TY - JOUR
T1 - Förster resonance energy transfer (FRET) correlates of altered subunit stoichiometry in Cys-loop receptors, exemplified by nicotinic α4β2
AU - Srinivasan, Rahul
AU - Richards, Christopher I.
AU - Dilworth, Crystal
AU - Moss, Fraser J.
AU - Dougherty, Dennis A.
AU - Lester, Henry A.
PY - 2012/8
Y1 - 2012/8
N2 - 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.
AB - 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.
KW - Cytisine
KW - Ion channels
KW - NFRET
KW - Nicotine
KW - Nicotine addiction
KW - Parkinson's disease
UR - https://www.scopus.com/pages/publications/84865017895
UR - https://www.scopus.com/pages/publications/84865017895#tab=citedBy
U2 - 10.3390/ijms130810022
DO - 10.3390/ijms130810022
M3 - Review article
C2 - 22949846
AN - SCOPUS:84865017895
SN - 1661-6596
VL - 13
SP - 10022
EP - 10040
JO - International Journal of Molecular Sciences
JF - International Journal of Molecular Sciences
IS - 8
ER -