Coupling between GABAA-R ligand-binding activity and membrane organization in -cyclodextrin-treated synaptosomal membranes from bovine brain cortex. New insights from EPR experiments
Anahí V. Turina1, Shirley Schreier2, & María A. Perillo1*
1Biofísica-Química, Departamento de Química, Facultad de Ciencias Exactas, Físicas y Naturales. Universidad Nacional de Córdoba. Av. Vélez Sarsfield 1611, 5016 Córdoba, Argentina and 2Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brasil.
*Corresponding autor
SUPPLEMENTARY TEXT
1-Atomic force microscopy of Langmuir-Blodgett films supports the monolayer-form organization acquired after the spreading of synaptosomal membranes at the air-water interface.
2-Determination of the polarity- sensitive14N-isotropic hyperfine splitting constants
3-Determination of the DPH and TMA-DPH fluorescence anisotropy values in the presence of -CD.
SUPPLEMENTARY TEXT
1-Atomic force microscopy of Langmuir-Blodgett films supports the monolayer-form organization acquired after the spreading of synaptosomal membranes at the air-water interface.
It has been demonstrated that monolayers formed by the spreading of natural membranes at the air-water interface can be transferred to an alkylated glass to obtain a planar-supported, Langmuir-Blodgett (LB) film conserving the molecular organization of the original floating monolayer(see Rosetti et al, 2008).
We obtained LB films from SM spread at the air-water interface and transferred to alkylated glasses at a constant surface pressure of 35 mN/m (LB35) and analyzed them by atomic force microscopy (AFM). In the Fig. 1S, it can be observed that the surface pattern obtained with LB35 had a roughness significantly different from that of the Control (alkylated glass without monolayer). The mean height H=20 nm in LB35 was 15 nm higher than that of the Control (H=5 nm). Moreover, AFM images exhibited a regular coverage of the surface. We present these experiments in another paper in preparation.
Although the existence of some remaining intact vesicles could not be excluded(Clop and Perillo, 2010), results from AFM indicated that the material was transferred from a layer at the air-water interface and the evidence from the literature supports that these LB films reflect the situation present at the air-water interface.
2-Determination of the polarity- sensitive14N-isotropic hyperfine splitting constants
In order to confirm the partitioning of 5-SASL and 12-SASL inside -CD, the isotropic hyperfine splitting constant () evidencing the environmental polarity sensed by the spin probe (0.15 mM) was measure in the presence and in the absence of 40 mM -CD either by applying Eq.1S
(1S)
where A|| and A are the outer and inner extrema, respectively, measured in the 5-SASL spectra, or from the baseline-crossing points of the first-derivative EPR spectra (Hoffman et al., 2000).
was measured in conditions of high isotropic motion to ensure that the parameter reflect only the contribution of the environment polarity. In the presence of -CD, values of increased as a function of temperature until reaching a plateau (Fig. 2S). This reflected a temperature-dependent minimization of slow-motional effects. Hence, the value of was determined at the plateau region. In the absence of -CD, values were maximum and temperature independent, reflecting that water is an isotropic media which polarity is the highest as compared with -CD or membrane environments. -CD offered an environment less polar than bulk water but more polar than phospholipid bilayers. Furthermore, from the lower values it was concluded that, in -CD, similarly to what had been observed in phospholipid bilayers, the nitroxide moiety of 12-SASL was located more deeply than that of 5-SASL.
Determination of the DPH and TMA-DPH fluorescence anisotropy values in the presence of -CD.
To support the hypothesis that the probe immobilization inside β-CD trapped in the membrane might be contributing to the increase in ADPH and ATMA-DPH in CD-PI samples compared with CON, the ADPH and ATMA-DPH values in aqueous solution, in the absence or in the presence of -CD, were determined as described in the main text.
Steady state fluorescence anisotropy values of both DPH and TMA-DPH probes (4M) were calculated in the presence and in the absence of 40 M -CD, according to Eqs.2S and 3S.
(2S) (3S)
where IVV, IHH, IVH and IHVare the values of the different measurements of fluorescence intensity taken with both polarizers in the vertical (VV) or in the horizontal (HH) orientation or with the excitation polarizer vertical and the emission polarizer horizontal (VH) or vice-versa (HV). G is a correction factor for differences in sensitivity of the detection system for the vertically and the horizontally polarized light.
It was observed that the presence of -CD induced an increase in the A values (Table 1S), suggesting the complex formation between -CD and both of the probes tested. These results and data from NMR spectroscopy (Li and McGown, 1994) contributed to explain the increase in the anisotropy values of DPH and TMA-DPH in synaptosomal membranes submitted to the CD-PI treatment (see main text).
REFERENCES
Clop EM and Perillo MA.(2010). Langmuir Films from Human Placental Membranes: reparation, Rheology, Transfer to Alkylated Glasses, and Sigmoidal Kinetics of Alkaline Phosphatase in the Resultant Langmuir-Blodgett Film. Cell Biochem Biophys, 56, 91–107.
Hoffman P, Sandhoff K,Marsh D. (2000). Comparative dynamics and location of chain spin-labelled sphingomyelin and phosphatidylcholine in dimyristoyl phosphatidylcholine membranes studied by EPR spectroscopy. Biochim Biophys Acta, 1468, 359-366.
Rosetti CM, Maggio B, Oliveira RG. (2008). The self-organization of lipids and proteins of myelin at the membrane interface. Molecular factors underlying the microheterogeneity of domain segregation. Biochim Biophys Acta, 1778, 1665-75.
Li, G. and L.B. McGown, (1994) Molecular Nanotube Aggregates of beta- and gama-Cyclodextrins Linked by Diphenylhexatrienes. Science, 264, 249-251.
LEGENDS TO THE SUPPLEMENTARY FIGURES
Suppl. Fig. 1S. Atomic force microscopy of clean (Control) and monolayer covered alkylated (LB35)glasses. See details in the text.
Suppl. Fig. 2S. Determination of the polarity-sensitive14N-isotropic hyperfine splitting constants. EPR spectra of spin probes (0.15 mM) were recorded in the presence ( and ) and in the absence ( and ) of 40 mM -CD. ( and ), correspond to the same probes in lipid bilayers (taken from Hoffman et al., 1468, 359-366,2000).See details in the text.
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