Curariform antagonists bind in different orientations to the nicotinic receptor ligand binding domain

Curariform alkaloids competitively inhibit muscle acetylcholine receptors (AChR) by bridging the alpha and non-alpha subunits that form the ligand-binding site. Here we delineate bound orientations of d-tubocurarine (d-TC) and its methylated derivative metocurine using mutagenesis, ligand binding measurements, and computational methods. When tested against a series of lysine mutations in the epsilon subunit, the two antagonists show marked differences in the consequences of the mutations on binding affinity. The mutations epsilon L117K, epsilon Y111K, and epsilon L109K decrease affinity of metocurine by up to 3 orders of magnitude but only slightly alter affinity of d-TC. At the alpha subunit face of the binding site, the mutation alpha Y198T decreases affinity of both antagonists, but alpha Y198F preferentially enhances affinity of d-TC. Computation of antagonist docking orientations, based on our structural model of the alpha-epsilon site of the human AChR, indicates distinct orientations of each antagonist; the flatter metocurine fits into a pocket formed principally by the epsilon subunit, whereas the more compact d-TC spans the narrower crevasse between alpha and epsilon subunits. The side chains of epsilon Tyr-111 and epsilon Thr-117 juxtapose one of two quaternary nitrogens in metocurine but are remote from the equivalent quaternary nitrogen in d-TC, which instead closely approaches alpha Tyr-198. The different docked orientations arise through tilt of the curariform scaffold by approximately 60 degrees normal to the nitrogen-nitrogen axis, together with a 20 degrees rotation about the axis. The overall mutagenesis and computational results show that despite their similar structures, d-TC and metocurine bind in distinctly different orientations to the adult human AChR.     

Mutation in the M1 Domain of the Acetylcholine Receptor α Subunit Decreases the Rate of Agonist Dissociation

We describe the kinetic consequences of the mutation N217K in the M1 domain of the acetylcholine receptor (AChR) α subunit that causes a slow channel congenital myasthenic syndrome (SCCMS). We previously showed that receptors containing αN217K expressed in 293 HEK cells open in prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Here we use single channel kinetic analysis to show that the prolonged activation episodes result primarily from slowing of the rate of acetylcholine (ACh) dissociation from the binding site. Rate constants for channel opening and closing are also slowed but to much smaller extents. The rate constants derived from kinetic analysis also describe the concentration dependence of receptor activation, revealing a 20-fold shift in the EC₅₀ to lower agonist concentrations for αN217K. The apparent affinity of ACh binding, measured by competition against the rate of ¹²⁵I-α-bungarotoxin binding, is also enhanced 20-fold by αN217K. Both the slowing of ACh dissociation and enhanced apparent affinity are specific to the lysine substitution, as the glutamine and glutamate substitutions have no effect. Substituting lysine for the equivalent asparagine in the β, ε, or δ subunits does not affect the kinetics of receptor activation or apparent agonist affinity. The results show that a mutation in the amino-terminal portion of the M1 domain produces a localized perturbation that stabilizes agonist bound to the resting state of the AChR.     

Mutations affecting agonist sensitivity of the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor (AChR) is a pentameric transmembrane protein (alpha 2 beta gamma delta) that binds the neurotransmitter acetylcholine (ACh) and transduces this binding into the opening of a cation selective channel. The agonist, competitive antagonist, and snake toxin binding functions of the AChR are associated with the alpha subunit (Kao et al., 1984; Tzartos and Changeux, 1984; Wilson et al., 1985; Kao and Karlin, 1986; Pederson et al., 1986). We used site-directed mutagenesis and expression of AChR in Xenopus oocytes to identify amino acid residues critical for ligand binding and channel activation. Several mutations in the alpha subunit sequence were constructed based on information from sequence homology and from previous biochemical (Barkas et al., 1987; Dennis et al., 1988; Middleton and Cohen, 1990) and spectroscopic (Pearce and Hawrot, 1990; Pearce et al., 1990) studies. We have identified one mutation, Tyr190 to Phe (Y190F), that had a dramatic effect on ligand binding and channel activation. These mutant channels required more than 50-fold higher concentrations of ACh for channel activation than did wild type channels. This functional change is largely accounted for by a comparable shift in the agonist binding affinity, as assessed by the ability of ACh to compete with alpha-bungarotoxin binding. Other mutations at nearby conserved positions of the alpha subunit (H186F, P194S, Y198F) produce less dramatic changes in channel properties. Our results demonstrate that ligand binding and channel gating are separable properties of the receptor protein, and that Tyr190 appears to play a specific role in the receptor site for acetylcholine.     

A distinct contribution of the delta subunit to acetylcholine receptor channel activation revealed by mutations of the M2 segment.

Acetylcholine receptor (AChR) channels with proline (P) mutations in the putative pore-forming domain (at the 12' position of the M2 segment) were examined at the single-channel level. For all subunits (alpha, beta, epsilon, and delta), a 12'P mutation increased the open channel lifetime >5-fold. To facilitate the estimation of binding and gating rate constants, subunits with 12'P mutations were co-expressed with alpha subunits having a binding site mutation that slows channel opening (alphaD200N). In these AChRs, a 12'P mutation in epsilon or beta slowed the closing rate constant approximately 6-fold but had no effect on either the channel opening rate constant or the equilibrium dissociation constant for ACh (Kd). In contrast, a 12'P mutation in delta slowed the channel closing rate constant only approximately 2-fold and significantly increased both the channel opening rate constant and the Kd. Pairwise expression of 12'P subunits indicates that mutations in epsilon and beta act nearly independently, but one in delta reduces the effect of a homologous mutation in epsilon or beta. The results suggest that a 12'P mutation in epsilon and beta has mainly local effects, whereas one in delta has both local and distributed effects that influence both agonist binding and channel gating.     

Activation kinetics of recombinant mouse nicotinic acetylcholine receptors: mutations of alpha-subunit tyrosine 190 affect both binding and gating.

Affinity labeling and mutagenesis studies have demonstrated that the conserved tyrosine Y190 of the acetylcholine receptor (AChR) alpha-subunit is a key determinant of the agonist binding site. Here we describe the binding and gating kinetics of embryonic mouse AChRs with mutations at Y190. In Y190F the dissociation constant for ACh binding to closed channels was reduced approximately 35-fold at the first binding site and only approximately 2-fold at the second site. At both binding sites the association and dissociation rate constants were decreased by the mutation. Compared with wildtype AChRs, doubly-liganded alpha Y190F receptors open 400 times more slowly but close only 2 times more rapidly. Considering the overall activation reaction (vacant-closed to fully occupied-open), there is an increase of approximately 6.4 kcal/mol caused by the Y-to-F mutation, of which at least 2.1 and 0.3 kcal/mol comes from altered agonist binding to the first and second binding sites, respectively. The closing rate constant of alpha Y190F receptors was the same with ACh, carbamoylcholine, or tetramethylammonium as the agonist. This rate constant was approximately 3 times faster in ACh-activated S, W, and T mutants. The equilibrium dissociation constant for channel block by ACh was approximately 2-fold lower in alpha Y190F receptors compared with in wildtype receptors, suggesting that there are changes in the pore region of the receptor as a consequence of the mutation. The activation reaction is discussed with regard to energy provided by agonist-receptor binding contacts, and by the intrinsic folding energy of the receptor.     

Initial coupling of binding to gating mediated by conserved residues in the muscle nicotinic receptor

We examined functional consequences of intrasubunit contacts in the nicotinic receptor alpha subunit using single channel kinetic analysis, site-directed mutagenesis, and structural modeling. At the periphery of the ACh binding site, our structural model shows that side chains of the conserved residues alphaK145, alphaD200, and alphaY190 converge to form putative electrostatic interactions. Structurally conservative mutations of each residue profoundly impair gating of the receptor channel, primarily by slowing the rate of channel opening. The combined mutations alphaD200N and alphaK145Q impair channel gating to the same extent as either single mutation, while alphaK145E counteracts the impaired gating due to alphaD200K, further suggesting electrostatic interaction between these residues. Interpreted in light of the crystal structure of acetylcholine binding protein (AChBP) with bound carbamylcholine (CCh), the results suggest in the absence of ACh, alphaK145 and alphaD200 form a salt bridge associated with the closed state of the channel. When ACh binds, alphaY190 moves toward the center of the binding cleft to stabilize the agonist, and its aromatic hydroxyl group approaches alphaK145, which in turn loosens its contact with alphaD200. The positional changes of alphaK145 and alphaD200 are proposed to initiate the cascade of perturbations that opens the receptor channel: the first perturbation is of beta-strand 7, which harbors alphaK145 and is part of the signature Cys-loop, and the second is of beta-strand 10, which harbors alphaD200 and connects to the M1 domain. Thus, interplay between these three conserved residues relays the initial conformational change from the ACh binding site toward the ion channel.     

The role of loop 5 in acetylcholine receptor channel gating

Nicotinic acetylcholine receptor channel (AChR) gating is an organized sequence of molecular motions that couples a change in the affinity for ligands at the two transmitter binding sites with a change in the ionic conductance of the pore. Loop 5 (L5) is a nine-residue segment (mouse alpha-subunit 92-100) that links the beta4 and beta5 strands of the extracellular domain and that (in the alpha-subunit) contains binding segment A. Based on the structure of the acetylcholine binding protein, we speculate that in AChRs L5 projects from the transmitter binding site toward the membrane along a subunit interface. We used single-channel kinetics to quantify the effects of mutations to alphaD97 and other L5 residues with respect to agonist binding (to both open and closed AChRs), channel gating (for both unliganded and fully-liganded AChRs), and desensitization. Most alphaD97 mutations increase gating (up to 168-fold) but have little or no effect on ligand binding or desensitization. Rate-equilibrium free energy relationship analysis indicates that alphaD97 moves early in the gating reaction, in synchrony with the movement of the transmitter binding site (Phi = 0.93, which implies an open-like character at the transition state). alphaD97 mutations in the two alpha-subunits have unequal energetic consequences for gating, but their contributions are independent. We conclude that the key, underlying functional consequence of alphaD97 perturbations is to increase the unliganded gating equilibrium constant. L5 emerges as an important and early link in the AChR gating reaction which, in the absence of agonist, serves to increase the relative stability of the closed conformation of the protein.     

Localization of agonist and competitive antagonist binding sites on nicotinic acetylcholine receptors

Identification of all residues involved in the recognition and binding of cholinergic ligands (e.g. agonists, competitive antagonists, and noncompetitive agonists) is a primary objective to understand which structural components are related to the physiological function of the nicotinic acetylcholine receptor (AChR). The picture for the localization of the agonist/competitive antagonist binding sites is now clearer in the light of newer and better experimental evidence. These sites are located mainly on both alpha subunits in a pocket approximately 30-35 A above the surface membrane. Since both alpha subunits are identical, the observed high and low affinity for different ligands on the receptor is conditioned by the interaction of the alpha subunit with other non-alpha subunits. This molecular interaction takes place at the interface formed by the different subunits. For example, the high-affinity acetylcholine (ACh) binding site of the muscle-type AChR is located on the alphadelta subunit interface, whereas the low-affinity ACh binding site is located on the alphagamma subunit interface. Regarding homomeric AChRs (e.g. alpha7, alpha8, and alpha9), up to five binding sites may be located on the alphaalpha subunit interfaces. From the point of view of subunit arrangement, the gamma subunit is in between both alpha subunits and the delta subunit follows the alpha aligned in a clockwise manner from the gamma. Although some competitive antagonists such as lophotoxin and alpha-bungarotoxin bind to the same high- and low-affinity sites as ACh, other cholinergic drugs may bind with opposite specificity. For instance, the location of the high- and the low-affinity binding site for curare-related drugs as well as for agonists such as the alkaloid nicotine and the potent analgesic epibatidine (only when the AChR is in the desensitized state) is determined by the alphagamma and the alphadelta subunit interface, respectively. The case of alpha-conotoxins (alpha-CoTxs) is unique since each alpha-CoTx from different species is recognized by a specific AChR type. In addition, the specificity of alpha-CoTxs for each subunit interface is species-dependent.In general terms we may state that both alpha subunits carry the principal component for the agonist/competitive antagonist binding sites, whereas the non-alpha subunits bear the complementary component. Concerning homomeric AChRs, both the principal and the complementary component exist on the alpha subunit. The principal component on the muscle-type AChR involves three loops-forming binding domains (loops A-C). Loop A (from mouse sequence) is mainly formed by residue Y(93), loop B is molded by amino acids W(149), Y(152), and probably G(153), while loop C is shaped by residues Y(190), C(192), C(193), and Y(198). The complementary component corresponding to each non-alpha subunit probably contributes with at least four loops. More specifically, the loops at the gamma subunit are: loop D which is formed by residue K(34), loop E that is designed by W(55) and E(57), loop F which is built by a stretch of amino acids comprising L(109), S(111), C(115), I(116), and Y(117), and finally loop G that is shaped by F(172) and by the negatively-charged amino acids D(174) and E(183). The complementary component on the delta subunit, which corresponds to the high-affinity ACh binding site, is formed by homologous loops. Regarding alpha-neurotoxins, several snake and alpha-CoTxs bear specific residues that are energetically coupled with their corresponding pairs on the AChR binding site. The principal component for snake alpha-neurotoxins is located on the residue sequence alpha1W(184)-D(200), which includes loop C. In addition, amino acid sequence 55-74 from the alpha1 subunit (which includes loop E), and residues gammaL(119) (close to loop F) and gammaE(176) (close to loop G) at the low-affinity binding site, or deltaL(121) (close to the homologous region of loop G) at the high-affinity binding site, are i     

Invariant aspartic Acid in muscle nicotinic receptor contributes selectively to the kinetics of agonist binding

We examined functional contributions of interdomain contacts within the nicotinic receptor ligand binding site using single channel kinetic analyses, site-directed mutagenesis, and a homology model of the major extracellular region. At the principal face of the binding site, the invariant alphaD89 forms a highly conserved interdomain contact near alphaT148, alphaW149, and alphaT150. Patch-clamp recordings show that the mutation alphaD89N markedly slows acetylcholine (ACh) binding to receptors in the resting closed state, but does not affect rates of channel opening and closing. Neither alphaT148L, alphaT150A, nor mutations at both positions substantially affects the kinetics of receptor activation, showing that hydroxyl side chains at these positions are not hydrogen bond donors for the strong acceptor alphaD89. However substituting a negative charge at alphaT148, but not at alphaT150, counteracts the effect of alphaD89N, demonstrating that a negative charge in the region of interdomain contact confers rapid association of ACh. Interpreted within the structural framework of ACh binding protein and a homology model of the receptor ligand binding site, these results implicate main chain amide groups in the domain harboring alphaW149 as principal hydrogen bond donors for alphaD89. The specific effect of alphaD89N on ACh association suggests that interdomain hydrogen bonding positions alphaW149 for optimal interaction with ACh.     

Mapping the structural requirements for nicotinic acetylcholine receptor activation by using tethered alkyltrimethylammonium agonists and antagonists

A molecule as simple in structure as tetramethylammonium gates the nicotinic acetylcholine receptor (nAChR) with high efficacy. To compare the structure of the nAChR transmitter binding site in the open channel state with that of the ACh binding protein, we determined the efficacy of nAChR gating by -S(CH(2))(n)N(CH(3))(3)(+) (n = 1-4) tethered to substituted cysteines at positions in the alpha subunits or gamma and delta subunits predicted to contribute to the ACh binding sites in mutant Torpedo nAChRs expressed in Xenopus oocytes. For tethered thiocholine [-S(CH(2))(2)N(CH(3))(3)(+)], we previously reported that within alpha195-201 gating was observed only at alphaY198C while at alphaY93C it acted as an antagonist. We now show that within alpha191-194, thiocholine activates when tethered at alphaCys192 or alphaCys193. Thiocholine also activates when tethered at alphaY190C or alphaW149C in nAChRs containing a beta subunit mutation (betaL257S) that destabilizes the closed channel, but not from gammaW55C/deltaW57C, where longer adducts can activate. When tethered at positions in binding site segment E, thiocholine activates only from gammaL119C/deltaL121C, where the shorter -S(CH(2))(1)N(CH3)(3)(+) acts as an antagonist. Longer adducts tethered at gammaL109C/deltaL111C or gammaL119C/deltaL121C also activate, but less efficiently. The length requirements for efficient gating by tethered agonists agree closely with predictions based upon the structure of the agonist site in a nAChR homology model derived from the ACh binding protein structure, which suggests that this structure is an excellent model of the nAChR agonist binding site in the open channel conformation. The inability of thiocholine to activate from alphaY93C, which is not predicted by the model, is discussed in terms of the structure of the nAChR in the closed state.     

Fundamental gating mechanism of nicotinic receptor channel revealed by mutation causing a congenital myasthenic syndrome

We describe the genetic and kinetic defects in a congenital myasthenic syndrome due to the mutation epsilonA411P in the amphipathic helix of the acetylcholine receptor (AChR) epsilon subunit. Myasthenic patients from three unrelated families are either homozygous for epsilonA411P or are heterozygous and harbor a null mutation in the second epsilon allele, indicating that epsilonA411P is recessive. We expressed human AChRs containing wild-type or A411P epsilon subunits in 293HEK cells, recorded single channel currents at high bandwidth, and determined microscopic rate constants for individual channels using hidden Markov modeling. For individual wild-type and mutant channels, each rate constant distributes as a Gaussian function, but the spread in the distributions for channel opening and closing rate constants is greatly expanded by epsilonA411P. Prolines engineered into positions flanking residue 411 of the epsilon subunit greatly increase the range of activation kinetics similar to epsilonA411P, whereas prolines engineered into positions equivalent to epsilonA411 in beta and delta subunits are without effect. Thus, the amphipathic helix of the epsilon subunit stabilizes the channel, minimizing the number and range of kinetic modes accessible to individual AChRs. The findings suggest that analogous stabilizing structures are present in other ion channels, and possibly allosteric proteins in general, and that they evolved to maintain uniformity of activation episodes. The findings further suggest that the fundamental gating mechanism of the AChR channel can be explained by a corrugated energy landscape superimposed on a steeply sloped energy well.     

CFTR gating II: Effects of nucleotide binding on the stability of open states

Previously, we demonstrated that ADP inhibits cystic fibrosis transmembrane conductance regulator (CFTR) opening by competing with ATP for a binding site presumably in the COOH-terminal nucleotide binding domain (NBD2). We also found that the open time of the channel is shortened in the presence of ADP. To further study this effect of ADP on the open state, we have used two CFTR mutants (D1370N and E1371S); both have longer open times because of impaired ATP hydrolysis at NBD2. Single-channel kinetic analysis of DeltaR/D1370N-CFTR shows unequivocally that the open time of this mutant channel is decreased by ADP. DeltaR/E1371S-CFTR channels can be locked open by millimolar ATP with a time constant of approximately 100 s, estimated from current relaxation upon nucleotide removal. ADP induces a shorter locked-open state, suggesting that binding of ADP at a second site decreases the locked-open time. To test the functional consequence of the occupancy of this second nucleotide binding site, we changed the [ATP] and performed similar relaxation analysis for E1371S-CFTR channels. Two locked-open time constants can be discerned and the relative distribution of each component is altered by changing [ATP] so that increasing [ATP] shifts the relative distribution to the longer locked-open state. Single-channel kinetic analysis for DeltaR/E1371S-CFTR confirms an [ATP]-dependent shift of the distribution of two locked-open time constants. These results support the idea that occupancy of a second ATP binding site stabilizes the locked-open state. This binding site likely resides in the NH2-terminal nucleotide binding domain (NBD1) because introducing the K464A mutation, which decreases ATP binding affinity at NBD1, into E1371S-CFTR shortens the relaxation time constant. These results suggest that the binding energy of nucleotide at NBD1 contributes to the overall energetics of the open channel conformation.     

Acetylcholine and epibatidine binding to muscle acetylcholine receptors distinguish between concerted and uncoupled models.

The muscle acetylcholine receptor (AChR) has served as a prototype for understanding allosteric mechanisms of neurotransmitter-gated ion channels. The phenomenon of cooperative agonist binding is described by the model of Monod et al. (Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118; MWC model), which requires concerted switching of the two binding sites between low and high affinity states. The present study examines binding of acetylcholine (ACh) and epibatidine, agonists with opposite selectivity for the two binding sites of mouse muscle AChRs. We expressed either fetal or adult AChRs in 293 HEK cells and measured agonist binding by competition against the initial rate of 125I-alpha-bungarotoxin binding. We fit predictions of the MWC model to epibatidine and ACh binding data simultaneously, taking as constants previously determined parameters for agonist binding and channel gating steps, and varying the agonist-independent parameters. We find that the MWC model describes the apparent dissociation constants for both agonists but predicts Hill coefficients that are far too steep. An Uncoupled model, which relaxes the requirement of concerted state transitions, accurately describes binding of both ACh and epibatidine and provides parameters for agonist-independent steps consistent with known aspects of AChR function.     

Nicotinic receptor fourth transmembrane domain: hydrogen bonding by conserved threonine contributes to channel gating kinetics

The fourth transmembrane domain (M4) of the nicotinic acetylcholine receptor (AChR) contributes to the kinetics of activation, yet its close association with the lipid bilayer makes it the outermost of the transmembrane domains. To investigate mechanistic and structural contributions of M4 to AChR activation, we systematically mutated alphaT422, a conserved residue that has been labeled by hydrophobic probes, and evaluated changes in rate constants underlying ACh binding and channel gating steps. Aromatic and nonpolar mutations of alphaT422 selectively affect the channel gating step, slowing the rate of opening two- to sevenfold, and speeding the rate of closing four- to ninefold. Additionally, kinetic modeling shows a second doubly liganded open state for aromatic and nonpolar mutations. In contrast, serine and asparagine mutations of alphaT422 largely preserve the kinetics of the wild-type AChR. Thus, rapid and efficient gating of the AChR channel depends on a hydrogen bond involving the side chain at position 422 of the M4 transmembrane domain.     

The extracellular linker of muscle acetylcholine receptor channels is a gating control element

We describe the functional consequences of mutations in the linker between the second and third transmembrane segments (M2-M3L) of muscle acetylcholine receptors at the single-channel level. Hydrophobic mutations (Ile, Cys, and Phe) placed near the middle of the linker of the alpha subunit (alphaS269) prolong apparent openings elicited by low concentrations of acetylcholine (ACh), whereas hydrophilic mutations (Asp, Lys, and Gln) are without effect. Because the gating kinetics of the alphaS269I receptor (a congenital myasthenic syndrome mutant) in the presence of ACh are too fast, choline was used as the agonist. This revealed an approximately 92-fold increased gating equilibrium constant, which is consistent with an approximately 10-fold decreased EC(50) in the presence of ACh. With choline, this mutation accelerates channel opening approximately 28-fold, slows channel closing approximately 3-fold, but does not affect agonist binding to the closed state. These ratios suggest that, with ACh, alphaS269I acetylcholine receptors open at a rate of approximately 1.4 x 10(6) s(-1) and close at a rate of approximately 760 s(-1). These gating rate constants, together with the measured duration of apparent openings at low ACh concentrations, further suggest that ACh dissociates from the diliganded open receptor at a rate of approximately 140 s(-1). Ile mutations at positions flanking alphaS269 impair, rather than enhance, channel gating. Inserting or deleting one residue from this linker in the alpha subunit increased and decreased, respectively, the apparent open time approximately twofold. Contrary to the alphaS269I mutation, Ile mutations at equivalent positions of the beta, straightepsilon, and delta subunits do not affect apparent open-channel lifetimes. However, in beta and straightepsilon, shifting the mutation one residue to the NH(2)-terminal end enhances channel gating. The overall results indicate that this linker is a control element whose hydrophobicity determines channel gating in a position- and subunit-dependent manner. Characterization of the transition state of the gating reaction suggests that during channel opening the M2-M3L of the alpha subunit moves before the corresponding linkers of the beta and straightepsilon subunits.     

The I182 region of k(ir)6.2 is closely associated with ligand binding in K(ATP) channel inhibition by ATP

The ATP-inhibited potassium (K(ATP)) channel is assembled from four inward rectifier potassium (K(ir)6.x) subunits and four sulfonylurea receptor (SURx) subunits. The inhibitory action of ATP is mediated by at least two distinct functional domains within the C-terminal cytoplasmic tail of K(ir)6.2. The G334D mutation of K(ir)6.2 virtually eliminates ATP-dependent gating with no effect on ligand-independent gating, suggesting a role in linkage of the site to the gate or in the ATP binding site, itself. The T171A mutation of K(ir)6.2 strongly disrupts both ATP-dependent and ligand-independent gating, suggesting a role for T171 in the gating step. A neighboring mutation, I182Q, virtually eliminates ATP inhibition, but its effect on ligand-independent gating remained unknown. We have now characterized both the K(i) values for inhibition by ATP and the ligand-independent gating kinetics of 15 substitutions at position 182. All substitutions decreased ATP-dependent inhibition gating as measured by the K(i), many profoundly so, yet had little or no effect on ligand-independent gating kinetics. Thus, substitutions at position 182 are unlikely to act by disrupting inhibition gate movement. Our results indicate an indispensable role for I182 in a step of the ATP binding mechanism, the linkage mechanism coupling the ATP binding site to the inhibition gate, or both.     

Identification of binding sites in the nicotinic acetylcholine receptor for [3H]azietomidate, a photoactivatable general anesthetic

To identify binding domains in a ligand-gated ion channel for etomidate, an intravenous general anesthetic, we photolabeled nicotinic acetylcholine receptor (nAChR)-rich membranes from Torpedo electric organ with a photoactivatable analog, [(3)H]azietomidate. Based upon the inhibition of binding of the noncompetitive antagonist [(3)H]phencyclidine, azietomidate and etomidate bind with 10-fold higher affinity to nAChRs in the desensitized state (IC(50) = 70 microm) than in the closed channel state. In addition, both drugs between 0.1 and 1 mm produced a concentration-dependent enhancement of [(3)H]ACh equilibrium binding affinity, but they inhibited binding at higher concentrations. UV irradiation resulted in preferential [(3)H]azietomidate photoincorporation into the nAChR alpha and delta subunits. Photolabeled amino acids in both subunits were identified in the ion channel domain and in the ACh binding sites by Edman degradation. Within the nAChR ion channel in the desensitized state, there was labeling of alphaGlu-262 and deltaGln-276 at the extracellular end and deltaSer-258 and deltaSer-262 toward the cytoplasmic end. Within the acetylcholine binding sites, [(3)H]azietomidate photolabeled alphaTyr-93, alphaTyr-190, and alphaTyr-198 in the site at the alpha-gamma interface and deltaAsp-59 (but not the homologous position, gammaGlu-57). Increasing [(3)H]azietomidate concentration from 1.8 to 150 microm increased the efficiency of incorporation into amino acids within the ion channel by 10-fold and in the ACh sites by 100-fold, consistent with higher affinity binding within the ion channel. The state dependence and subunit selectivity of [(3)H]azietomidate photolabeling are discussed in terms of the structures of the nAChR transmembrane and extracellular domains.     

Gamma- and delta-subunits regulate the affinity and the cooperativity of ligand binding to the acetylcholine receptor.

The acetylcholine receptor (AChR) from vertebrate skeletal muscle is a pentamer composed of two ligand-binding alpha-subunits and one beta-, gamma-, and delta-subunit. To examine the functional roles of the non-alpha-subunits, we have expressed, in stable cell lines, AChRs lacking either a gamma- or a delta-subunit. Most previous work has examined how these changes in subunit composition affect single channel properties. Here, we take advantage of the stable expression system to produce large amounts of AChR and, for the first time, examine ligand binding to altered AChRs on intact cells. The changes in subunit composition affect both ligand affinity and cooperativity of the receptor, suggesting important roles for the gamma- and delta-subunits, both in shaping the ligand binding site and maintaining cooperative interactions between alpha-subunits.     

Estimating binding affinities of the nicotinic receptor for low-efficacy ligands using mixtures of agonists and two-dimensional concentration-response relationships

The phenomenon of ligand-induced ion channel gating hinges upon the ability of a receptor channel to bind ligand molecules with conformation-specific affinities. However, our understanding of this fundamental phenomenon is notably limited, not only because the changes in binding site structure and ligand conformation that occur upon gating are largely unknown but, also, because the strength of these ligand-receptor interactions are experimentally elusive. Both high- and low-efficacy ligands pose a number of analytical and experimental challenges that can render the estimation of their conformation-specific binding affinities impossible. In this paper, we present a novel assay that overcomes some of the hurdles presented by weak agonists of the muscle nicotinic receptor and allows the estimation of their closed-state affinities. The method, which we have termed the "activation-competition" assay, consists of a single-channel concentration-response assay performed in the presence of a binary mixture of ligands of widely different efficacies. By plotting the channel response (i.e., the open probability) as a function of the concentration of each agonist in the mixture, interpreting the observed response in the framework of a plausible kinetic scheme, and fitting the open probability surface with the corresponding function, the affinities of the closed receptor for the two agonists can be simultaneously extracted as free parameters. Here, we applied this methodology to estimate the closed-state affinity of the muscle nicotinic receptor for choline (a very weak agonist) using acetylcholine (ACh) as the partner in the mixture. We estimated the dissociation equilibrium constant of choline (K(D)) from the wild type's closed state to be 4.1 +/- 0.5 mM (and that of ACh to be 106 +/- 6 microM). We also discuss the use of accurate estimates of affinities for low-efficacy agonists as a tool to discriminate between binding and gating effects of mutations, and in the context of the rational design of therapeutic drugs.     

The two ATP binding sites of cystic fibrosis transmembrane conductance regulator (CFTR) play distinct roles in gating kinetics and energetics

Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC (ATP binding cassette) transporter family, is a chloride channel whose activity is controlled by protein kinase-dependent phosphorylation. Opening and closing (gating) of the phosphorylated CFTR is coupled to ATP binding and hydrolysis at CFTR's two nucleotide binding domains (NBD1 and NBD2). Recent studies present evidence that the open channel conformation reflects a head-to-tail dimerization of CFTR's two NBDs as seen in the NBDs of other ABC transporters (Vergani et al., 2005). Whether these two ATP binding sites play an equivalent role in the dynamics of NBD dimerization, and thus in gating CFTR channels, remains unsettled. Based on the crystal structures of NBDs, sequence alignment, and homology modeling, we have identified two critical aromatic amino acids (W401 in NBD1 and Y1219 in NBD2) that coordinate the adenine ring of the bound ATP. Conversion of the W401 residue to glycine (W401G) has little effect on the sensitivity of the opening rate to [ATP], but the same mutation at the Y1219 residue dramatically lowers the apparent affinity for ATP by >50-fold, suggesting distinct roles of these two ATP binding sites in channel opening. The W401G mutation, however, shortens the open time constant. Energetic analysis of our data suggests that the free energy of ATP binding at NBD1, but not at NBD2, contributes significantly to the energetics of the open state. This kinetic and energetic asymmetry of CFTR's two NBDs suggests an asymmetric motion of the NBDs during channel gating. Opening of the channel is initiated by ATP binding at the NBD2 site, whereas separation of the NBD dimer at the NBD1 site constitutes the rate-limiting step in channel closing.