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Coupling Between GABAA-R Ligand-Binding Activity and Membrane Organization in β-Cyclodextrin-Treated Synaptosomal Membranes from Bovine Brain Cortex: New Insights from EPR Experiments

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Abstract

Correlations between GABAA receptor (GABAA-R) activity and molecular organization of synaptosomal membranes (SM) were studied along the protocol for cholesterol (Cho) extraction with β-cyclodextrin (β-CD). The mere pre-incubation (PI) at 37°C accompanying the β-CD treatment was an underlying source of perturbations increasing [3H]-FNZ maximal binding (70%) and K d (38%), plus a stiffening of SMs’ hydrocarbon core region. The latter was inferred from an increased compressibility modulus (K) of SM-derived Langmuir films, a blue-shifted DPH fluorescence emission spectrum and the hysteresis in DPH fluorescence anisotropy (A DPH) in SMs submitted to a heating–cooling cycle (4–37–4°C) with A DPH,heating < A DPH,cooling. Compared with PI samples, the β-CD treatment reduced B max by 5% which correlated with a 45%-decrement in the relative Cho content of SM, a decrease in K and in the order parameter in the EPR spectrum of a lipid spin probe labeled at C5 (5-SASL), and significantly increased A TMA-DPH. PI, but not β-CD treatment, could affect the binding affinity. EPR spectra of 5-SASL complexes with β-CD-, SM-partitioned, and free in solution showed that, contrary to what is usually assumed, β-CD is not completely eliminated from the system through centrifugation washings. It was concluded that β-CD treatment involves effects of at least three different types of events affecting membrane organization: (a) effect of PI on membrane annealing, (b) effect of residual β-CD on SM organization, and (c) Cho depletion. Consequently, molecular stiffness increases within the membrane core and decreases near the polar head groups, leading to a net increase in GABAA-R density, relative to untreated samples.

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Abbreviations

β-CD:

β-Cyclodextrin

B max :

Maximal binding

BSA:

Bovine serum albumin

CD-PI:

Synaptosomal membranes treated with β-CD

Cho:

Cholesterol

CON-PI:

Synaptosomal membranes submitted to the pre-incubation necessary for the β-CD-mediated Cho extraction but in absence of β-CD

DPH:

1,6-diphenyl-1,3,5-hexatriene

CSM:

Center of spectral mass

DZ:

Diazepam

EPR:

Electron paramagnetic resonance

FNZ:

Flunitrazepam

I:

Immobile (protein bound) component in 12-SASL EPR spectra

K :

Compressibility modulus

K d :

Equilibrium dissociation constant

Lo:

Liquid-ordered phase

M:

Mobile component (partitioned in the lipid phase) in 12-SASL EPR spectra

MF:

Membrane-free

PI:

Pre-incubation

GABAA-R:

GABAA receptor

SEM:

Standard error of the mean

SM:

Synaptosomal membranes

12-SASL:

12-doxylstearic acid spin label

5-SASL:

5-doxylstearic acid spin label

So:

Solid-ordered phase

TMA-DPH:

1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate

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Acknowledgments

This study was partially financed by Grants from CONICET, SECyT-Universidad Nacional de Córdoba, Mincyt-Pcia.Córdoba and ANPCyT from Argentina and Fundacão de Amparo à Pesquisa do Estado de São Paulo, Brasil. A.V.T. and M.A.P. are Career Investigators from CONICET, Argentina. S.S. holds a research fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brasil.

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  1. IIBYT, CONICET - Biofísica-Química, Departamento de Química, Facultad de Ciencias Exactas, Físicas y Naturales, Universidad Nacional de Córdoba, Av. Vélez Sarsfield 1611, 5016, Córdoba, Argentina

    Anahí V. Turina & María A. Perillo

  2. Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes 748, São Paulo, 05508-900, Brazil

    Shirley Schreier

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  1. Anahí V. Turina
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Correspondence to María A. Perillo.

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Turina, A.V., Schreier, S. & Perillo, M.A. Coupling Between GABAA-R Ligand-Binding Activity and Membrane Organization in β-Cyclodextrin-Treated Synaptosomal Membranes from Bovine Brain Cortex: New Insights from EPR Experiments. Cell Biochem Biophys 63, 17–33 (2012). https://doi.org/10.1007/s12013-012-9338-1

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