Critical elements determining diversity in agonist binding and desensitization of neuronal nicotinic acetylcholine receptors

To identify the molecular determinants underlying the pharmacological diversity of neuronal nicotinic acetylcholine receptors, we compared the alpha7 homo-oligomeric and alpha4beta2 hetero-oligomeric receptors. Sets of residues from the regions initially identified within the agonist binding site of the alpha4 subunit were introduced into the alpha7 agonist binding site, carried by the homo-oligomeric alpha7-V201-5HT3 chimera. Introduction of the alpha4 residues 183-191 into alpha7 subunit sequence (chimera C2) selectively increased the apparent affinities for equilibrium binding and for ion channel activation by acetylcholine, resulting in a receptor that no longer displays differences in the responses to acetylcholine and nicotine. Introduction of the alpha4 residues 151-155 (chimera B) produced a approximately 100-fold increase in the apparent affinity for both acetylcholine and nicotine in equilibrium binding measurements. In both cases electrophysiological recordings revealed a much smaller increase (three- to sevenfold) in the apparent affinity for activation, but the concentrations required to desensitize the mutant chimeras parallel the shifts in apparent binding affinity. The data were fitted by a two-state concerted model, and an alteration of the conformational isomerization constant leading to the desensitized state accounts for the chimera B phenotype, whereas alteration of the ligand binding site accounts for the chimera C2 phenotype. Point mutation analysis revealed that several residues in both fragments contribute to the phenotypes, with a critical effect of the G152K and T183N mutations. Transfer of alpha4 amino acids 151-155 and 183-191 into the alpha7-V201-5HT3 chimera thus confers physiological and pharmacological properties typical of the alpha4beta2 receptor.     

A capsaicin-receptor antagonist, capsazepine, reduces inflammation-induced hyperalgesic responses in the rat: evidence for an endogenous capsaicin-like substance

Substance P is known to noncompetitively inhibit activation of muscle and neuronal nicotinic acetylcholine receptors. Neuronal nicotinic receptors formed from different combinations of alpha and beta subunits exhibited differential sensitivity to substance P, with those containing beta-4 subunits having a 25-fold higher affinity than those having beta-2 subunits. To identify the regions and/or amino acid residues of the beta subunit responsible for this difference, chimeric beta subunits were coexpressed with alpha-3 in Xenopus oocytes and the IC50 values for substance P were determined. Amino acid residues between 105 and 109 (beta4 numbering), in the middle of the N-terminal domain, and between 214 and 301, between the extracellular side of M1 and the intracellular side of M3, were identified as major contributors to the apparent affinity of substance P. The affinity of acetylcholine was only affected by residue changes between 105 and 109. Site-directed mutagenesis revealed two amino acids that are important determinants of the affinity of substance P, beta4(V108)/beta2(F106), which is in the middle of the first extracellular domain, and beta4(F255)/beta2(V253), which is within the putative channel lining transmembrane domain M2. However, other residues within these domains must be making subtle but significant contributions, since simultaneous mutation of both these amino acids did not cause complete interconversion of the beta subunit-dependent differences in the receptor affinity for substance P.     

Residues at the subunit interfaces of the nicotinic acetylcholine receptor that contribute to alpha-conotoxin M1 binding

The two binding sites in the pentameric nicotinic acetylcholine receptor of subunit composition alpha2 beta gamma delta are formed by nonequivalent alpha-gamma and alpha-delta subunit interfaces, which produce site selectivity in the binding of agonists and antagonists. We show by sedimentation analysis that 125I-alpha-conotoxin M1 binds with high affinity to the alpha-delta subunit dimers, but not to alpha-gamma dimers, nor to alpha, gamma, and delta monomers, a finding consistent with alpha-conotoxin M1 selectivity for the alpha delta interface in the intact receptor measured by competition against alpha-bungarotoxin binding. We also extend previous identification of alpha-conotoxin M1 determinants in the gamma and delta subunits to the alpha subunit interface by mutagenesis of conserved residues in the alpha subunit. Most mutations of the alpha subunit affect affinity similarly at the two sites, but Tyr93Phe, Val188Lys, Tyr190Thr, Tyr198Thr, and Asp152Asn affect affinity in a site-selective manner. Mutant cycle analysis reveals only weak or no interactions between mutant alpha and non-alpha subunits, indicating that side chains of the alpha subunit do not interact with those of the gamma or delta subunits in stabilizing alpha-conotoxin M1. The overall findings suggest different binding configurations of alpha-conotoxin M1 at the alpha-delta and alpha-gamma binding interfaces.     

Expression and transcriptional control of the Salmonella typhimurium Ipf fimbrial operon by phase variation

Functional effects of human alpha 5 nicotinic ACh receptor (AChR) subunits coassembled with alpha 3 and beta 2 or with alpha 3 and beta 4 subunits, were investigated in Xenopus oocytes. The presence of alpha 5 subunits altered some properties of both alpha 3 AChRs and differentially altered other properties of alpha 3 beta 2 AChRs vs. alpha 3 beta 4 AChRs. alpha 5 subunits increased desensitization and Ca++ permeability of all alpha 3 AChRs. The Ca++ permeabilities of both alpha 3 beta 2 alpha 5 and alpha 3 beta 4 alpha 5 AChRs were comparable to that of alpha 7 AChRs. As we have shown previously, alpha 5 subunits increased the ACh sensitivity of alpha 3 beta 2 AChRs 50-fold but had little effect on alpha 3 beta 4 AChRs. alpha 5 caused only subtle changes in the activation potencies of alpha 3 AChRs for nicotine, cytisine and 1,1-dimethyl-4-plenylpiperazinium (DMPP). However, alpha 5 increased the efficacies of nicotine and DMPP on alpha 3 beta 2 AChRs but decreased them on alpha 3 beta 4 AChRs. Immunoisolation of cloned human AChRs expressed in oocytes showed that alpha 5 efficiently coassembled with alpha 3 plus beta 2 and/or beta 4 subunits. As expected, human AChRs immunoisolated from SH-SY5Y neuroblastoma cells showed that AChRs containing alpha 3 and probably alpha 5 subunits were present, but alpha 4 AChRs were not. In brain, by contrast, alpha 4 beta 2 AChRs were shown to predominate over alpha 3 AChRs. Some of the brain alpha 4 beta 2 AChRs were found to contain alpha 5 subunits.     

Four pharmacologically distinct subtypes of alpha4beta2 nicotinic acetylcholine receptor expressed in Xenopus laevis oocytes

Nicotinic receptors generally are presumed to consist of two alpha and three non-alpha subunits. We varied the relative levels of expression of the neuronal nicotinic alpha4 and beta2 receptor subunits in Xenopus laevis oocytes by nuclear injection of cDNAs coding for these subunits in alpha:beta ratios of 9:1, 1:1, and 1:9. The sensitivities of the receptors to acetylcholine and d-tubocurarine were investigated in voltage-clamp experiments. For receptors expressed at the 9:1 and 1:1 alpha:beta ratios, the EC50 value of acetylcholine is approximately 60 microM. For the majority of the receptors expressed at the 1:9 alpha:beta ratio, the sensitivity to acetylcholine is enhanced 30-fold. No evidence for more than one type of acetylcholine binding site in a single receptor is obtained. The sensitivity to d-tubocurarine decreases with decreasing alpha:beta ratio. IC50 values of d-tubocurarine are 0.2, 0.5, and 2 microM for the 9:1, 1:1, and 1:9 alpha:beta ratios, respectively. At the 1:9 alpha:beta ratio, additional receptors with an IC50 value of 163 microM d-tubocurarine are expressed. At least two components with distinct sensitivities to d-tubocurarine are required to account for the shift in IC50. The combined agonist and antagonist effects reveal four distinct subtypes of alpha4beta2 nicotinic receptors. The results imply that the subunit stoichiometry of heteromeric alpha4beta2 acetylcholine receptors is not restricted to 2alpha:3beta.     

Role of the putative transmembrane segment M3 in gating of neuronal nicotinic receptors

The involvement of some structural domains in the gating of the neuronal nicotinic acetylcholine receptor (AChR) was studied by expressing functional alpha7/alpha3 chimeric subunits in Xenopus oocytes. Substitution of the M3 transmembrane segment in the alpha7 subunit modifies the kinetic properties of the chimeric AChRs as follows: (a) a 6-fold reduction in the maximal current evoked by nicotinic agonists, (b) a 10-fold decrease in the macroscopic desensitization rate, (c) an increase of almost 1 order of magnitude in the apparent affinity for acetylcholine and nicotine, and (d) a decrease in the affinity for alpha-bungarotoxin. Computer simulations showed that the first three effects could be accounted for by a simple kinetic model in which chimeric AChRs presented a smaller ratio of the gating rates, beta/alpha, and a slightly slower desensitization rate. It is concluded that the M3 domain influences the gating of neuronal AChRs.     

A reporter mutation approach shows incorporation of the "orphan" subunit beta3 into a functional nicotinic receptor

We have investigated whether the neuronal nicotinic subunit beta3 can participate in the assembly of functional recombinant receptors. Although beta3 is expressed in several areas of the central nervous system, it does not form functional receptors when expressed heterologously together with an alpha or another beta nicotinic subunit. We inserted into the human beta3 subunit a reporter mutation (V273T), which, if incorporated into a functional receptor, would be expected to increase its agonist sensitivity and maximum response to partial agonists. Expressing the mutant beta3(V273T) in Xenopus oocytes together with both the alpha3 and the beta4 subunits resulted in the predicted changes in the properties of the resulting nicotinic receptor when compared with those of alpha3 beta4 receptors. This indicated that some of the receptors incorporated the mutant beta3 subunit, as part of a "triplet" alpha3 beta4 beta3 receptor. The proportion of triplet receptors was dependent on the ratios of the alpha3:beta4:beta3 cRNA injected. We conclude that, like the related alpha5 subunit, the beta3 subunit can form functional receptors only if expressed together with both alpha and beta subunits.     

Nine L-type amino acid residues confer full 1,4-dihydropyridine sensitivity to the neuronal calcium channel alpha1A subunit. Role of L-type Met1188

Pharmacological modulation by 1,4-dihydropyridines is a central feature of L-type calcium channels. Recently, eight L-type amino acid residues in transmembrane segments IIIS5, IIIS6, and IVS6 of the calcium channel alpha1 subunit were identified to substantially contribute to 1,4-dihydropyridine sensitivity. To determine whether these eight L-type residues (Thr1066, Gln1070, Ile1180, Ile1183, Tyr1490, Met1491, Ile1497, and Ile1498; alpha1C-a numbering) are sufficient to form a high affinity 1,4-dihydropyridine binding site in a non-L-type calcium channel, we transferred them to the 1, 4-dihydropyridine-insensitive alpha1A subunit using site-directed mutagenesis. 1,4-Dihydropyridine agonist and antagonist modulation of barium inward currents mediated by the mutant alpha1A subunits, coexpressed with alpha2delta and beta1a subunits in Xenopus laevis oocytes, was investigated with the two-microelectrode voltage clamp technique. The resulting mutant alpha1A-DHPi displayed low sensitivity for 1,4-dihydropyridines. Analysis of the 1,4-dihydropyridine binding region of an ancestral L-type alpha1 subunit previously cloned from Musca domestica body wall muscle led to the identification of Met1188 (alpha1C-a numbering) as an additional critical constituent of the L-type 1,4-dihydropyridine binding domain. The introduction of this residue into alpha1A-DHPi restored full sensitivity for 1,4-dihydropyridines. It also transferred functional properties considered hallmarks of 1, 4-dihydropyridine agonist and antagonist effects (i.e. stereoselectivity, voltage dependence of drug modulation, and agonist-induced shift in the voltage-dependence of activation). Our gain-of-function mutants provide an excellent model for future studies of the structure-activity relationship of 1, 4-dihydropyridines to obtain critical structural information for the development of drugs for neuronal, non-L-type calcium channels.     

Potentiation and inhibition of neuronal nicotinic receptors by atropine: competitive and noncompetitive effects

Atropine, the classic muscarinic receptor antagonist, inhibits ion currents mediated by neuronal nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes. At the holding potential of -80 mV, 1 microM atropine inhibits 1 mM acetylcholine-induced inward currents mediated by rat alpha2beta2, alpha2beta4, alpha3beta2, alpha3beta4, alpha4beta2, alpha4beta4, and alpha7 nicotinic receptors by 12-56%. Inward currents induced with a low agonist concentration are equally inhibited (alpha3beta2, alpha3beta4), less inhibited (alpha2beta4, alpha7), or potentiated (alpha4beta2, alpha4beta4) by 1 microM atropine. Effects on the more sensitive alpha4beta4 nicotinic receptors were investigated in detail by systematic variation of acetylcholine and atropine concentrations and of membrane potential. At high agonist concentration, atropine inhibits alpha4beta4 nicotinic receptor-mediated ion current in a noncompetitive, voltage-dependent way with IC50 values of 655 nM at -80 mV and of 4.5 microM at -40 mV. At low agonist concentration, 1 microM atropine potentiates alpha4beta4 nicotinic receptor-mediated ion current. This potentiating effect is surmounted by high concentrations of acetylcholine, indicating a competitive interaction of atropine with the nicotinic receptor, and potentiation is also reversed at high atropine concentrations. Steady state effects of acetylcholine and atropine are accounted for by a model for combined receptor occupation and channel block, in which atropine acts on two distinct sites. The first site is associated with noncompetitive ion channel block. The second site is associated with competitive potentiation, which appears to occur when the agonist recognition sites of the receptor are occupied by acetylcholine and atropine. The apparent affinity of atropine for the agonist recognition sites of the alpha4beta4 nicotinic acetylcholine receptor is estimated to be 29.9 microM.     

Differential roles for disulfide bonds in the structural integrity and biological activity of kappa-Bungarotoxin, a neuronal nicotinic acetylcholine receptor antagonist

kappa-Bungarotoxin, a kappa-neurotoxin derived from the venom of the banded Krait, Bungarus multicinctus, is a homodimeric protein composed of subunits of 66 amino acid residues containing five disulfide bonds. kappa-Bungarotoxin is a potent, selective, and slowly reversible antagonist of alpha3 beta2 neuronal nicotinic acetylcholine receptors. kappa-Bungarotoxin is structurally related to the alpha-neurotoxins, such as alpha-bungarotoxin derived from the same snake, which are monomeric in solution and which effectively antagonize muscle type receptors (alpha1 beta1 gamma delta) and the homopentameric neuronal type receptors (alpha7, alpha8, and alpha9). Like the kappa-neurotoxins, the long alpha-neurotoxins contain the same five conserved disulfide bonds, while the short alpha-neurotoxins only contain four of the five. Systematic removal of single disulfide bonds in kappa-bungarotoxin by site-specific mutagenesis reveals a differential role for each of the disulfide bonds. Removal of either of the two disulfides connecting elements of the carboxy terminal loop of this toxin (Cys 46-Cys 58 and Cys 59-Cys 64) interferes with the ability of the toxin to fold. In contrast, removal of each of the other three disulfides does not interfere with the general folding of the toxin and yields molecules with biological activity. In fact, when either C3-C21 or C14-C42 are removed individually, no loss in biological activity is seen. However, removing both produces a polypeptide chain which fails to fold properly. Removal of the C27-C31 disulfide only reduces the activity of the toxin 46.6-fold. This disulfide may play a role in specific interaction of the toxin with specific neuronal receptors.     

A two-amino acid insertion in the Cys146- Cys167 loop of the alphaIIb subunit is associated with a variant of Glanzmann thrombasthenia. Critical role of Asp163 in ligand binding

The ligand binding site(s) of the alpha subunit of integrin alphaIIb beta3 (GPIIb-IIIa), a prototypic non-I domain integrin, remains elusive. In this study, we have characterized a Japanese variant of Glanzmann thrombasthenia, KO, whose platelets express normal amounts of alphaIIb beta3. KO platelets failed to bind the activation-independent ligand-mimetic mAb OP-G2 and did not bind fibrinogen or the activation-dependent ligand-mimetic mAb PAC-1 following activation of alphaIIb beta3 under any condition examined. Sequence analysis of PCR fragments derived from KO platelet mRNA revealed a 6-bp insertion leading to a 2-amino-acid insertion (Arg-Thr) between residues 160 and 161 of the alphaIIb subunit. Introduction of the insertion into wild-type recombinant alphaIIb beta3 expressed in 293 cells led to the normal expression of alphaIIb beta3 having the defect in ligand binding function. The insertion is located within the small loop (Cys146-Cys167) in the third NH2-terminal repeat of the alphaIIb subunit. Alanine substitution of each of the oxygenated residues within the loop (Thr150, Ser152, Glu157, Asp159, Ser161, and Asp163) did not significantly affect expression of alphaIIbbeta3, and only Asp163AlaalphaIIb beta3 abolished the ligand binding function. In addition, Asp163AlaalphaIIb beta3 as well as KO mutant alphaIIb beta3 constitutively expressed the PMI-1 epitope. Our present data suggest that Asp163 of the alphaIIb subunit is one of the critical residues for ligand binding.     

Molecular diversity of the calcium channel alpha2delta subunit

Sequence database searches with the alpha2delta subunit as probe led to the identification of two new genes encoding proteins with the essential properties of this calcium channel subunit. Primary structure comparisons revealed that the novel alpha2delta-2 and alpha2delta-3 subunits share 55.6 and 30.3% identity with the alpha2delta-1 subunit, respectively. The number of putative glycosylation sites and cysteine residues, hydropathicity profiles, and electrophysiological character of the alpha2delta-3 subunit indicates that these proteins are functional calcium channel subunits. Coexpression of alpha2delta-3 with alpha1C and cardiac beta2a or alpha1E and beta3 subunits shifted the voltage dependence of channel activation and inactivation in a hyperpolarizing direction and accelerated the kinetics of current inactivation. The kinetics of current activation were altered only when alpha2delta-1 or alpha2delta-3 was expressed with alpha1C. The effects of alpha2delta-3 on alpha1C but not alpha1E are indistinguishable from the effects of alpha2delta-1. Using Northern blot analysis, it was shown that alpha2delta-3 is expressed exclusively in brain, whereas alpha2delta-2 is found in several tissues. In situ hybridization of mouse brain sections showed mRNA expression of alpha2delta-1 and alpha2delta-3 in the hippocampus, cerebellum, and cortex, with alpha2delta-1 strongly detected in the olfactory bulb and alpha2delta-3 in the caudate putamen.     

Characterization of human recombinant neuronal nicotinic acetylcholine receptor subunit combinations alpha2beta4, alpha3beta4 and alpha4beta4 stably expressed in HEK293 cells

Human embryonic kidney (HEK293) cells were transfected with cDNA encoding the human beta4 neuronal nicotinic acetylcholine (ACh) receptor subunit in pairwise combination with human alpha2, alpha3 or alpha4 subunits. Cell lines A2B4, A3B4.2 and A4B4 were identified that stably express mRNA and protein corresponding to alpha2 and beta4, to alpha3 and beta4 and to alpha4 and beta4 subunits, respectively. Specific binding of [3H]epibatidine was detected in A2B4, A3B4.2 and A4B4 cells with Kd (mean +/- S.D. in pM) values of 42 +/- 10, 230 +/- 12 and 187 +/- 29 and with Bmax (fmol/mg protein) values of 1104 +/- 338, 2010 +/- 184 and 3683 +/- 1450, respectively. Whole-cell patch-clamp recordings in each cell line demonstrated that (-)nicotine (Nic), ACh, cytisine (Cyt) and 1, 1-dimethyl-4-phenylpiperazinium iodide (DMPP) elicit transient inward currents. The current-voltage (I-V) relation of these currents showed strong inward rectification. Pharmacological characterization of agonist-induced elevations of intracellular free Ca++ concentration revealed a distinct rank order of agonist potency for each subunit combination as follows: alpha2beta4, (+)epibatidine (Epi) > Cyt > suberyldicholine (Sub) = Nic = DMPP; alpha3beta4, Epi > DMPP = Cyt = Nic = Sub; alpha4beta4, Epi > Cyt = Sub > Nic > DMPP. The noncompetitive antagonists mecamylamine and d-tubocurarine did not display subtype selectivity. In contrast, the Kb value for the competitive antagonist dihydro-beta-erythroidine (DHbetaE) was highest at alpha3beta4 compared with alpha2beta4 or alpha4beta4 receptors. These data illustrate that the A2B4, A3B4.2 and A4B4 stable cell lines are powerful tools for examining the functional and pharmacological properties of human alpha2beta4, alpha3beta4 and alpha4beta4 neuronal nicotinic receptors.     

alpha-Conotoxin ImI exhibits subtype-specific nicotinic acetylcholine receptor blockade: preferential inhibition of homomeric alpha 7 and alpha 9 receptors

Through a study of cloned nicotinic receptors expressed in Xenopus oocytes, we provide evidence that alpha-conotoxin ImI, a peptide marine snail toxin that induces seizures in rodents, selectively blocks subtypes of nicotinic acetylcholine receptors. alpha-Conotoxin ImI blocks homomeric alpha 7 nicotinic receptors with the highest apparent affinity and homomeric alpha 9 receptors with 8-fold lower affinity. This toxin has no effect on receptors composed of alpha 2 beta 2, alpha 3 beta 2, alpha 4 beta 2, alpha 2 beta 4, alpha 3 beta 4, or alpha 4 beta 4 subunit combinations. In contrast to alpha-bungarotoxin, which has high affinity for alpha 7, alpha 9, and alpha 1 beta 1 gamma delta receptors, alpha-conotoxin ImI has low affinity for the muscle nAChR. Related Conus peptides, alpha-conotoxins MI and GI, exhibit a distinct specificity, strictly targeting the muscle subtype receptor but not alpha 7 or alpha 9 receptors. alpha-Conotoxins thus represent selective tools for the study of neuronal nicotinic acetylcholine receptors.     

A randomized controlled study comparing elemental diet and steroid treatment in Crohn''s disease

To elucidate the mechanism underlying the interaction between the L-type Ca2+ channel and the dihydropyridines (DHPs), contribution of the repeat III was studied by constructing chimeras between the DHP-sensitive alpha1C and DHP-insensitive alpha1E subunits. The chimeras were transiently expressed in human embryonic kidney 293 cells and the whole-cell Ba2+ current (IBa) was recorded. Mutating Thr1061 to Tyr in IIIS5 of the alpha1C sequence completely abolished the inhibition and stimulation of IBa by the antagonist (+)-isradipine and agonist (-)-Bay K 8644, whereas mutating Gln1065 to Met in IIIS5 decreased the affinity for isradipine 100-fold without affecting the stimulating effect of Bay K 8644. The conserved amino acid residue Tyr1174 in IIIS6 of the alpha1C subunit was necessary for the high affinity DHP block. The DHP-dependent block and stimulation of IBa were transferred to the alpha1E channel by the mutation of two amino acid residues in IIIS5 (Y1295T, M1299Q), three residues in IIIS6 (F1406I, F1409I, V1414M) and three residues in IVS6 (I1706Y, F1707M, L1714I). The mutated alpha1E channel was stimulated 2.8-fold by 1 microM Bay K 8644 and blocked by isradipine with an IC50 value of 60 nM. These results show that mutation of Thr1061 in the alpha1C sequence results in a DHP-insensitive L-type channel and that transfer of the high affinity DHP sensitivity requires mutation of eight amino acid residues in the alpha1E sequence.