111 research outputs found
Potentiation of neuronal nicotinic receptors by 17β-estradiol: Roles of the carboxy-terminal and the amino-terminal extracellular domains
The endogenous steroid 17β-estradiol (βEST) potentiates activation of neuronal nicotinic receptors containing α4 subunits. Previous work has shown that the final 4 residues of the α4 subunit are required for potentiation. However, receptors containing the α2 subunit are not potentiated although it has these 4 residues, and only one amino acid difference in the C-terminal tail (FLAGMI vs. WLAGMI). Previous work had indicated that the tryptophan residue was involved in binding an analog of βEST, but not in potentiation by βEST. To determine the structural basis for the loss of potentiation we analyzed data from chimeric subunits, which indicated that the major factor underlying the difference between α2 and α4 is the tryptophan/phenylalanine difference, while the N-terminal extracellular domain is a less significant factor. When the tryptophan in α4 was mutated, both phenylalanine and tyrosine conferred lower potentiation while lysine and leucine did not. The reduction reflected a reduced maximal magnitude of potentiation, indicating that the tryptophan is involved in transduction of steroid effects. The regions of the α4 N-terminal extracellular domain involved in potentiation lie near the agonist-binding pocket, rather than close to the membrane or the C-terminal tail, and appear to be involved in transduction rather than binding. These observations indicate that the C-terminal region is involved in both steroid binding (AGMI residues) and transduction (W). The role of the N-terminus appears to be independent of the C-terminal tryptophan and likely reflects an influence on conformational changes caused during channel activation by agonist and potentiation by estradiol
Introduced amino terminal epitopes can reduce surface expression of neuronal nicotinic receptors
Steady-state activation and modulation of the synaptic-type α1β2γ2L GABAA receptor by combinations of physiological and clinical ligands
The synaptic α1β2γ2 GAB
The Nicotinic a5 Subunit Can Replace Either an Acetylcholine-Binding or Nonbinding Subunit in the a4b2* Neuronal Nicotinic Receptor
ABSTRACT Heteropentameric neuronal nicotinic receptors assemble so that the canonical acetylcholine-binding sites are located at the interfaces between two pairs of subunits, while the fifth subunit does not participate in a canonical transmitter-binding site. Several subunits are considered to be unable to participate in forming a functional receptor when they occupy a position that would contribute to such a site, including the a5 subunit. The a5 subunit is of interest because of its apparent involvement in nicotine dependence and in the control of dopamine release. We have examined this question using a4 and b2 subunits in concatemeric constructs with the a5 subunit, expressed in Xenopus oocytes. Using dimeric constructs of a4 and b2 subunits expressed with free a5 and pentameric constructs incorporating a single copy of a5, we find that the a5 subunit can occupy the position of a nonbinding subunit, or replace a b2 subunit participating in a canonical binding site. The resulting receptors functionally resemble pentamers assembled with two copies of a4 and three copies of b2. Functional receptors apparently cannot be formed with a5 subunits in both canonical binding sites. These observations extend the present ideas on the possible positions in the pentamer that may be occupied by the a5 subunit, and suggest that additional physiologic or pharmacological subtypes of neuronal nicotinic receptors may be present in neurons
Energetic contributions to channel gating of residues in the muscle nicotinic receptor β1 subunit
In the pentameric ligand-gated ion channel family, transmitter binds in the extracellular domain and conformational changes result in channel opening in the transmembrane domain. In the muscle nicotinic receptor and other heteromeric members of the family one subunit does not contribute to the canonical agonist binding site for transmitter. A fundamental question is whether conformational changes occur in this subunit. We used records of single channel activity and rate-equilibrium free energy relationships to examine the β1 (non-ACh-binding) subunit of the muscle nicotinic receptor. Mutations to residues in the extracellular domain have minimal effects on the gating equilibrium constant. Positions in the channel lining (M2 transmembrane) domain contribute strongly and relatively late during gating. Positions thought to be important in other subunits in coupling the transmitter-binding to the channel domains have minimal effects on gating. We conclude that the conformational changes involved in channel gating propagate from the binding-site to the channel in the ACh-binding subunits and subsequently spread to the non-binding subunit
Chemogenetic isolation reveals synaptic contribution of δ GABAA receptors in mouse dentate granule neurons
Functional characterization improves associations between rare non-synonymous variants in CHRNB4 and smoking behavior
Smoking is the leading cause of preventable death worldwide. Accordingly, effort has been devoted to determining the genetic variants that contribute to smoking risk. Genome-wide association studies have identified several variants in nicotinic acetylcholine receptor genes that contribute to nicotine dependence risk. We previously undertook pooled sequencing of the coding regions and flanking sequence of the CHRNA5, CHRNA3, CHRNB4, CHRNA6 and CHRNB3 genes and found that rare missense variants at conserved residues in CHRNB4 are associated with reduced risk of nicotine dependence among African Americans. We identified 10 low frequency (<5%) non-synonymous variants in CHRNB4 and investigated functional effects by co-expression with normal α3 or α4 subunits in human embryonic kidney cells. Voltage-clamp was used to obtain acetylcholine and nicotine concentration-response curves and qRT-PCR, western blots and cell-surface ELISAs were performed to assess expression levels. These results were used to functionally weight genetic variants in a gene-based association test. We find that there is a highly significant correlation between carrier status weighted by either acetylcholine EC50 (β = -0.67, r2 = 0.017, P = 2 × 10(-4)) or by response to low nicotine (β = -0.29, r2 = 0.02, P = 6 × 10(-5)) when variants are expressed with the α3 subunit. In contrast, there is no significant association when carrier status is unweighted (β = -0.04, r2 = 0.0009, P = 0.54). These results highlight the value of functional analysis of variants and the advantages to integrating such data into genetic studies. They also suggest that an increased sensitivity to low concentrations of nicotine is protective from the risk of developing nicotine dependence
Pharmacological and biophysical characteristics of picrotoxin-resistant, δsubunit-containing GABAA receptors
GAB
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