Functional contribution of the α7 subunit to multiple subtypes of nicotinic receptors in embryonic chick sympathetic neurones

Many studies of the α7 subunit of the neuronal nicotinic acetylcholine receptor (nAChR) family have demonstrated that this α-bungarotoxin (α-BgTx)-binding neuronal receptor can participate in ACh-gated channels. Heterologous expression studies reveal that α7 subunits form homomeric channels of unusually high Ca2+ permeability. However, the physiological role of the α7 subunit in native neuronal nAChR channels is less clear. We present evidence that the α7 subunit contributes to the function of at least three subtypes of native nAChR expressed by embryonic chick sympathetic neurones. These subtypes are functionally distinct from heterologously expressed homomeric α7 nAChRs as well as homomeric-like currents described in studies of hippocampal and parasympathetic neurones. The proposed nAChRs differ from one another and from homomeric α7 nAChRs in their sensitivity to block by α7 subunit-specific antagonists: α-BgTx and methyllycaconitine (MLA). While MLA blocks 60 % of the macroscopic ACh response, α-BgTx inhibits a small component of the macroscopic current described by slow-on and slow-off kinetics. Functional deletion of the α7 subunit by antisense oligonucleotide treatment eliminates the susceptibility of the nAChRs to block by both MLA and α-BgTx. Single channel recordings combined with pharmacological and antisense-mediated ‘deletion’ techniques reveal that α-BgTx-sensitive α7-containing nAChRs have a small unitary conductance (18 pS), brief open time kinetics and relatively low open probability (Po). MLA-sensitive α7 nAChRs are characterized by a conductance of ≈35 pS, intermediate burst duration, and a relatively high Po. The third nAChR subtype deleted by α7 antisense treatment is characterized by a unitary conductance of 50 pS and prolonged opening duration. We propose that these three populations of native α7-containing nAChRs are distinct heteromeric complexes that include other α and/or β nAChR subunits. Neurotransmission at interneuronal synapses is mediated in part by activation of ligand-gated ion channels. In general, such channels are composed of one or more subunit types encoded by a family of receptor subunit genes. The diversity of subunit expression correlates with the diversity of receptor channels detected in individual neurones. Although considerable effort has elucidated key molecular, biochemical and biophysical characteristics of numerous neuronal ligand-gated receptors, the composition of native channels at neuronal synapses is still largely unknown. The neuronal nicotinic acetylcholine receptors (nAChRs) constitute a subfamily of ligand-gated ion channels that are widely distributed in both the peripheral and central nervous system (PNS and CNS, respectively). To date, eleven genes of this family have been cloned. These genes share a number of structural and functional features, and encode α- and β-subunits (eight α-subunits: α2-α9; three β-subunits: β2-β4; for review see Sargent, 1993; McGehee & Role, 1995). Correspondingly, a vast array of receptor subtypes have been detected and characterized in specific neuronal systems (for reviews see Sargent, 1993; McGehee & Role, 1995). Neuronal nAChRs were initially identified as two major groups in the brain, high affinity nicotine binding sites and α-bungarotoxin (α-BgTx) binding sites. Despite the wide distribution of α-BgTx binding sites throughout the vertebrate nervous system, the physiological role of α-BgTx binding nAChRs remained elusive (Clarke, Schwartz, Paul & Pert, 1985; Clarke, 1992; Keyser et al. 1993; Lindstrom, Anand, Peng, Gerzanich, Wang & Li, 1995). The isolation of the α7 nAChR subunit gene and the demonstration that expression of the α7 subunit in heterologous systems yields α-BgTx-blockable nAChR channels transformed the nAChR field (Couturier et al. 1990). α7 homomeric channels, when gated by agonist, mediate rapidly activating and desensitizing currents with strong inward rectification at hyperpolarized membrane potentials. nAChRs composed solely of α7 are blocked by α-BgTx at nanomolar concentrations (Couturier et al. 1990; Bertrand, Bertrand & Ballivet, 1992), and are highly permeable to calcium (Seguela, Wadiche, Dineley-Miller, Dani & Patrick, 1993). Several recent reports describe similarly rapid, α-BgTx-sensitive nicotine-gated currents in specific populations of both CNS and PNS neurones, most notably rat hippocampal neurones (Alkondon & Albuquerque, 1993; Castro & Albuquerque, 1995; Alkondon, Pereira, Barbosa & Albuquerque, 1997a), olfactory bulb (Alkondon & Albuquerque, 1994) and parasympathetic ganglion neurones (Vijayaraghavan, Pugh, Zhang, Rathouz & Berg, 1992; Zhang, Vijayaraghavan & Berg, 1994). The biophysical and pharmacological characteristics of these native nAChRs are comparable to those of the α7 homomeric nAChRs, supporting the contention that these neurones express α7 homomeric channels. Despite the demonstration of these ‘α7 homomeric-like’ currents, many neurones express high levels of both α7 mRNA and protein, and yet α-BgTx-sensitive currents are not detected, even with the use of rapid drug delivery systems. The broad expression of α7 mRNA and protein throughout the CNS and PNS, compared with the relative paucity of reports of α7 homomeric-like currents, may reflect the participation of α7 in functionally distinct nAChR complexes. Indeed, studies by Lindstrom and colleagues suggest that chick retinal ganglion neurones express heteromeric channels of α7 and α8 that may include other α and/or β nAChR subunits (Britto, Hamassaki-Britto, Ferro, Keyser, Karten & Lindstrom, 1992; Anand, Peng, Ballesta & Lindstrom, 1993a; Anand, Peng & Lindstrom, 1993b). Biochemical studies also support the possibility of α7 heteromeric nAChRs as α7 protein can be immunoprecipitated with other subunits, although the identity of these subunits remains to be determined (Pugh, Corriveau, Conroy & Berg, 1995). Furthermore, recent results demonstrate α7 can form heteromeric complexes with α5 and β2 or β4 (G. Crabtree, J. Ramirez & L. W. Role, unpublished results). In this study we examine the contribution of α7 subunits to ACh-induced currents in embryonic chick sympathetic neurones. These neurones express the α7 gene and α7 protein as indicated from Northern blot, in situ, immunohistochemical and immunoprecipitation studies (Devay, Qu & Role, 1994; Pugh et al. 1995). Furthermore, the levels of expression of α7 mRNA and protein are developmentally regulated by chronic depolarization, presynaptic input and target interaction (Margiotta & Gurantz, 1989; Jacob, 1991; Devay et al. 1994; De Koninck & Cooper, 1995). Despite the prominent expression of α7 and the use of rapid perfusion techniques, α7 homomeric channels are not detected in rat or chick sympathetic neurones (De Koninck & Cooper, 1995; authors’ unpublished results). This study demonstrates that ACh elicits currents with distinct kinetics and pharmacological properties that are blocked by α-BgTx or methyllycaconitine (MLA) in chick sympathetic neurones. The α-BgTx-sensitive component of the macroscopic currents is characterized by both slow-on and slow-off kinetics, and by an 18 pS unitary conductance. The MLA-sensitive component is a relatively rapid current (though considerably slower than that of α7 homomeric channels) and is mediated by a 35 pS, intermediate burst duration channel. Confirmation of these results by antisense oligonucleotide-mediated deletion of α7 also reveals that the α7 subunit is present in a 50 pS conductance channel distinguished by prolonged channel opening kinetics.