The neuronal α₆ subunit forms functional heteromeric acetylcholine receptors in human transfected cells

Abstract

We examine some of the biological and physiological properties of the avian alpha6 neuronal nicotinic acetylcholine receptor (nAChR) subunit. We show here that, beginning at embryonic day 5, alpha6 mRNA is abundantly expressed in the developing chick neuroretina, where it coexists with other nicotinic receptor subunit mRNAs such as alpha3, beta2 and beta4. In contrast, alpha6 mRNA is absent from the optic tectum and from the peripheral ganglia. Despite numerous efforts, the alpha6 subunit has long failed the critical test of functional reconstitution. Here we use patch-clamp techniques and confocal laser microscopy to measure ACh-activated currents and nicotine-elicited Ca2+ transients in human BOSC 23 cells transfected with chick alpha6 in combination with other chick nAChR neuronal subunits. Heterologously expressed alpha6 and beta4 subunits form functional heteromeric nAChRs, which are permeable to Ca2+ ions and blocked by the nicotinic antagonist methyllycaconitine (10 microM). Likewise, ACh elicits measurable currents in cells transfected with alpha6 and beta2. Hill analysis of the dose-response curves in cells transfected with alpha3, beta4 and alpha6 cDNAs, suggests the assembly of functional alpha3beta4alpha6 receptor, with an apparent affinity for ACh threefold lower than alpha3beta4. Our results indicate that alpha6-containing nAChRs assemble in heterologous expression systems and are probably present in retinal cells.

Identification of N-terminal Extracellular Domain Determinants in Nicotinic Acetylcholine Receptor (nAChR) α6 Subunits That Influence Effects of Wild-type or Mutant β3 Subunits on Function of α6β2*- or α6β4*-nAChR

“…Moreover, and although these studies were done using oocytes manipulated to express disproportionate ratios of subunits, incorporation of nAChR h␤3 subunits present in presumed excess have a dominant-negative effect on the function of all-human ␣2␤2*-, ␣2␤4*-, ␣3␤2*-, ␣3␤4*-, ␣4␤2*-, or ␣4␤4*-nAChR that is reversed upon substitution of mutant, h␤3 V9ЈS for wild-type ␤3 subunits (12). Similar effects are mentioned (but not described in detail) of effects of wild-type or mutant ␤3 subunits on function of ␣6␤2*-and ␣6␤4*-nAChR (11). Interestingly, other studies using chimeric (␣6/␣3) subunits (containing the N-terminal domain of the nAChR ␣6 subunit substituting for that of the otherwise ␣3 subunit) instead of wild-type ␣6 subunits showed potentiating effects of wild-type ␤3 subunit coexpression on ␣6*-nAChR (8).…”

“…Chick or rat nAChR ␣6 subunits form functional channels when coexpressed with h␤4 subunits in Xenopus oocytes, but the ␣6 plus ␤2 subunit combination fails to form functional channels (10). Chick ␣6 subunits also formed functional receptors when coexpressed with chick ␤2 or ␤4 subunits in BOSC 23 cells (11). More recently, Kuryatov et al (8) reported functional expression of human ␣6␤4-nAChR in Xenopus oocytes and formation of ligand-binding (nonfunctional) aggregates of ␣6␤2-nAChR.…”

Human neuronal threonine-for-leucine-248 alpha 7 mutant nicotinic acetylcholine receptors are highly Ca2+ permeable

“…Transient transfections were carried out in the cell line Bosc 23 as described (28)(29)(30). Subunit cDNAs were added to 35-mm dishes in equivalent amounts (2 g of L248T␣7 subunit cDNA and 1 g each of all other subunits) as described (28)(29)(30). More than the 50% of the cells died after transfection with L248T␣7 subunit cDNA, and the survival increased to Ϸ90% when the cells were incubated with the nicotinic antagonist methyllycaconitine (MLA, 2 M).…”

“…Whole-cell patch-clamp recordings of AcCho-or nicotine-induced currents (I AcCho and I nic , respectively) were performed on cells continuously superfused with control or agonist solutions via independent tubes positioned 50-100 m from the patched cell and connected to a fast exchanger system. Recordings were performed as described (28)(29)(30). Unless otherwise indicated, cells were voltageclamped at Ϫ50 mV.…”

“…The full-length cDNAs encoding mouse muscle nAcChoR ␣-, ␤-, -, and ␦-subunits were cloned in the simian virus 40-based pSM expression vector. Transient transfections were carried out in the cell line Bosc 23 as described (28)(29)(30). Subunit cDNAs were added to 35-mm dishes in equivalent amounts (2 g of L248T␣7 subunit cDNA and 1 g each of all other subunits) as described (28)(29)(30).…”