Förster resonance energy transfer (FRET) correlates of altered subunit stoichiometry in Cys-loop receptors, exemplified by nicotinic α4β2

Rahul Srinivasan; Christopher I. Richards; Crystal Dilworth; Fraser J. Moss; Dennis A. Dougherty; Henry A. Lester

doi:10.3390/ijms130810022

Förster resonance energy transfer (FRET) correlates of altered subunit stoichiometry in Cys-loop receptors, exemplified by nicotinic α4β2

Rahul Srinivasan, Christopher I. Richards, Crystal Dilworth, Fraser J. Moss, Dennis A. Dougherty, Henry A. Lester

Chemistry

Research output: Contribution to journal › Review article › peer-review

19 Scopus citations

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.

Original language	English
Pages (from-to)	10022-10040
Number of pages	19
Journal	International Journal of Molecular Sciences
Volume	13
Issue number	8
DOIs	https://doi.org/10.3390/ijms130810022
State	Published - Aug 2012

Keywords

Cytisine
Ion channels
NFRET
Nicotine
Nicotine addiction
Parkinson's disease

ASJC Scopus subject areas

Catalysis
Molecular Biology
Spectroscopy
Computer Science Applications
Physical and Theoretical Chemistry
Organic Chemistry
Inorganic Chemistry

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10.3390/ijms130810022

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@article{2dd0a27870764e59874653289ab99bd8,

title = "F{\"o}rster resonance energy transfer (FRET) correlates of altered subunit stoichiometry in Cys-loop receptors, exemplified by nicotinic α4β2",

abstract = "We provide a theory for employing F{\"o}rster 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.",

keywords = "Cytisine, Ion channels, NFRET, Nicotine, Nicotine addiction, Parkinson's disease",

author = "Rahul Srinivasan and Richards, {Christopher I.} and Crystal Dilworth and Moss, {Fraser J.} and Dougherty, {Dennis A.} and Lester, {Henry A.}",

year = "2012",

month = aug,

doi = "10.3390/ijms130810022",

language = "English",

volume = "13",

pages = "10022--10040",

number = "8",

}

Förster resonance energy transfer (FRET) correlates of altered subunit stoichiometry in Cys-loop receptors, exemplified by nicotinic α4β2. / Srinivasan, Rahul; Richards, Christopher I.; Dilworth, Crystal et al.
In: International Journal of Molecular Sciences, Vol. 13, No. 8, 08.2012, p. 10022-10040.

Research output: Contribution to journal › Review article › peer-review

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

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UR - http://www.scopus.com/inward/citedby.url?scp=84865017895&partnerID=8YFLogxK

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 -

Förster resonance energy transfer (FRET) correlates of altered subunit stoichiometry in Cys-loop receptors, exemplified by nicotinic α4β2

Abstract

Keywords

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