Amino Acid Determinants of α7 Nicotinic Acetylcholine Receptor Surface Expression

Abstract

Transient transfection has not been a successful method to express the ␣7 nicotinic acetylcholine receptor such that these receptors are detected on the cell surface. This is not the case for all ligand-gated ion channels. Transient transfection with the 5-hydroxytryptamine type 3 subunit cDNA results in detectable surface receptor expression. Cell lines stably expressing the ␣7 nicotinic acetylcholine receptor produce detectable, albeit variable, levels of surface receptor expression. ␣7 nicotinic acetylcholine receptor surface expression is dependent, at least in part, on cell-specific factors. In addition to factors provided by the cells used for receptor expression, we hypothesize that the surface expression level in transfected cells is an intrinsic property of the receptor protein under study. Employing a set of ␣7-5-hydroxytryptamine type 3 chimeric receptor subunit cDNAs, we expressed these constructs in a transient transfection system and quantified surface receptor expression. We have identified amino acids that control receptor distribution between surface and intracellular pools; surface receptor expression can be manipulated without affecting the total number of receptors. These determinants function independently of the cell line used for expression and the transfection method employed. How these surface expression determinants in the ␣7 nicotinic acetylcholine receptor might influence synaptic efficacy is discussed.The ␣7 nicotinic acetylcholine receptor (nAChR) 1 is a member of the ligand-gated ion channel superfamily of neurotransmitter receptors that includes the nAChRs, the cation-permeable 5-hydroxytryptamine type 3 (5HT 3 ) receptor, and the inhibitory glycine and GABA A receptors. All members possess the conserved subunit topology of a large extracellular aminoterminal domain followed by four transmembrane domains, the third and fourth of which are separated by the cytoplasmic loop, a highly variable stretch of amino acids both in composition and length (for reviews, see Refs. 1-3). ␣7 subunits are capable of forming homooligomeric receptors for which ␣-bungarotoxin is a specific and high affinity label (4, 5). Upon activation, ␣7 nAChRs conduct a significant amount of Ca 2ϩ (P Ca 2ϩ /PNa ϭ 20) (5). Thus, depending on their anatomical location, ␣7 nAChRs may influence neurotransmitter release and synaptic integration. In the mammalian brain, small populations (compared with predictions from ␣-bungarotoxin binding density) of this receptor have been functionally located to presynaptic and postsynaptic sites by recording from individual neurons under conditions of rapid drug application and removal (6 -13).Heterologous expression of the ␣7 nAChR would facilitate investigations of the structure-function relationships of this receptor; however, transient transfection of neuronal and nonneuronal cell lines with ␣7 cDNA results in a surface receptor expression level that is below the detection limit of an 125 I-␣-bungarotoxin binding assay. The failure to detect receptors with this assay indica...

Dual Role of the RIC-3 Protein in Trafficking of Serotonin and Nicotinic Acetylcholine Receptors

“…Moreover, we have previously shown that an arginine (Arg 205 according to the numbering system used here) located between Ile 202 and the latter motif and conserved in all ligand-gated channel subunits is required for transport of assembled ␣7 receptors to the cell surface (31). Also in this region certain domains have been found that affect surface expression of ␣7-5HT 3 chimeric receptors (32). All these data suggest that this region, adjacent to the first transmembrane segment, constitutes a "hot spot" with potential retention/retrieval signals involved in receptor trafficking.…”

Rapid Upregulation of α7 Nicotinic Acetylcholine Receptors by Tyrosine Dephosphorylation

“…The cellular processes that control the number of functional membrane ␣7 nAChRs are not fully understood. The normal delivery of ␣7 receptors from intracellular pools to the surface is clearly an important but not well defined process (Dineley and Patrick, 2000), and it is likely that, at least for this nAChR subtype, trafficking may depend on neuronspecific factors involved in the membrane targeting of proteins (Kassner and Berg, 1997;Sweileh et al, 2000). Indeed, during assembly in the endoplasmic reticulum, palmitoylation at cysteine residues in the intracellular loop of the ␣7 subunit is critical for surface receptor expression and fails to occur in certain nonneuronal cells (Drisdel et al, 2004).…”

SSR591813, a Novel Selective and Partial α4β2 Nicotinic Receptor Agonist with Potential as an Aid to Smoking Cessation

“…The pellet was suspended in p-aminobenzamidine-free HEPES buffer, incubated at room temperature for 15 min, and centrifuged once again for 15 min at 4°C (40,000g). Marks et al (1992) and Dineley and Patrick (2000) with slight modifications. Samples containing rat brain membranes (300 -350 g of protein), the test compound and 1 nM [ 3 H]␣-bungarotoxin (60 Ci/mmol) were incubated in a final volume of 250 l for 1 h at 37°C.…”