Role of key aromatic residues in the ligand-binding domain of alpha7 nicotinic receptors in the agonist action of beta-amyloid

Soluble β-amyloid (Aβ) resides in certain regions of the brain at or near picomolar concentration, rising in level during the prodromic stage of Alzheimer disease. Recently, we identified the homomeric α7 nicotinic acetylcholine receptor (α7-nAChR) as one possible functional target for picomolar Aβ. This study was aimed at addressing which residues in α7-nAChRs potentially interact with Aβ to regulate the presynaptic function of this receptor. Site-directed mutagenesis was carried out to study the key aromatic residues in the mouse α7-nAChR agonist-binding pocket. Mutations of tyrosine188 resulted in a decrease in activation of presynaptic α7-nAChRs by ACh and Aβ but with no change in response to nicotine, indicating the critical role of Tyr-188 in presynaptic regulation by Aβ. Coimmunoprecipitation additionally revealed direct binding of Aβ to α7-nAChRs and to the Tyr-188 mutant receptor. In contrast, mutations of Tyr-195 in α7-nAChR led to decreased activation by nicotine without apparent effects on ACh- or Aβ-induced responses. Agonist-induced responses of Tyr-93 mutant α7-nAChRs indicated possible interactions of nicotine and Aβ with its hydroxyl group, but there was no change in presynaptic responses after mutation of Trp-149. All of the mutants were shown to be expressed on the plasma membrane using cell surface labeling. Together, these results directly demonstrate an essential role for the aromatic residue Tyr-188 as a key component in the agonist binding domain for the activation of α7-nAChRs by Aβ.     

A physiological study on acetylcholine receptor expressed in Xenopus oocytes from cloned cDNAs

Nicotinic ACh receptor was expressed in Xenopus oocytes by injecting mRNAs produced from cloned cDNAs encoding the four subunits of ACh receptor of Torpedo californica. ACh responses recorded from oocytes 3 days after injection of the mRNAs were reversibly blocked by d-tubocurarine (1-2 microM), indicating that the newly synthesized receptor is of nicotinic type. The reversal potential of ACh response was found at around -1 - -5 mV. The reversal potential was not changed by removal of extracellular C1-, suggesting that the ionic channel of the newly expressed ACh receptor is permeable only to cations. Repetitive applications of ACh caused desensitization of the receptor. The rate of the desensitization was greater when the membrane potential was more negative. Subunit deletion studies showed that all four subunits are required for the formation of ACh receptors with normal ACh sensitivity. However, ACh receptors without delta subunit responded to ACh with low sensitivity. Studies on ACh receptor mutants with -subunits altered by site directed mutagenesis of the cDNA suggest that the anphipathic segment is involved in the channel function of the receptor as well as the four hydrophobic segments since partial deletion of amino acids in these segments essentially abolished ACh sensitivity with relatively little change in 125I-alpha-bungarotoxin binding activity.     

An extracellular RRR motif flanking the M1 transmembrane domain governs the biogenesis of homomeric neuronal nicotinic acetylcholine receptors

We have previously demonstrated that the highly conserved R209, that flanks the M1 transmembrane segment of nicotinic acetylcholine (ACh) receptors, is required for the transport of assembled homomeric neuronal α7 nicotinic ACh receptors to the cell surface. In the present paper we show that basic residues at positions 208 and 210 are necessary for the assembly of α7 receptors. On the contrary, a basic residue at position 210 of α3 subunit decreases the assembly of heteromeric neuronal α3β4 nicotinic ACh receptors. A basic residue at position 210 of the β4 subunit slightly decreases α3β4 receptor expression. We conclude that a pre-M1 RRR motif is necessary for the biogenesis of homomeric α-bungarotoxin-sensitive neuronal α7 nicotinic ACh receptors.     

Expression and functional properties of α7 acetylcholine nicotinic receptors are modified in the presence of other receptor subunits

Although α7 nicotinic receptors are predominantly homopentamers, previous reports have indicated that α7 and β2 subunits are able to form heteromers. We have studied whether other nicotinic receptor subunits can also assemble with α7 subunits and the effect of this potential association. Coexpression of α7 with α2, α3, or β4 subunits reduced to about half, surface α‐bungarotoxin binding sites and acetylcholine‐gated currents. This is probably because of inhibition of membrane trafficking, as the total amount of α7 subunits was similar in all cases and a significant proportion of mature α7 receptors was present inside the cell. Only β4 subunits appeared to directly associate with α7 receptors at the membrane and these heteromeric receptors showed some kinetic and pharmacological differences when compared with homomeric α7 receptors. Finally, we emulated the situation of bovine chromaffin cells in Xenopus laevis oocytes by using the same proportion of α3, β4, α5, and α7 mRNAs, finding that α‐bungarotoxin binding was similarly reduced in spite of increased currents, apparently mediated by α3β4(α5) receptors.     

Analysis of the third transmembrane domain of the human type 1 angiotensin II receptor by cysteine scanning mutagenesis

Activation of G protein-coupled receptors by agonists involves significant movement of transmembrane domains (TMD) following agonist binding. The underlying structural mechanism by which receptor activation takes place is largely unknown but can be inferred by detecting variability within the environment of the ligand-binding pocket, which is a water-accessible crevice surrounded by the seven TMD helices. Using the substituted-cysteine accessibility method, we identified the residues within the third TMD of the wild-type angiotensin II (AT1) receptor that contribute to the formation of the binding site pocket. Each residue within the Ile103-Tyr127 region was mutated one at a time to a cysteine. Treating the A104C, N111C, and L112C mutant receptors with the charged sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA) strongly inhibited ligand binding, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT1 receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD3 reporter cysteines engineered in a constitutively active AT1 receptor. Indeed, two additional mutants (S109C and V116C) were found to be sensitive to MTSEA treatment. Our results suggest that constitutive activation of the AT1 receptor causes a minor counterclockwise rotation of TMD3, thereby exposing residues, which are not present in the inactive state, to the binding pocket. This pattern of accessibility of residues in the TMD3 of the AT1 receptor parallels that of homologous residues in rhodopsin. This study identified key elements of TMD3 that contribute to the activation of class A G protein-coupled receptors through structural rearrangements.     

Binding-gating coupling in a nondesensitizing α7 nicotinic receptor: A single channel pharmacological study

The highly conserved αLys145 has been suggested to play an important role in the early steps of activation of the nicotinic acetylcholine receptor (nAChR) by acetylcholine. Both macroscopic and single channel currents were recorded in the slowly desensitizing mutants L248T- and K145A-L248T-α7 receptors expressed in Xenopus oocytes. On ACh-evoked currents, substitution of Lys145 by alanine showed the same effects that in wild type receptors: moderately decreased gating function and a more-than-expected loss of ACh potency, thus validating the experimental model. Single channel analysis quantitatively agreed with macroscopic data and revealed that impaired gating function in the double mutant α7K145A/L248T is the consequence of a slower opening rate, β. Several nicotinic agonists were also studied, showing important features. Particularly, dimethylphenylpiperazinium (DMPP), acting as an antagonist in α7K145A, became a full agonist in α7K145A/L248T. Single channel analysis of DMPP-evoked currents showed effects of Lys145 removal similar to those observed with ACh. Data suggest that α7Lys145 facilitates the early steps of channel activation. Moreover, the slowly desensitizing mutant α7L248T could be an interesting tool for the study of channel activation in α7 receptors. Nevertheless, its extensively altered pharmacology precludes the simple extrapolation of pharmacological data obtained in singly mutated α7 receptors.     

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     

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.     

Delimiting the Binding Site for Quaternary Ammonium Lidocaine Derivatives in the Acetylcholine Receptor Channel

The triethylammonium QX-314 and the trimethylammonium QX-222 are lidocaine derivatives that act as open-channel blockers of the acetylcholine (ACh) receptor. When bound, these blockers should occlude some of the residues lining the channel. Eight residues in the second membrane-spanning segment (M2) of the mouse-muscle α subunit were mutated one at a time to cysteine and expressed together with wild-type β, γ, and δ subunits in Xenopus oocytes. The rate constant for the reaction of each substituted cysteine with 2-aminoethyl methanethiosulfonate (MTSEA) was determined from the time course of the irreversible effect of MTSEA on the ACh-induced current. The reactions were carried out in the presence and absence of ACh and in the presence and absence of QX-314 and QX-222. These blockers had no effect on the reactions in the absence of ACh. In the presence of ACh, both blockers retarded the reaction of extracellularly applied MTSEA with cysteine substituted for residues from αVal255, one third of the distance in from the extracellular end of M2, to αGlu241, flanking the intracellular end of M2, but not with cysteine substituted for αLeu258 or αGlu262, at the extracellular end of M2. The reactions of MTSEA with cysteines substituted for αLeu258 and αGlu262 were considerably faster in the presence of ACh than in its absence. That QX-314 and QX-222 did not protect αL258C and αE262C against reaction with MTSEA in the presence of ACh implies that protection of the other residues was due to occlusion of the channel and not to the promotion of a less reactive state from a remote site. Given the 12-Å overall length of the blockers and the α-helical conformation of M2 in the open state, the binding site for both blockers extends from αVal255 down to αSer248.     

Reversal of agonist selectivity by mutations of conserved amino acids in the binding site of nicotinic acetylcholine receptors

Homomeric alpha7 and heteromeric alpha4beta2 nicotinic acetylcholine receptors (nAChR) can be distinguished by their pharmacological properties, including agonist specificity. We introduced point mutations of conserved amino acids within the C loop, a region of the receptor critical for agonist binding, and we examined the expression of the mutant receptors in Xenopus oocytes. Mutation of either a conserved C loop tyrosine (188) to phenylalanine or a nearby conserved aspartate (197) to alanine resulted in alpha7 receptors for which the alpha7-selective agonist 3-(4-hydroxy, 2-methoxybenzylidene) anabaseine (4OH-GTS-21) had roughly the same potency as for wild-type receptors, whereas the physiologic agonist acetylcholine (ACh) showed drastically reduced potency for these mutant receptors. Corresponding mutations in alpha4 receptors co-expressed with beta2 resulted in alpha4beta2 receptors for which ACh potency was relatively unchanged, although the efficacy of the alpha7-selective agonist 4OH-GTS-21 was increased greatly relative to that of ACh. We also investigated the significance of a conserved lysine (145 in alpha7), proposed to form a stable salt bridge with Asp-197 in the resting state of the receptor. Mutations of this residue in both alpha7 and alpha4 resulted in receptors that were largely unresponsive to both ACh and 4OH-GTS-21. Our results suggest that initiation of gating depends both on specific interactions between residues in the C loop domain and, depending on receptor subtype, the physiochemical properties of the agonist, so that in the altered environment of the alpha4Y190F-binding site, large hydrophobic benzylidene anabaseines may close the C loop and initiate channel gating more effectively than the polar agonist ACh.     

Effect of secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1) on airway epithelial cells

Acetylcholine (ACh) exerts various anti-inflammatory effects through α7 nicotinic ACh receptors (nAChRs). We have previously shown that secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1), a positive allosteric modulator of α7 nAChR signaling, is down-regulated both in an animal model of asthma and in human epithelial cells treated with an inflammatory cytokine related to asthma. Our aim of this study was to explore the effect of SLURP-1, signal through α7 nAChR, in the pathophysiology of airway inflammation. Cytokine production was examined using human epithelial cells. Ciliary beat frequency of murine trachea was measured using a high speed camera. The IL-6 and TNF-α production by human epithelial cells was augmented by siRNA of SLURP-1 and α7 nicotinic ACh receptor. The cytokine production was also dose-dependently suppressed by human recombinant SLURP-1 (rSLURP-1). The ciliary beat frequency and amplitude of murine epithelial cells were augmented by PNU282987, a selective α7 nAChR agonist. Those findings suggested that SLURP-1 and stimulus through α7 nicotinic ACh receptors actively controlled asthmatic condition by stimulating ciliary beating and also by suppressing airway inflammation.     

Voltage-jump relaxation kinetics for wild-type and chimeric beta subunits of neuronal nicotinic receptors

We have studied the voltage-jump relaxation currents for a series of neuronal nicotinic acetylcholine receptors resulting from the coexpression of wild-type and chimeric beta 4/beta 2 subunits with alpha 3 subunits in Xenopus oocytes. With acetylcholine as the agonist, the wild-type alpha 3 beta 4 receptors displayed five- to eightfold slower voltage-jump relaxations than did the wild-type alpha 3 beta 2 receptors. In both cases, the relaxations could best be described by two exponential components of approximately equal amplitudes over a wide range of [ACh]'s. Relaxation rate constants increased with [ACh] and saturated at 20- to 30-fold lower concentrations for the alpha 3 beta 2 receptor than for the alpha 3 beta 4 receptor, as observed previously for the peak steady state conductance. Furthermore, the chimeric beta 4/beta 2 subunits showed a transition in the concentration dependence of the rate constants in the region between residues 94 and 109, analogous to our previous observation with steady state conductances. However, our experiments with a series of beta- subunit chimeras did not localize residues that govern the absolute value of the kinetic parameters. Hill coefficients for the relaxations also differed from those previously measured for steady state responses. The data reinforce previous conclusions that the region between residues 94 and 109 on the beta subunit plays a role in binding agonist but also show that other regions of the receptor control gating kinetics subsequent to the binding step.     

The seventh transmembrane domains of the delta and kappa opioid receptors have different accessibility patterns and interhelical interactions

We applied the substituted cysteine accessibility method (SCAM) to map the residues of the transmembrane helices (TMs) 7 of delta and kappa opioid receptors (deltaOR and kappaOR) that are on the water-accessible surface of the binding-site crevices. A total of 25 consecutive residues (except C7.38) in the TMs 7 were mutated to Cys, one at a time, and each mutant was expressed in HEK 293 cells. Most mutants displayed similar binding affinity for [(3)H]diprenorphine, an antagonist, as the wild types. Pretreatment with (2-aminoethyl)methanethiosulfonate (MTSEA) inhibited [(3)H]diprenorphine binding to eight deltaOR and eight kappaOR mutants. All mutants except deltaOR L7.52(317)C were protected by naloxone from the MTSEA effect, indicating that the side chains of V7.31(296), A7.34(299), I7.39(304), L7.41(306), G7.42(307), P7.50(315), and Y7.53(318) of deltaOR and S7.34(311), F7.37(314), I7.39(316), A7.40(317), L7.41(318), G7.42(319), Y7.43(320), and N7.49(326) of kappaOR are on the water-accessible surface of the binding pockets. Combining the SCAM data with rhodopsin-based molecular models of the receptors led to the following conclusions. (i) The residues of the extracellular portion of TM7 predicted to face TM1 are sensitive to MTSEA in kappaOR but are not in deltaOR. Thus, TM1 may be closer to TM7 in deltaOR than in kappaOR. (ii) MTSEA-sensitive mutants start at position 7.31(296) in deltaOR and at 7.34(311) in kappaOR, suggesting that TM7 in deltaOR may have an additional helical turn (from 7.30 to 7.33). (iii) There is a conserved hydrogen-bond network linking D2.50 of the NLxxxD motif in TM2 with W6.48 of the CWxP motif in TM6. (iv) The NPxxY motif in TM7 interacts with TM2, TM6, and helix 8 to maintain receptors in inactive states. To the best of our knowledge, this represents the first such comparison of the structures of two highly homologous GPCRs.     

Mutants of β-strand β3 and the loop B in the interface between α7 subunits of a homomeric acetylcholine receptor show functional and pharmacological alterations

Activation of nicotinic acetylcholine receptors (nAChR) requires a global conformational change involving a number of domains of the protein. Structural data from Torpedo nAChR suggest that adjacent subunits might be functionally coupled at the interface between the β-strand β3 and the loop B through a salt bridge between α1Asp152 and γArg78. We have checked this hypothesis in homomeric α7 nAChRs by mutating residues at these (Gly152 and Arg79) and neighboring locations and analyzing the results obtained after expression of single and double mutants in Xenopus oocytes. We found that Arg79 mutants showed a decreased gating function when challenged with different agonists, being the reduction more important for dimethylphenylpiperazinium. EC(50) values in these mutants were also increased up to 30-fold. In contrast, mutating Gly152 only showed significant higher EC(50) values for ACh. However, all Gly153 mutants presented increased gating function and lower EC(50) values with no significant differences among them. When analyzing several mutant cycles it is concluded that Arg79 is functionally coupled to Gly152, but neither to Gly153 nor to Asp157. These data suggest an involvement of the minus side of homomeric α7 nAChRs in their gating function, reinforcing the significance of complementary subunits in the gating of neuronal nAChRs.     

A Novel Agonist Binding Site on Nicotinic Acetylcholine Receptors

Abstract

This report provides evidence that physostigmine (Phy) and benzoquinonium (BZQ) are able to activate nicotinic acetylcholine receptors (nAChRs) through binding site(s) distinct from those of the natural transmitter, ACh. Such findings are in agreement with a second pathway of activation of nAChRs. Receptor activation may be modulated through the novel site, and, consequently, physiological processes involving nicotinic synapses could be controlled. Using patch clamp techniques, single channel currents activated by ACh and anatoxin were recorded from frog interosseal muscle fibers under cell-attached condition and outside-out patches excised from cultured rat hippocampal neurons. Whole cell nicotinic currents were also studied in the cultured neurons. In most of the neurons, nicotinic responses were blocked by the nicotinic antagonists methyllycaconitine (MLA) and α-bungarotoxin (α-BGT). Evaluation of the effects of Phy and BZQ on the muscle and on the α-BGT- and MLA-sensitive neuronal nAChRs demonstrated that both compounds were open channel blockers at these receptors. Furthermore, at low micromolar concentrations, Phy and BZQ activated the nAChRs of all preparations tested, such an effect being unexpectedly resistant to α-BGT or MLA. Thus, the nAChRs could be activated via two distinct binding sites: one for ACh and the other for Phy and BZQ. These findings and previous biochemical results led us to suggest that a putative endogenous ligand could bind to the new site and thereby regulate the activation of nAChRs in nicotinic synapses.     

The activation and inhibition of human nicotinic acetylcholine receptor by RJR-2403 indicate a selectivity for the alpha4beta2 receptor subtype

Human nicotinic acetylcholine (ACh) receptor subtypes expressed in Xenopus oocytes were characterized in terms of their activation by the experimental agonist RJR-2403. Responses to RJR-2403 were compared with those evoked by ACh and nicotine. These agonists were also characterized in terms of whether application of the drugs had the effect of producing a residual inhibition that was manifest as a decrease in subsequent control responses to ACh measured 5 min after the washout of the drug. For the activation of alpha4beta2 receptors, RJR-2403 had an efficacy equivalent to that of ACh and was more potent than ACh. RJR-2403 was less efficacious than ACh for other human receptor subtypes, suggesting that it is a partial agonist for all these receptors. Nicotine activated peak currents in human alpha4beta2 and alpha3beta2 receptors that were 85 and 50% of the respective ACh maximum responses. Nicotine was an efficacious activator of human alpha7 receptors, with a potency similar to ACh, whereas RJR-2403 had very low potency and efficacy for these receptors. At concentrations of <1 mM, RJR-2403 did not produce any residual inhibition of subsequent ACh responses for any receptor subtype. In contrast, nicotine produced profound residual inhibition of human alpha4beta2, alpha3beta2, and alpha7 receptors with IC(50) values of 150, 200, and 150 microM, respectively. Co-expression of the human alpha5 subunit with alpha3 and beta2 subunits had the effect of producing protracted responses to ACh and increasing residual inhibition by ACh and nicotine but not RJR-2403. In conclusion, our results, presented in the context of the complex pharmacology of nicotine for both activating and inhibiting neuronal nicotinic receptor subtypes, suggest that RJR-2403 will be a potent and relatively selective activator of human alpha4beta2 receptors.     

The functional unit of the renal type IIa Na+/Pi cotransporter is a monomer

The composition of the functional unit of the rat renal type IIa Na(+)/P(i) cotransporter (NaPi-IIa) was investigated by using two approaches based on the differential sensitivities of the wild type (WT) and mutant S460C proteins to 2-aminoethylmethanethiosulfonate hydrobromide (MTSEA), a charged cysteine modifier. Transport activity of S460C is completely blocked after incubation in MTSEA, whereas that of the WT remains unaffected. First, Xenopus laevis oocytes were coinjected with cRNAs coding for the WT and S460C in different proportions, and the transport inhibition after MTSEA incubation was assayed by electrophysiology. The relationship between MTSEA inhibition and proportion of cRNA was consistent with that for a functional monomer. Second, concatameric proteins were constructed that either comprised two WT proteins (WT-WT), two S460C mutants (S460C-S460C), or one of each (WT-S460C). Western blots of oocytes injected with fusion protein cRNA showed bands at approximately 200 kDa, whereas a main band at approximately 90 kDa was obtained for the WT cRNA alone. The kinetic properties of concatamers were the same as for the single proteins. Transport activity of the WT-WT concatamer was unaffected by MTSEA incubation, fully inhibited for S460C-S460C, but 50% inhibited for WT-S460C. This behavior was also consistent with NaPi-IIa being a functional monomer.     

Human α3β4 neuronal nicotinic receptors show different stoichiometry if they are expressed in Xenopus oocytes or mammalian HEK293 cells

Background

The neuronal nicotinic receptors that mediate excitatory transmission in autonomic ganglia are thought to be formed mainly by the α3 and β4 subunits. Expressing this composition in oocytes fails to reproduce the properties of ganglionic receptors, which may also incorporate the α5 and/or β2 subunits. We compared the properties of human α3β4 neuronal nicotinic receptors expressed in Human embryonic kidney cells (HEK293) and in Xenopus oocytes, to examine the effect of the expression system and α∶β subunit ratio.

Methodology/Principal Findings

Two distinct channel forms were observed: these are likely to correspond to different stoichiometries of the receptor, with two or three copies of the α subunit, as reported for α4β2 channels. This interpretation is supported by the pattern of change in acetylcholine (ACh) sensitivity observed when a hydrophilic Leu to Thr mutation was inserted in position 9′ of the second transmembrane domain, as the effect of mutating the more abundant subunit is greater. Unlike α4β2 channels, for α3β4 receptors the putative two-α form is the predominant one in oocytes (at 1∶1 α∶β cRNA ratio). This two-α form has a slightly higher ACh sensitivity (about 3-fold in oocytes), and displays potentiation by zinc. The putative three-α form is the predominant one in HEK cells transfected with a 1∶1 α∶β DNA ratio or in oocytes at 9∶1 α∶β RNA ratio, and is more sensitive to dimethylphenylpiperazinium (DMPP) than to ACh. In outside-out single-channel recordings, the putative two-α form opened to distinctive long bursts (100 ms or more) with low conductance (26 pS), whereas the three-α form gave rise to short bursts (14 ms) of high conductance (39 pS).

Conclusions/Significance

Like other neuronal nicotinic receptors, the α3β4 receptor can exist in two different stoichiometries, depending on whether it is expressed in oocytes or in mammalian cell lines and on the ratio of subunits transfected.     

Probing the agonist domain of the nicotinic acetylcholine receptor by cysteine scanning mutagenesis reveals residues in proximity to the alpha-bungarotoxin binding site

We have constructed a series of cysteine-substitution mutants in order to identify residues in the mouse muscle nicotinic acetylcholine receptor (AChR) that are involved in alpha-bungarotoxin (alpha-Bgtx) binding. Following transient expression in HEK 293-derived TSA-201 cells, covalent modification of the introduced cysteines with thiol-specific reagents reveals that alpha subunit residues W187, V188, F189, Y190, and P194 are solvent accessible and are in a position to contribute to the alpha-Bgtx binding site in native receptors. These results with the intact receptor are consistent with NMR studies of an alpha-Bgtx/receptor-dodecapeptide complex [Basus, V., Song., G., and Hawrot, E. (1993) Biochemistry 32, 12290-12298]. We pursued a more detailed analysis of the F189C mutant as this site varies substantially between AChRs that bind Bgtx and certain neuronal AChRs that do not. Treatment of intact cells expressing F189C with either bromoacetylcholine (BrACh) or [2-(trimethylammonium)ethyl] methane-thiosulfonate (MTSET), both methylammonium-containing thiol-modifying reagents with agonist properties, results in a marked decrease ( approximately 55-70%) in the number of alpha-Bgtx binding sites, as measured under saturating conditions. The decrease in sites appears to affect both alpha/gamma and alpha/delta sites to the same extent, as shown for alphaW187C and alphaF189C which were the two mutants examined on this issue. In contrast to the results obtained with MTSET and BrACh, modification with reagents that lack the alkylammonium entity, such as methylmethanethiosulfonate (MMTS), the negatively charged 2-sulfonatoethyl methane-thiosulfonate (MTSES), or the positively charged aminoethyl methylthiosulfonate (MTSEA), has little or no effect on the maximal binding of alpha-Bgtx to the alphaW187C, alphaV188C, or alphaF189C mutant receptors. The striking alkylammonium dependency suggests that an interaction of the tethered modifying group with the negative subsite within the agonist binding domain is primarily responsible for the observed blockade of toxin binding.     

The conserved cysteine 7.38 residue is differentially accessible in the binding-site crevices of the mu, delta, and kappa opioid receptors

Binding pockets of the opioid receptors are presumably formed among the transmembrane domains (TMDs) and are accessible from the extracellular medium. In this study, we determined the sensitivity of binding of [(3)H]diprenorphine, an antagonist, to mu, delta, and kappa opioid receptors to charged methanethiosulfonate (MTS) derivatives and identified the cysteine residues within the TMDs that conferred the sensitivity. Incubation of the mu opioid receptor expressed in HEK293 cells with MTS ethylammonium (MTSEA), MTS ethyltrimethylammonium (MTSET), or MTS ethylsulfonate (MTSES) inhibited [(3)H]diprenorphine binding with the potency order of MTSEA > MTSET > MTSES. Pretreatment of mu, delta, and kappa opioid receptors with MTSEA dose-dependently inhibited [(3)H]diprenorphine binding with MTSEA sensitivity in the order of kappa > mu > delta. The effects of MTSEA occurred rapidly, reaching the maximal inhibition in 10 min. (-)-Naloxone, but not (+)-naloxone, prevented the MTSEA effect, demonstrating that the reaction occurs within or in the vicinity of the binding pockets. Each cysteine residue in the TMDs of the three receptors was mutated singly, and the effects of MTSEA treatment were examined. The mutants had similar affinities for [(3)H]diprenorphine, and C7. 38(321)S, C7.38(303)S, and C7.38(315)S mutations rendered mu, delta, and kappa opioid receptors less sensitive to the effect of MTSEA, respectively. These results indicate that the conserved Cys7.38 is differentially accessible in the binding-site crevice of these receptors. The second extracellular loop of the kappa receptor, which contains several acidic residues, appears to play a role, albeit small, in its higher sensitivity to MTSEA, whereas the negative charge of Glu6.58(297) did not. To the best of our knowledge, this is the first report to show that a conserved residue among highly homologous G protein-coupled receptors is differentially accessible in the binding-site crevice. In addition, this represents the first successful generation of MTSEA-insensitive mutants of mu, delta, and kappa opioid receptors, which will allow determination of residues accessible in the binding-site crevices of these receptors by the substituted cysteine accessibility method.