NACHO and 14-3-3 promote expression of distinct subunit stoichiometries of the α4β2 acetylcholine receptor

Stoichiometry‐selective modulation of α4β2 nicotinic ACh receptors by divalent cations

“…Herein, we investigate the functional consequences of physiological concentrations of Ca 2+ and Mg 2+ on single-channel currents through the two predominant stoichiometric forms of the α4β2 nAChR. To generate enriched populations of each stoichiometric form, we take advantage of the stoichiometry-selective chaperones, NACHO and 14-3-3η, which promote expression of enriched populations of either the (α4) 2 (β2) 3 or the (α4) 3 (β2) 2 stoichiometry, respectively (Mazzaferro et al, 2021). We first present the effects of the divalent cations on the amplitude of ACh-elicited single-channel currents for each stoichiometric form and then describe the effects on single-channel open and closed-dwell times.…”

“…2.1 | Expression of human (α4) 3 (β2) 2 and (α4) 2 (β2) 3 AChRs cDNAs encoding human α4 and β2 subunits, and the chaperones 14-3-3η and nAChR regulator chaperone (NACHO), were individually subcloned into a modified pCI mammalian expression vector (Promega), as previously described (Carbone et al, 2009;Mazzaferro et al, 2011Mazzaferro et al, , 2021. 14-3-3η and NACHO were used to bias the expression of (α4) 3 (β2) 2 and (α4) 2 (β2) 3 AChRs, respectively (Carbone et al, 2009;Exley et al, 2006;Jeanclos et al, 2001;Mazzaferro et al, 2021).…”

“…2.1 | Expression of human (α4) 3 (β2) 2 and (α4) 2 (β2) 3 AChRs cDNAs encoding human α4 and β2 subunits, and the chaperones 14-3-3η and nAChR regulator chaperone (NACHO), were individually subcloned into a modified pCI mammalian expression vector (Promega), as previously described (Carbone et al, 2009;Mazzaferro et al, 2011Mazzaferro et al, , 2021. 14-3-3η and NACHO were used to bias the expression of (α4) 3 (β2) 2 and (α4) 2 (β2) 3 AChRs, respectively (Carbone et al, 2009;Exley et al, 2006;Jeanclos et al, 2001;Mazzaferro et al, 2021). BOSC 23 cells, a cell line derived from HEK 293 cells (CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045) (Pear et al, 1993), were maintained in DMEM (Gibco) containing 10% FBS and transfected by calcium phosphate precipitation, as previously described (Bouzat et al, 1994;Sine, 1993;Sine et al, 1994).…”

“…An excess of α4 over β2 cDNAs has been shown to increase expression of (α4) 3 (β2) 2 over the (α4) 2 (β2) 3 stoichiometry in BOSC 23 cells (Carignano et al, 2016). To ensure a pure population of the (α4) 3 (β2) 2 stoichiometry, the cDNAencoding 14-3-3η was added to the transfection mix (Mazzaferro et al, 2017(Mazzaferro et al, , 2019(Mazzaferro et al, , 2021. The amounts of transfected α4 and β2…”

“…Single-channel recordings were obtained in the cell-attached patch configuration at a membrane potential of À70 mV and a temperature of 20 C, as previously described (Mazzaferro et al, 2017(Mazzaferro et al, , 2019(Mazzaferro et al, , 2021. For all experiments, pipette and extracellular bathing solutions…”