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.
Highlights
A superfamily of ligand-gated ion channels comprises homo- and heteropentameric combinations of subunits
We define the average Förster resonance energy transfer (FRET) values as E3, 2 and E 2,3 respectively, so that the desired ratio is E3, 2 / E 2,3. We extend these calculations to normalized FRET (NFRET) and calculate the analogous ratios, NFRET3,2/NFRET2,3
We generally omit the description of the fluorophore when discussing stoichiometry; in most cases (α4)2(β2)3 implies (α4-mCherry)2(β2-enhanced green fluorescent protein (EGFP))3
Summary
A superfamily of ligand-gated ion channels comprises homo- and heteropentameric combinations of subunits. Permeability [4], their sensitivity to upregulation by chronic nicotine and other drugs [5,6], and possibly their subcellular localization Some of these characteristics may provide signatures for determining the stoichiometry of nAChRs when they appear on the plasma membrane. In addition to the obvious connection with nicotine addiction, pharmacological chaperoning may provide the mechanistic basis for the inverse correlation between a person’s history of smoking and his/her susceptibility to Parkinson’s disease [12] These points increase our interest in intracellular measurements of alterations in α4β2 nAChR stoichiometry. The theory is of interest because the measurements have yielded estimates about changes in stoichiometry that are consistent with known structural information and with chaperoning by nicotine, as well as a novel effect of chaperoning by cytisine
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