Single channel properties of human α3 AChRs: impact of β2, β4 and α5 subunits

Neuronal nicotinic acetylcholine receptors (AChRs) are expressed throughout the central and peripheral nervous system where their subunits are encoded by eleven genes identified as α2-9 and β2-4 (Papke, 1993; McGehee & Role, 1995; Lindstrom, 1996). The large variety of possible subunit combinations resulting from the multitude of nicotinic genes has led to considerable functional diversity for nicotinic AChRs expressed in tissues throughout the nervous system and in heterologous expression systems. Genes for multiple AChR subunits are often transcribed within the same cell. For example, neurons of peripheral ganglia have been shown to express α3, α5, α7, β2 and β4 subunits (McGehee & Role, 1995; Lindstrom, 1996). AChRs that are expressed in ganglia represent the best-characterized source of native neuronal nicotinic AChRs at many levels including functional, structural and histological (reviewed in the following: Papke, 1993; Sargent, 1993; McGehee & Role, 1995; Lindstrom, 1996). AChRs expressed in ganglia have been attributed mainly to two types, the α7 type that may be homomeric, and the α3 type that consists of α3 subunits assembled with α5, β2 and/or β4 subunits. α7 AChRs are characterized by their sensitivity to inhibition by α-bungarotoxin (α-BuTX), rapid desensitization and relatively low sensitivity to activation by acetylcholine (ACh), while α3 AChRs are α-BuTX insensitive, desensitize more slowly and are activated by ACh with more potency. The subunit composition of AChRs has been shown to determine a number of pharmacological and functional properties. These include apparent affinities for both agonists and antagonists, agonist efficacy and desensitization rates (Luetje et al. 1990; Cachelin & Jaggi, 1991; Luetje & Patrick, 1991; Papke & Heinemann, 1994). Determining the subunit composition of human ganglionic AChRs is crucial to understanding the physiological roles played by these AChRs and to developing effective therapeutic agents that may target particular AChRs. The dependence of AChR function on subunit composition has been studied extensively through expression of invitro transcribed RNA in Xenopus oocytes coupled with electrophysiological approaches. Early studies that utilized this approach quickly established the functionality of pairwise combinations of α2, α3 or α4 with β2 or β4 subunits (Papke, 1993, and references therein). On the other hand, early attempts to establish the functionality of α5, α6 and β3 were unsuccessful when these were coinjected with any known α or β subunit. Much later studies established functionality for the α6 subunit when coexpressed with the β2 or β4 subunits (Gerzanich et al. 1996; Fucile et al. 1998). Recent reports suggest that the α5 subunit of chicken or human does not contribute to the agonist binding site but serves a structural role, since assembly into surface AChRs requires both an α3 and a β subunit (Conroy et al. 1992; Wang et al. 1996). Assembly of the human α5 subunit into α3 AChRs altered many of the functional and pharmacological properties of the resulting AChRs (Gerzanich et al. 1998). Similarly, the α5 subunit altered the functional properties of α4β2 AChRs (Ramirez-Latorre et al. 1996; Kuryatov et al. 1997). Finally, the β3 subunit has recently been shown to assemble with α4 AChRs in rat brain (Forsayeth & Kobrin, 1997) and to assemble into functional AChRs when expressed heterologously with α3 and β4 subunits (Groot-Kormelink et al. 1998). Even though the functional properties of ganglionic α3 AChRs have been well described, questions concerning the subunit composition of native α3 AChRs remain (McGehee & Role, 1995; Lindstrom, 1996). To address these questions many studies have relied on approaches that utilize pharmacological fingerprinting of heterologously expressed AChRs. Pharmacological profiles established for these subunit-defined AChRs and for AChRs studied in native tissues provide a basis for comparison. Matches between profiles substantiate subunit identity. However, no exact match has been found between heterologous AChRs and native ganglionic AChRs (Covernton et al. 1994; Sivilotti et al. 1997). This might reflect differences in the expression environment between native cells and heterologous systems. Here, we have used single channel analysis to characterize the impact of the various AChR subunits that have been identified in ganglia on human α3 AChR function, i.e. AChRs formed from coexpression of α3, α5, β2 and β4 subunits in pairs or as triplets. We have found that for human α3 AChRs, substituting β4 for β2 resulted in an increase in open and burst times, with a slight change in channel conductance. When the α5 subunit was coexpressed with α3β2, the resulting α3α5β2 AChRs had a new open time that was not observed for α3β2 AChRs. When the α5 subunit was coexpressed with α3β4, the α3α5β4 AChRs had a new open time, altered burst times and an additional conductance state that were not observed for α3β4 AChRs. Comparison of oocyte-expressed AChR single channel properties with those of AChRs in the human neuroblastoma cell line IMR-32 suggests that the predominant functional AChR expressed in these cells is probably composed of α3 and β4 subunits. Functional properties that were linked to the presence of β2 and α5 subunits in heterologous AChRs were not observed in AChRs recorded from IMR-32 cells. This was consistent with the low level of β2- and α5-containing AChRs identified by radioimmunoassay of these cells.