Förster Resonance Energy Transfer (FRET) Correlates of Altered Subunit Stoichiometry in Cys-Loop Receptors, Exemplified by Nicotinic α4β2

Abstract

We provide a theory for employing Förster resonance energy transfer (FRET) measurements to determine altered heteropentameric ion channel stoichiometries in intracellular compartments of living cells. We simulate FRET within nicotinic receptors (nAChRs) whose α4 and β2 subunits contain acceptor and donor fluorescent protein moieties, respectively, within the cytoplasmic loops. We predict FRET and normalized FRET (NFRET) for the two predominant stoichiometries, (α4)3(β2)2 vs. (α4)2(β2)3. Studying the ratio between FRET or NFRET for the two stoichiometries, minimizes distortions due to various photophysical uncertainties. Within a range of assumptions concerning the distance between fluorophores, deviations from plane pentameric geometry, and other asymmetries, the predicted FRET and NFRET for (α4)3(β2)2 exceeds that of (α4)2(β2)3. The simulations account for published data on transfected Neuro2a cells in which α4β2 stoichiometries were manipulated by varying fluorescent subunit cDNA ratios: NFRET decreased monotonically from (α4)3(β2)2 stoichiometry to mostly (α4)2(β2)3. The simulations also account for previous macroscopic and single-channel observations that pharmacological chaperoning by nicotine and cytisine increase the (α4)2(β2)3 and (α4)3(β2)2 populations, respectively. We also analyze sources of variability. NFRET-based monitoring of changes in subunit stoichiometry can contribute usefully to studies on Cys-loop receptors.

The Duplicated α7 Subunits Assemble and Form Functional Nicotinic Receptors with the Full-length α7

“…When the fluorescent proteins are fused into the M3-M4 loop of nicotinic receptor subunits, subunits within pentamers undergo FRET. We have previously developed experimental approaches, well supported by theory, to analyze the assembly and subunit stoichiometry of such subunits within Cys loop receptors (23,28,(31)(32)(33). For the present experiments, important guidelines arise from the strong distance dependence of FRET.…”

“…Potential Stoichiometry of ␣7dup␣7 and ␣7dup⌬␣7 Heteromers, FLIM Measurements-DRAP is not optimal for determining subunit stoichiometry in the present experiments because there are many more full-length ␣7 subunits than duplicated subunits when they are co-expressed (32). We also employed FLIM to measure FRET.…”

The Nicotine Metabolite, Cotinine, Alters the Assembly and Trafficking of a Subset of Nicotinic Acetylcholine Receptors

“…Previous studies have altered the primary stoichiometry of ␣4␤2 expressed using biased transfection, with one subunit expressed at higher levels than the other (4,5,19,46). These types of studies have primarily used Xenopus oocyte expression systems with changes in stoichiometry determined from changes in the biphasic dose response based on whole cell current measurements.…”

“…Cell Culture and Transfection-Undifferentiated mouse neuroblastoma 2a (N2a) cells were cultured using standard tissue culture techniques and maintained in growth media consisting of DMEM and Opti-MEM, supplemented with 10% fetal bovine serum, penicillin, and streptomycin (19,36). Cells were plated by adding 90,000 cells to poly-D-lysine-coated 35-mm glass bottom dishes (In Vitro Scientific).…”

“…The current pharmacological chaperoning hypothesis suggests that these changes only require concentrations of nicotine to be high enough to interact with the specific subtype in the endoplasmic reticulum, meaning surface activation is not required (8,18) Other nAChR ligands, including drugs that have been evaluated as smoking cessation agents, have also been shown to up-regulate nAChRs. These drugs include the partial agonists cytisine (19) and varenicline (20) as well as the antagonist mecamylamine (21). Although nicotine exposure increases the assembly of the high sensitivity receptor, (␣4) 2 (␤2) 3 (16,22), cytisine has been shown to alter the stoichiometry of ␣4␤2 with a preference for the low sensitivity receptor, (␣4) 3 (␤2) 2 , potentially due to the existence of an additional binding site for cytisine at the ␣-␣ interface (23,24).…”