of CFTR in NBD1 where F508 normally resides and small molecule binders of the domain increase the thermal stability of the mutant. These manipulations also stabilize the wild-type protein. Here we have applied ΔF508-stabilizing changes and other modifications to generate wild-type constructs that express at much higher levels in scaled-up suspension cultures of mammalian cells. After purification and reconstitution into liposomes these proteins are active in a locked-open conformation at temperatures as high as 50°C and remain monodisperse at 4°C in detergent or lipid for at least a week. The availability of adequate amounts of these and related stable active preparations of homogeneous CFTR will enable stalled structural and ligand binding studies to proceed.

On the interactions between nucleotide binding domains and membrane spanning domains in cystic fibrosis transmembrane regulator: A molecular dynamic study.

The Cystic Fibrosis Transmembrane Regulator (CFTR) is a membrane protein whose mutations cause cystic fibrosis, a lethal genetic disease. We performed a molecular dynamic (MD) study of the properties of the nucleotide binding domains (NBD) whose conformational changes, upon ATP binding, are the direct responsible of the gating mechanisms of CFTR. This study was done for the wild type (WT) CFTR and for the two most common mutations, ΔF508, that produces a traffic defect of the protein, and the mutation G551D, that causes a gating defect on CFTR. Using an homology model of the open channel conformation of the CFTR we thus introduced the mutations to the structure. Although the overall structures of the G551D and ΔF508 are quite well conserved, the NBD1-NBD2 interactions are severely modified in both mutants. NBD1 and NBD2 are indeed destabilized with a higher internal energy (Ei) in the ΔF508-CFTR. Differently, Ei does not change in the NBDs of G551D but, while the number of close contacts between NBD1 and NBD2 in ΔF508 is increased, a significant reduction of close contacts is found in the G551D mutated form. Hydrogen bonds formation between NBDs of the two mutated forms is also altered and it is slightly increased for the ΔF508, while are severely reduced in G551D. A consequent modification of the NBDs-ICLs interactions between residues involved in the transduction of the ATP binding and the channel gating is also registered. Indeed, while a major interaction is noticed between NBDs interface and ICL2 and ICL4 in the WT, this interaction is somehow altered in both mutated forms plausibly with effect on channel gating. Thus, single point mutations of the CFTR protein can reasonably results in channel gating defects due to alteration of the interaction mechanisms between the NBDs and NBDs-ICLs interfaces upon ATP-binding process.

ATP Binding and Channel Activation in P2X Receptors

Purinergic P2X receptors are a family of nonselective cation channels gated by extracellular adenosine 5′-triphosphate. They are important drug targets primarily because of their involvement in neuropathic pain and inflammation. ATP binds allows Na + and Ca 2+ to pass through the channel pore, thus causing membrane depolarization and affecting various downstream Ca 2+ -dependent signaling processes. A concerted effort by investigators over the last two decades has culminated in significant advances in our understanding of where ATP binds and how ATP binding leads to channel opening and ion flux. The recent publication of the crystal structures for both the closed and open channel conformations of the zebrafish P2X4 receptor sheds new light on how P2X receptors work. In this review, we will attempt to present the existing functional data regarding ATP binding with the available crystal structure data and different experimental approaches that have been used to explore the ATP-binding sites.

Engineered Ionizable Side Chains.

One of the great challenges of mechanistic ion-channel biology is to obtain structural information from well-defined functional states. In the case of neurotransmitter-gated ion channels, the open-channel conformation is particularly elusive owing to its transient nature and brief mean lifetime. In this Chapter, we show how the analysis of single-channel currents recorded from mutants engineered to contain single ionizable side chains in the transmembrane region can provide specific information about the open-channel conformation without any interference from the closed or desensitized conformations. The method takes advantage of the fact that the alternate binding and unbinding of protons to and from an ionizable side chain causes the charge of the protein to fluctuate by 1 unit. We show that, in mutant muscle acetylcholine nicotinic receptors (AChRs), this fluctuating charge affects the rate of ion conduction in such a way that individual proton-transfer events can be identified in a most straightforward manner. From the extent to which the single-channel current amplitude is reduced every time a proton binds, we can learn about the proximity of the engineered side chain to the lumen of the pore. And from the kinetics of proton binding and unbinding, we can calculate the side-chain's affinity for protons (pK a), and hence, we can learn about the electrostatic properties of the microenvironment around the introduced ionizable group. The application of this method to systematically mutated AChRs allowed us to identify unambiguously the stripes of the M1, M2 and M3 transmembrane α-helices that face the pore's lumen in the open-channel conformation in the context of a native membrane.

Thermal stability of purified and reconstituted CFTR in a locked open channel conformation

Thermal stability of purified and reconstituted CFTR in a locked open channel conformation

Conformational changes opening and closing the CFTR chloride channel: insights from cysteine scanning mutagenesis.

Cystic fibrosis, the most common lethal genetic disease affecting young people in North America, is caused by failure of the chloride ion channel known as CFTR (cystic fibrosis transmembrane conductance regulator). CFTR belongs to the large family of ATP-binding cassette (ABC) membrane transporters. In CFTR, ATP-driven events at the nucleotide-binding domains (NBDs) open and close a gate that controls chloride permeation. However, the conformational changes concomitant with opening and closing of the CFTR gate are unknown. Diverse techniques including substituted cysteine accessibility method, disulfide cross-linking, and patch-clamp recording have been used to explore CFTR channel structure. Here, we consider the architecture of both the open and the closed CFTR channel. We review how CFTR channel structure changes between the closed and the open channel conformations and portray the relative function of both cytoplasmic and vestigial gates during the gating cycle. Understanding how the CFTR channel gates chloride permeation is central for understanding how CFTR defects lead to CF. Such knowledge opens the door for novel ways to maximize CFTR channel activity in a CF setting.

The membrane protein Pannexin1 forms two open-channel conformations depending on the mode of activation.

Pannexin1 (Panx1) participates in several signaling events that involve adenosine triphosphate (ATP) release, including the innate immune response, ciliary beat in airway epithelia, and oxygen supply in the vasculature. The view that Panx1 forms a large ATP release channel has been challenged by the association of a low-conductance, small anion-selective channel with the presence of Panx1. We showed that Panx1 membrane channels can function in two distinct modes with different conductances and permeabilities when heterologously expressed in Xenopus oocytes. When stimulated by potassium ions (K(+)), Panx1 formed a high-conductance channel of ~500 pS that was permeable to ATP. Various physiological stimuli can induce this ATP-permeable conformation of the channel in several cell types. In contrast, the channel had a low conductance (~50 pS) with no detectable ATP permeability when activated by voltage in the absence of K(+). The two channel states were associated with different reactivities of the terminal cysteine of Panx1 to thiol reagents, suggesting different conformations. Single-particle electron microscopic analysis revealed that K(+) stimulated the formation of channels with a larger pore diameter than those formed in the absence of K(+). These data suggest that different stimuli lead to distinct channel structures with distinct biophysical properties.

Combined effects of VX-770 and VX-809 on several functional abnormalities of F508del-CFTR channels

BackgroundThe most common cystic fibrosis-associated mutation, the deletion of phenylalanine 508 (F508del), results in channels with poor membrane expression and impaired function. VX-770, a clinically approved drug for treatment of CF patients carrying the G551D mutation, and VX-809, a corrector shown in vitro to increase membrane expression of mutant channels, are currently undergoing clinical trials, but functional data at the molecular level is still lacking. MethodsThe effect of VX-770 and VX-809 on the multiple functional defects of F508del-CFTR was assessed via excised inside-out patch-clamp experiments. ResultsVX-770 completely restores the low opening-rate of F508del-CFTR, with smaller open-time increase, in temperature-corrected and VX-809-treated channels. The shorter locked-open time of hydrolysis-deficient F508del-CFTR is also prolonged by VX-770. VX-809 does not improve channel function by itself as previously reported. ConclusionsThe results from these studies can be interpreted as an equilibrium shift toward the open-channel conformation of F508del-CFTR channels.

Energy Maps of Acetylcholine Receptor Gating

Occupancy of an AChR binding site by an agonist (A) shifts a pre-existing equilibrium from the Closed-channel towards the Open-channel conformation. We estimated energy changes in adult mouse A2C↔A2O gating (adult mouse) from single-channels. Mutations mainly cause local energy changes that can be mapped onto structures of related proteins. We made maps showing residue reaction progress (phi values) and approximate extents of energy changes in the channel-opening isomerization. Phi values range from 1 to 0, are approximately constant for each position and have a modal distribution (n=5 populations). Residues having similar phi values are clustered, with phi decreasing approximately longitudinally between the binding site and the gate. Two separated regions in the alpha subunits - the transmitter binding sites and M2-M3 linkers in the membrane domain - have the highest phi values (∼0.95), followed by the rest of the extracellular domain (∼0.8), most of the membrane domain (∼0.6) and the gate region of the pore (0.3). Residues at the extracellular-transmembrane domain interface undergo large energy changes in C→O. Largest energy changes occur at the binding sites, and in the alpha subunit at the M2-M3 linker, M1 and the gate. All of these energy changes, including those associated with the affinity change for the agonist, appear to arise from local ‘resettling’ events that do not transfer significant energy over distance. Expansion and flexure of the M2-M3 linkers in the alpha subunit appears to trigger the global allosteric transition, and the hydrophobic gate appears to unlock in three steps. The local character of side chain energy changes and the similar phi values of the binding sites and distant M2-M3 linkers suggest that the gating transition is not strictly a mechanical process.

Novel Trans-Membrane Mutation Switches Orai1 to a Constitutively Active and Ca2+ Selective Channel

The endoplasmic reticulum Ca2+ sensor STIM1 forms together with the Ca2+ channel Orai1 the molecular basis for Ca2+ release activated Ca2+ (CRAC) channels. The recent crystal structure of Orai from Drosophila melanogaster shows a unique Ca2+ channel composed of a hexameric subunit complex. The pore structure is formed by transmembrane (TM) 1 helices, surrounded by two ring-like structures, formed by TM2 and TM3 as well as TM4. Employing a combined approach of patch-clamp, molecular biology, biochemical techniques, molecular modeling and structure guided mutagenesis; we discovered a novel key mutation in the second trans-membrane helix of Orai1 that results in a Ca2+ selective, STIM1 independent, constitutively active current. Substitution of this essential residue to a hydrophobic amino-acid retained store-operated activation, yet with largely reduced Orai1 currents. In addition, we took advantage of the constitutively active Orai1 mutant, to evaluate reorientation of the gate located within the cytosolic region of TM1 helices. Cysteine scanning mutagenesis within the TM1 helix enabled identification of gating residues, the dimerization of which was altered in the constitutively opened and closed Orai1 channel conformation. In molecular dynamic simulations of an all-atom model of human Orai1 we will show the interaction network of these identified residues and predict implication of mutations on conformational changes. Our experiments will be summarized in a unique gating model, and we will moreover discuss how STIM1 binding might trigger the open channel conformation. This work was supported by the Austrian Science Foundation (FWF): P26067 to R.S. and P25172 to C.R. Irene Frischauf is an Elise Richter Scholarship holder: V286.

Principles and properties of ion flow in P2X receptors

P2X receptors are a family of trimeric ion channels that are gated by extracellular adenosine 5′-triphosphate (ATP). These receptors have long been a subject of intense research interest by virtue of their vital role in mediating the rapid and direct effects of extracellular ATP on membrane potential and cytosolic Ca2+ concentration, which in turn underpin the ability of ATP to regulate a diverse range of clinically significant physiological functions, including those associated with the cardiovascular, sensory, and immune systems. An important aspect of an ion channel's function is, of course, the means by which it transports ions across the biological membrane. A concerted effort by investigators over the last two decades has culminated in significant advances in our understanding of how P2X receptors conduct the inward flux of Na+ and Ca2+ in response to binding by ATP. However, this work has relied heavily on results from current recordings of P2X receptors altered by site-directed mutagenesis. In the absence of a 3-dimensional channel structure, this prior work provided only a vague and indirect appreciation of the relationship between structure, ion selectivity and flux. The recent publication of the crystal structures for both the closed and open channel conformations of the zebrafish P2X4 receptor has thus proved a significant boon, and has provided an important opportunity to overview the amassed functional data in the context of a working 3-dimensional model of a P2X receptor. In this paper, we will attempt to reconcile the existing functional data regarding ion permeation through P2X receptors with the available crystal structure data, highlighting areas of concordance and discordance as appropriate.

Mechanism and Site of Inhibition of AMPA Receptors: Pairing a Thiadiazole with a 2,3-Benzodiazepine Scaffold

2,3-Benzodiazepine compounds are synthesized as drug candidates for treatment of various neurological disorders involving excessive activity of AMPA receptors. Here we report that pairing a thiadiazole moiety with a 2,3-benzodiazepine scaffold via the N-3 position yields an inhibitor type with >28-fold better potency and selectivity on AMPA receptors than the 2,3-benzodiazepine scaffold alone. Using whole-cell recording, we characterized two thiadiazolyl compounds, that is, one contains a 1,3,4-thiadiazole moiety and the other contains a 1,2,4-thiadiazole-3-one moiety. These compounds exhibit potent, equal inhibition of both the closed-channel and the open-channel conformations of all four homomeric AMPA receptor channels and two GluA2R-containing complex AMPA receptor channels. Furthermore, these compounds bind to the same receptor site as GYKI 52466 does, a site we previously termed as the "M" site. A thiadiazole moiety is thought to occupy more fully the side pocket of the receptor site or the "M" site, thereby generating a stronger, multivalent interaction between the inhibitor and the receptor binding site. We suggest that, as a heterocycle, a thiadiazole can be further modified chemically to produce a new class of even more potent, noncompetitive inhibitors of AMPA receptors.

Gating of the proton-gated ion channel from Gloeobacter violaceus at pH 4 as revealed by X-ray crystallography

Cryoelectron microscopy and X-ray crystallography have recently been used to generate structural models that likely represent the unliganded closed-channel conformation and the fully liganded open-channel conformation of different members of the nicotinic-receptor superfamily. To characterize the structure of the closed-channel conformation in its liganded state, we identified a number of positions in the loop between transmembrane segments 2 (M2) and 3 (M3) of a proton-gated ortholog from the bacterium Gloeobacter violaceus (GLIC) where mutations to alanine reduce the liganded-gating equilibrium constant, and solved the crystal structures of two such mutants (T25'A and Y27'A) at pH ~4.0. At the level of backbone atoms, the liganded closed-channel model presented here differs from the liganded open-channel structure of GLIC in the pre-M1 linker, the M3-M4 loop, and much more prominently, in the extracellular half of the pore lining, where the more pronounced tilt of the closed-channel M2 α-helices toward the pore's long axis narrows the permeation pathway. On the other hand, no differences between the liganded closed-channel and open-channel models could be detected at the level of the extracellular domain, where conformational changes are expected to underlie the low-to-high proton-affinity switch that drives gating of proton-bound channels. Thus, the liganded closed-channel model is nearly indistinguishable from the recently described "locally closed" structure. However, because cross-linking strategies (which could have stabilized unstable conformations) and mutations involving ionizable side chains (which could have affected proton-gated channel activation) were purposely avoided, we favor the notion that this structure represents one of the end states of liganded gating rather than an unstable intermediate.

Propofol Binding to the Resting State of the Gloeobacter violaceus Ligand-gated Ion Channel (GLIC) Induces Structural Changes in the Inter- and Intrasubunit Transmembrane Domain (TMD) Cavities

General anesthetics exert many of their CNS actions by binding to and modulating membrane-embedded pentameric ligand-gated ion channels (pLGICs). The structural mechanisms underlying how anesthetics modulate pLGIC function remain largely unknown. GLIC, a prokaryotic pLGIC homologue, is inhibited by general anesthetics, suggesting anesthetics stabilize a closed channel state, but in anesthetic-bound GLIC crystal structures the channel appears open. Here, using functional GLIC channels expressed in oocytes, we examined whether propofol induces structural rearrangements in the GLIC transmembrane domain (TMD). Residues in the GLIC TMD that frame intrasubunit and intersubunit water-accessible cavities were individually mutated to cysteine. We measured and compared the rates of modification of the introduced cysteines by sulfhydryl-reactive reagents in the absence and presence of propofol. Propofol slowed the rate of modification of L240C (intersubunit) and increased the rate of modification of T254C (intrasubunit), indicating that propofol binding induces structural rearrangements in these cavities that alter the local environment near these residues. Propofol acceleration of T254C modification suggests that in the resting state propofol does not bind in the TMD intrasubunit cavity as observed in the crystal structure of GLIC with bound propofol (Nury, H., Van Renterghem, C., Weng, Y., Tran, A., Baaden, M., Dufresne, V., Changeux, J. P., Sonner, J. M., Delarue, M., and Corringer, P. J. (2011) Nature 469, 428-431). In silico docking using a GLIC closed channel homology model suggests propofol binds to intersubunit sites in the TMD in the resting state. Propofol-induced motions in the intersubunit cavity were distinct from motions associated with channel activation, indicating propofol stabilizes a novel closed state.

Loop C and the mechanism of acetylcholine receptor–channel gating

Agonist molecules at the two neuromuscular acetylcholine (ACh) receptor (AChR) transmitter-binding sites increase the probability of channel opening. In one hypothesis for AChR activation (“priming”), the capping of loop C at each binding site transfers energy independently to the distant gate over a discrete structural pathway. We used single-channel analyses to examine the experimental support for this proposal with regard to brief unliganded openings, the effects of loop-C modifications, the effects of mutations to residues either on or off the putative pathway, and state models for describing currents at low [ACh]. The results show that (a) diliganded and brief unliganded openings are generated by the same essential, global transition; (b) the radical manipulation of loop C does not prevent channel opening but impairs agonist binding; (c) both on- and off-pathway mutations alter gating by changing the relative stability of the open-channel conformation by local interactions rather than by perturbing a specific site–gate communication link; and (d) it is possible to estimate directly the rate constants for agonist dissociation from and association to both the low and high affinity forms of the AChR-binding site by using a cyclic kinetic model. We conclude that the mechanism of energy transfer between the binding sites and the gate remains an open question.

Asymmetric Ligand Binding Facilitates Conformational Transitions in Pentameric Ligand-Gated Ion Channels

The anesthetic propofol inhibits the currents of the homopentameric ligand-gated ion channel GLIC, yet the crystal structure of GLIC with five propofol molecules bound symmetrically shows an open-channel conformation. To address this dilemma and determine if the symmetry of propofol binding sites affects the channel conformational transition, we performed a total of 1.5 μs of molecular dynamics simulations for different GLIC systems with propofol occupancies of 0, 1, 2, 3, and 5. GLIC without propofol binding or with five propofol molecules bound symmetrically, showed similar channel conformation and hydration status over multiple replicates of 100-ns simulations. In contrast, asymmetric binding to one, two or three equivalent sites in different subunits accelerated the channel dehydration, increased the conformational heterogeneity of the pore-lining TM2 helices, and shifted the lateral and radial tilting angles of TM2 toward a closed-channel conformation. The results differentiate two groups of systems based on the propofol binding symmetry. The difference between symmetric and asymmetric groups is correlated with the variance in the propofol-binding cavity adjacent to the hydrophobic gate and the force imposed by the bound propofol. Asymmetrically bound propofol produced greater variance in the cavity size that could further elevate the conformation heterogeneity. The force trajectory generated by propofol in each subunit over the course of a simulation exhibits an ellipsoidal shape, which has the larger component tangential to the pore. Asymmetric propofol binding creates an unbalanced force that expedites the channel conformation transitions. The findings from this study not only suggest that asymmetric binding underlies the propofol functional inhibition of GLIC, but also advocate for the role of symmetry breaking in facilitating channel conformational transitions.

Probing the Pore Dimensions of Bacterial Nicotinic Receptor-Like Channels with Open-Channel Blockers

ELIC and GLIC, two bacterial nicotinic receptor-like channels, are remarkably amenable to both structural and functional studies. This observation, along with the notion that the overall structure and functional properties of these channels are very similar to those of their metazoan counterparts, amply justifies the use of ELIC and GLIC as models for the entire superfamily. At the level of the transmembrane pore, the structural model of ELIC shows a prominent constriction around the 16' position of the M2 alpha-helices, whereas that of GLIC displays a much wider, semi-conical cavity with helices tilted away from the long axis of the pore. As a result, the structural models of ELIC and GLIC are often considered to represent the closed-channel and open-channel conformations, respectively, of all members of the superfamily, a notion that assumes that the end states of the gating conformational change are conserved across orthologs. Thus far, attempts to capture the different physiologically relevant conformations of each of these channels in well-diffracting crystals have failed. Here, as an alternative approach to probe the transmembrane-pore domain in the open-channel conformation, we studied the interaction of ELIC, GLIC and the muscle nicotinic acetylcholine receptor (AChR) with pore blockers. From the comparative analysis of these results, it appears as though the muscle AChR and GLIC have very similar open-channel pore structures, whereas the open-channel pore of ELIC seems much narrower, and thus, closer to the structural model of ELIC in its closed state. Hence, it is likely that the closed-to-open conformational change of ELIC at the level of the pore domain is subtler than the one that could be predicted if ELIC were to adopt a GLIC-like structure in the open state.

Mechanisms of Channel Activation in GLIC

GLIC is a pentameric proton-gated ion channel with an EC50 at pH of 5.0. It remains unclear which regions of GLIC are responsible for channel activation. In this study, we explore two potential regions by combining mutagenesis/functional experiments with computations using the Perturbation-based Markovian Transmission (PMT) model. The first region involves inter-subunit salt bridges between the C-loop (E177 and D178) and β5 (D91) of the complementary side. The second region (N152, D153, and D154) was part of the cavity identified crystallographically for anesthetic ketamine binding (Pan, Chen, et.al, Structure, 20, 1463, 2012). Mutations in the first region (D91N, E177Q, D178N) removed salt bridges and resulted in an increase of the EC50 (decreased pH to 4.8), suggesting the importance of the salt bridges in stabilizing the open-channel conformation, as confirmed by the less closed C-loop in the X-ray crystal structure. The mutation in the second region (N152C) resulted in a decreased of the EC50 (increased the pH to 5.4), presumably due to more flexibility for the protonation of D153 and D154. Using the PMT model, which provides time dependent information on the pathway of signal propagation, we found that perturbations to the first and second regions propagated through two different paths to the pore-lining TM2. The perturbation to the first region travels mainly from loop C, down β10 and β7 to the Cys-loop, and on to TM1 and TM3, which together affect TM2. The perturbation in the second region starts in the middle of the F-loop, travels down along the F-loop into the TM2-TM3 linker, and then to TM2. Taken together, our results reveal residues important to channel activation and allosteric signal pathways. Supported by NIH (R01GM066358, R01GM056257, R37GM049202, and T32GM075770).

A gating motif in the translocation channel sets the hydrophobicity threshold for signal sequence function

A critical event in protein translocation across the endoplasmic reticulum is the structural transition between the closed and open conformations of Sec61, the eukaryotic translocation channel. Channel opening allows signal sequence insertion into a gap between the N- and C-terminal halves of Sec61. We have identified a gating motif that regulates the transition between the closed and open channel conformations. Polar amino acid substitutions in the gating motif cause a gain-of-function phenotype that permits translocation of precursors with marginally hydrophobic signal sequences. In contrast, hydrophobic substitutions at certain residues in the gating motif cause a protein translocation defect. We conclude that the gating motif establishes the hydrophobicity threshold for functional insertion of a signal sequence into the Sec61 complex, thereby allowing the wild-type translocation channel to discriminate between authentic signal sequences and the less hydrophobic amino acid segments in cytosolic proteins. Bioinformatic analysis indicates that the gating motif is conserved between eubacterial and archaebacterial SecY and eukaryotic Sec61.

Activation of α7 Nicotinic Receptors by Orthosteric and Allosteric Agonists: Influence on Single-Channel Kinetics and Conductance

Nicotinic acetylcholine receptors (nAChRs) are oligomeric transmembrane proteins in which five subunits coassemble to form a central ion channel pore. Conventional agonists, such as acetylcholine (ACh), bind to an orthosteric site, located at subunit interfaces in the extracellular domain. More recently, it has been demonstrated that nAChRs can also be activated by ligands binding to an allosteric transmembrane site. In the case of α7 nAChRs, ACh causes rapid activation and almost complete desensitization. In contrast, allosteric agonists such as 4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c] quin oline-8-sulfonamide (4BP-TQS) activate α7 nAChRs more slowly and cause only low levels of apparent desensitization. In the present study, single-channel patch-clamp recording has been used to investigate differences in the mechanism of activation of α7 nAChRs by ACh and 4BP-TQS. The most striking difference between activation by ACh and 4BP-TQS is in single-channel kinetics. In comparison with activation by ACh, single-channel open times and burst lengths are substantially longer (~160-800-fold, respectively), and shut times are shorter (~8-fold) when activated by 4BP-TQS. In addition, coapplication of ACh and 4BP-TQS results in a further increase in single-channel burst lengths. Mean burst lengths seen when the two agonists are coapplied (3099 ± 754 ms) are ~2.5-fold longer than with 4BP-TQS alone and ∼370-fold longer than with ACh alone. Intriguingly, the main single-channel conductance of α7 nAChRs, was significantly larger when activated by 4BP-TQS (100.3 ± 2.4 pS) than when activated by ACh (90.0 ± 2.7 pS), providing evidence that activation by allosteric and orthosteric agonists results in different α7 nAChRs open-channel conformations.