57 research outputs found
Genetic Elucidation of Human Hyperosmia to Isovaleric Acid
The genetic basis of odorant-specific variations in human olfactory thresholds, and in particular of enhanced odorant sensitivity (hyperosmia), remains largely unknown. Olfactory receptor (OR) segregating pseudogenes, displaying both functional and nonfunctional alleles in humans, are excellent candidates to underlie these differences in olfactory sensitivity. To explore this hypothesis, we examined the association between olfactory detection threshold phenotypes of four odorants and segregating pseudogene genotypes of 43 ORs genome-wide. A strong association signal was observed between the single nucleotide polymorphism variants in OR11H7P and sensitivity to the odorant isovaleric acid. This association was largely due to the low frequency of homozygous pseudogenized genotype in individuals with specific hyperosmia to this odorant, implying a possible functional role of OR11H7P in isovaleric acid detection. This predicted receptor–ligand functional relationship was further verified using the Xenopus oocyte expression system, whereby the intact allele of OR11H7P exhibited a response to isovaleric acid. Notably, we also uncovered another mechanism affecting general olfactory acuity that manifested as a significant inter-odorant threshold concordance, resulting in an overrepresentation of individuals who were hyperosmic to several odorants. An involvement of polymorphisms in other downstream transduction genes is one possible explanation for this observation. Thus, human hyperosmia to isovaleric acid is a complex trait, contributed to by both receptor and other mechanisms in the olfactory signaling pathway
Determinants of competitive antagonist sensitivity on neuronal nicotinic receptor �3 subunits
We constructed a series of chimeric and mutant neuronal nicotinic acetylcholine receptor p subunits to map amino acid residues that determine sensitivity to competitive antagonists. The p2 and p4 subunits form pharmacologically distinct receptors when expressed in combination with the a3 subunit in Xenopus oocytes. At equipotent acetylcholine concentrations, ot3p2 is 56-fold more sensitive to blockade by dihydro-Perythroidine than is a3P4. The c~3/32 combination is also sensitive to long-term blockade by neuronal bungarotoxin, whereas (r3p4 is not. Pharmacological analysis of receptors formed by chimeric p subunits reveals that amino acid residues that determine both dihydro-P-erythroidine and neuronal bungarotoxin sensitivity are located within several sequence segments. The major determinant of sensitivity to both competitive antagonists is located between residues 54 and 63. A mino
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Mapping of ligand binding sites of neuronal nicotinic acetylcholine receptors using chimeric alpha subunits
We constructed a series of chimeric neuronal nicotinic acetylcholine (ACh) receptor (nAChR) alpha subunits to map the location of amino acid residues that determine the pharmacological properties of these receptors. The alpha 2 and alpha 3 subunits form pharmacologically distinct nAChRs upon expression, in combination with the beta 2 subunit, in Xenopus oocytes. The alpha 2 beta 2 subunit combination is insensitive to the nicotinic antagonist neuronal bungarotoxin (NBT) and is much more sensitive to nicotine than to ACh. In contrast, the alpha 3 beta 2 subunit combination is potently inhibited by NBT and is much less sensitive to nicotine than to ACh. Chimeric subunits were constructed by replacing portions of alpha 2 or alpha 3 with the analogous portion of the other alpha subunit. Pharmacological analysis of receptors formed by these chimeric subunits, in combination with beta 2, revealed that amino acid residues involved in determining NBT sensitivity were located within sequence segments 84-121, 121-181, and 195-215. Amino acid residues that determine agonist sensitivity were located within sequence segments 1-84 and 195-215. Within region 195-215, we used site-directed mutagenesis to demonstrate the importance of Gln-198 of alpha 3 (proline in alpha 2) in determining both the antagonist sensitivity and the agonist sensitivity of neuronal nAChRs
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Determinants of agonist binding affinity on neuronal nicotinic receptor beta subunits
The alpha and beta subunits of heteromeric neuronal nicotinic acetylcholine receptors (nAChRs) are thought to contribute "principal" and "complementary" components to the agonist binding site, respectively. At least six loops of amino acid sequence (A, B, and C from alpha; D, E, and F from beta) are involved. We demonstrated previously that receptors containing the beta2 subunit had consistently higher affinities for a variety of agonists than beta4-containing receptors. For example, the affinity of the alpha2beta2 receptor for epibatidine, ACh, nicotine, and dimethylphenylpiperazinium (DMPP) exceeds that of alpha2beta4 by 9-, 61-, 87-, and 120-fold, respectively. Using saturation and competition analysis of receptors formed by chimeric beta subunits coexpressed with alpha2 in Xenopus laevis oocytes, we have now identified sequence segment 54-63 (corresponding to loop D) as a major determinant of affinity for epibatidine, ACh, nicotine, and DMPP. We then analyzed a series of mutant beta2 subunits in which each residue that differs between beta2 and beta4 in this region was changed from what occurs in beta2 to what occurs in beta4. The N55S, V56I, and E63T mutations each resulted in a loss of affinity for ACh and nicotine of 3- to 4-fold, whereas the T59K mutation resulted in a 7-fold loss of ACh and nicotine affinity. These mutations had little or no effect on epibatidine and DMPP affinity. The positive charge introduced by the T59K mutation does not appear to underlie loss of agonist affinity, because a similar loss of affinity was observed when a negative charge (T59D) was introduced at this position
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Glycosylation within the cysteine loop and six residues near conserved Cys192/Cys193 are determinants of neuronal bungarotoxin sensitivity on the neuronal nicotinic receptor alpha 3 subunit
Neuronal bungarotoxin (NBT) is a highly selective, slowly reversible, competitive antagonist of the alpha 3 beta 2 neuronal nicotinic receptor. Contributions to NBT sensitivity are made by both the alpha 3 and beta 2 subunits. We used a chimeric alpha subunit to demonstrate that the entire alpha 3 contribution lies within sequence segment 84-215. Construction and analysis of a series of mutant alpha 3 subunits identified seven amino acid residues (Thr143, Tyr184, Lys185, His186, Ile188, Gln198, Ser203) within this region that contribute to NBT sensitivity. Changing Thr143 to lysine, as in alpha 2, resulted in a similar to 1000-fold loss of NBT sensitivity. The effect on NBT sensitivity of changing each of the other six residues ranged from 1.8- to 40.5-fold. More extensive mutagenesis demonstrated that Thr143 serves as part of the consensus sequence for glycosylation at N141, and it is this glycosylation that is the determinant of NBT sensitivity. Only serine could substitute for threonine to maintain full NBT sensitivity, and changing Asn141 to alanine resulted in a similar to 300-fold loss of NBT sensitivity. The chimera alpha 2-181- alpha 3, containing all identified determinants except the glycosylation site, formed receptors insensitive to 300 nM NBT. Installation of threonine to complete the glycosylation consensus site in this chimera conferred NBT sensitivity only 10-fold less than that of wild-type alpha 3 beta 2. These seven determinants of NBT sensitivity are located in close proximity to a series of conserved residues that are common features of all nicotinic receptor binding sites
Varying Chirality Across Nicotinic Acetylcholine Receptor Subtypes: Selective Binding of Quinuclidine Triazole Compounds
Multiple Determinants of Dihydro‐β‐Erythroidine Sensitivity on Rat Neuronal Nicotinic Receptor α Subunits
: Neuronal nicotinic acetylcholine receptors are differentially sensitive to blockade by the competitive antagonist dihydro‐β‐erythroidine. Both α and β subunits participate in determining sensitivity to this antagonist. The α subunit contribution to dihydro‐β‐erythroidine sensitivity is illustrated by comparing the α4β4 receptor and the α3β4 receptor, which differ in sensitivity to dihydro‐β‐erythroidine by ∼120‐fold. IC50 values for blocking α4β4 and α3β4, responding to EC20 concentrations of acetylcholine, were 0.19 ± 0.06 and 23.1 ± 10.2 µM, respectively. To map the sequence segments responsible for this difference, we constructed a series of chimeric α subunits containing portions of the α4 and α3 subunits. These chimeras were coexpressed with β4, allowing pharmacological characterization. We found determinants of dihydro‐β‐erythroidine sensitivity to be distributed throughout the N‐terminal extracellular domain of the α subunit. These determinants were localized to sequence segments 1–94, 94–152, and 195–215. Loss of determinants within segment 1–94 had the largest effect, decreasing dihydro‐β‐erythroidine sensitivity by 4.3‐fold
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Differential inhibition by α-conotoxin-MII of the nicotinic stimulation of [3H]dopamine release from rat striatal synaptosomes and slices
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The postsynaptic 43k protein clusters muscle nicotinic acetylcholine receptors in xenopus oocytes
Nicotinic acetylcholine receptors (AChRs) are localized at high concentrations in the postsynaptic membrane of the neuromuscular junction. A peripheral membrane protein of M
r 43,000 (43K protein) is closely associated with AChRs and has been proposed to anchor receptors at postsynaptic sites. We have used the Xenopus oocyte expression system to test the idea that the 43K protein clusters AChRs. Mouse muscle AChRs expressed in oocytes after injection of RNA encoding receptor subunits are uniformly distributed in the surface membrane. Co-injection of AChR RNA and RNA encoding the mouse muscle 43K protein causes AChRs to form clusters of 0.5–1.5 μm diameter. AChR clustering is not a consequence of increased receptor expression in the surface membrane or nonspecific clustering of all membrane proteins. The 43K protein is colocalized with AChRs in clusters when the two proteins are expressed together and forms clusters of similar size even in the absence of AChRs. These results provide direct evidence that the 43K protein causes clustering of AChRs and suggest that regulation of 43K protein clustering may be a key step in neuromuscular synaptogenesis
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