Linda Lucero

Inducible, heterologous expression of human α7-nicotinic acetylcholine receptors in a native nicotinic receptor-null human clonal line

Tetracycline-regulated expression of recombinant nicotinic acetylcholine receptors (nAChR) composed of human alpha7 subunits is achieved in native nAChR-null SH-EP1 human epithelial cells. alpha7 subunits are heterologously expressed as messenger RNA and as components of 125I-labeled alpha-bungarotoxin (I-Bgt)-binding nAChR ( approximately 10 pmol per milligram of membrane protein) at levels sensitive to the amount of tetracycline in cell growth medium. I-Bgt-binding alpha7-nAChR appear on the cell surface pool and in intracellular pools. The pharmacological profile for drug competition toward I-Bgt binding to these recombinant alpha7-nAChR matches that of human native alpha7-nAChR naturally expressed in SH-SY5Y human neuroblastoma cells (rank order potency methyllycaconitine>1, 1-dimethyl-4-phenylpiperazinium>(-)nicotine>cytisine>carbamylch oli ne> /=d-tubocurarine). Chronic exposure to nicotine induces up-regulation of human recombinant alpha7-nAChR (80% up-regulation at 10 microM nicotine) just as it does native alpha7-nAChR in other human cell lines. These studies confirm expression of nAChR as homooligomers of human alpha7 subunits from transgenes, establish a native nAChR-null background for such expression, and demonstrate that this expression can be regulated to facilitate studies of human alpha7-nAChR.

Differential expression of nicotinic acetylcholine receptor subunits in fetal and neonatal mouse thymus

Studies were initiated to identify nicotinic acetylcholine receptor (nAChR) subunits and subtypes expressed in the developing immune system and cell types on which nAChR are expressed. Reported here are reverse transcription-polymerase chain reactions (RT-PCR) studies of nAChR alpha2-alpha7 and beta2-beta4 subunit gene expression using fetal or neonatal regular or scid/scid C57BL/6 mouse thymus. Findings are augmented with studies of murine fetal thymic organ cultures (FOTC) and of human peripheral lymphocytes. Novel partial cDNA sequences were derived for mouse nAChR alpha2, alpha3, beta3 and beta4 subunits, polymorphisms were identified in mouse nAChR alpha4, alpha7 and beta2 subunits, and recently derived sequences for mouse nAChR alpha5 and alpha6 subunits were confirmed. Thymic stromal cells appear to express nAChR alpha2, alpha3, alpha4, alpha7 and beta4 subunits, perhaps in addition to alpha5 and beta2 subunits, in a pattern reminiscent of expression in the developing brain. Immature T cells appear to express alpha3, alpha5, alpha7, beta2 and beta4 subunits, just as do neural crest-derived cells targeted by cholinergic innervation. Peripheral T cells seem to express an unusual profile of alpha2, alpha5 and alpha7 subunits, perhaps indicating that their nAChR express yet-to-be-identified assembly partners or that T cell nicotinic responsiveness occurs through homomeric nAChR composed of alpha7 subunits. Our findings are consistent with published work but show a much wider array of nAChR subunit gene expression in mouse thymic stromal and/or lymphoid cells and evidence for developmental regulation of nAChR subunit expression. These studies suggest important roles for nAChR in immune system development and function and in the neuroimmune network.

The Unique α4(+)/(−)α4 Agonist Binding Site in (α4)3(β2)2 Subtype Nicotinic Acetylcholine Receptors Permits Differential Agonist Desensitization Pharmacology versus the (α4)2(β2)3 Subtype

Selected nicotinic agonists were used to activate and desensitize high-sensitivity (HS) (α4)2(β2)3) or low-sensitivity (LS) (α4)3(β2)2) isoforms of human α4β2-nicotinic acetylcholine receptors (nAChRs). Function was assessed using 86Rb+ efflux in a stably transfected SH-EP1-hα4β2 human epithelial cell line, and two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing concatenated pentameric HS or LS α4β2-nAChR constructs (HSP and LSP). Unlike previously studied agonists, desensitization by the highly selective agonists A-85380 [3-(2(S)-azetidinylmethoxy)pyridine] and sazetidine-A (Saz-A) preferentially reduced α4β2-nAChR HS-phase versus LS-phase responses. The concatenated-nAChR experiments confirmed that approximately 20% of LS-isoform acetylcholine-induced function occurs in an HS-like phase, which is abolished by Saz-A preincubation. Six mutant LSPs were generated, each targeting a conserved agonist binding residue within the LS-isoform-only α4(+)/(−)α4 interface agonist binding site. Every mutation reduced the percentage of LS-phase function, demonstrating that this site underpins LS-phase function. Oocyte-surface expression of the HSP and each of the LSP constructs was statistically indistinguishable, as measured using β2-subunit–specific [125I]mAb295 labeling. However, maximum function is approximately five times greater on a “per-receptor” basis for unmodified LSP versus HSP α4β2-nAChRs. Thus, recruitment of the α4(+)/(−)α4 site at higher agonist concentrations appears to augment otherwise-similar function mediated by the pair of α4(+)/(−)β2 sites shared by both isoforms. These studies elucidate the receptor-level differences underlying the differential pharmacology of the two α4β2-nAChR isoforms, and demonstrate that HS versus LS α4β2-nAChR activity can be selectively manipulated using pharmacological approaches. Since α4β2 nAChRs are the predominant neuronal subtype, these discoveries likely have significant functional implications, and may provide important insights for drug discovery and development.

Neurotoxicity of channel mutations in heterologously expressed α7‐nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChR) composed of chick α7 subunits mutated to threonine at amino acid valine‐251 in the putative channel‐lining M2 domain were expressed heterologously in several neuron‐like and non‐neuronal mammalian cell lines. Expression of mutant α7‐nAChR is toxic to neuron‐like cells of the human neuroblastoma cell lines SH‐SY5Y and IMR‐32, but not to several other cell types. Growth in the presence of the α7‐nAChR antagonist methyllycaconitine (MLA) protects against neurotoxicity, as does gradual downregulation of functional, mutant α7‐nAChR in surviving transfected SH‐SY5Y cells. Relative to wild‐type α7‐nAChR, functional α7‐nAChR mutants show a higher affinity for agonists, slower rates of desensitization, and sensitivity to dihydro‐β‐erythroidine (DHβE) as an agonist, but they retain sensitivity to MLA as a competitive antagonist. These findings demonstrate that expression of hyperfunctional, mutant forms of Ca2+‐permeable α7‐nAChR is toxic to neuron‐like cells.

Differential α4(+)/(−)β2 Agonist-binding Site Contributions to α4β2 Nicotinic Acetylcholine Receptor Function within and between Isoforms

Two α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) isoforms exist with (α4)2(β2)3 and (α4)3(β2)2 subunit stoichiometries and high versus low agonist sensitivities (HS and LS), respectively. Both isoforms contain a pair of α4(+)/(−)β2 agonist-binding sites. The LS isoform also contains a unique α4(+)/(−)α4 site with lower agonist affinity than the α4(+)/(−)β2 sites. However, the relative roles of the conserved α4(+)/(−)β2 agonist-binding sites in and between the isoforms have not been studied. We used a fully linked subunit concatemeric nAChR approach to express pure populations of HS or LS isoform α4β2*-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of β2 subunit (−)-face E-loop residues to their non-conserved α4 subunit counterparts or vice versa (β2HQT and α4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. Acetylcholine concentration-response curves and maximum function were measured using two-electrode voltage clamp electrophysiology. Surface expression was measured with 125I-mAb 295 binding and was used to define function/nAChR. If the α4(+)/(−)β2 sites contribute equally to function, making identical β2HQT substitutions at either site should produce similar functional outcomes. Instead, highly differential outcomes within the HS isoform, and between the two isoforms, were observed. In contrast, α4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise equivalent α4(+)/(−)β2 agonist sites modifies their contributions to nAChR activation and that E-loop residues are an important contributor to this neighbor effect.

α-Conotoxins Identify the α3β4∗ Subtype as the Predominant Nicotinic Acetylcholine Receptor Expressed in Human Adrenal Chromaffin Cells

Ligands that selectively inhibit human α3β2 and α6β2 nicotinic acetylcholine receptor (nAChRs) and not the closely related α3β4 and α6β4 subtypes are lacking. Current α-conotoxins (α-Ctxs) that discriminate among these nAChR subtypes in rat fail to discriminate among the human receptor homologs. In this study, we describe the development of α-Ctx LvIA(N9R,V10A) that is 3000-fold more potent on oocyte-expressed human α3β2 than α3β4 and 165-fold more potent on human α6/α3β2β3 than α6/α3β4 nAChRs. This analog was used in conjuction with three other α-Ctx analogs and patch-clamp electrophysiology to characterize the nAChR subtypes expressed by human adrenal chromaffin cells. LvIA(N9R,V10A) showed little effect on the acetylcholine-evoked currents in these cells at concentrations expected to inhibit nAChRs with β2 ligand-binding sites. In contrast, the β4-selective α-Ctx BuIA(T5A,P6O) inhibited >98% of the acetylcholine-evoked current, indicating that most of the heteromeric receptors contained β4 ligand-binding sites. Additional studies using the α6-selective α-Ctx PeIA(A7V,S9H,V10A,N11R,E14A) indicated that the predominant heteromeric nAChR expressed by human adrenal chromaffin cells is the α3β4* subtype (asterisk indicates the possible presence of additional subunits). This conclusion was supported by polymerase chain reaction experiments of human adrenal medulla gland and of cultured human adrenal chromaffin cells that demonstrated prominent expression of RNAs for α3, α5, α7, β2, and β4 subunits and a low abundance of RNAs for α2, α4, α6, and α10 subunits.