Korean J Physiol Pharmacol.  2012 Aug;16(4):255-264. 10.4196/kjpp.2012.16.4.255.

The Effect of Methanol on the Structural Parameters of Neuronal Membrane Lipid Bilayers

• Joo HJ
• Ahn SH
• Lee HR
• Jung SW
• Choi CW
• Kim MS
• Bae MK
• Chung IK
• Bae SK
• Jang HO
• Yun I

• KMID: 2011160
• DOI: http://doi.org/10.4196/kjpp.2012.16.4.255

Abstract

The structures of the intact synaptosomal plasma membrane vesicles (SPMVs) isolated from bovine cerebral cortexs, and the outer and the inner monolayer separately, were evaluated with 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1,3-di(1-pyrenyl)propane (Py-3-Py) as fluorescent reporters and trinitrophenyl groups as quenching agents. The methanol increased bulk rotational and lateral mobilities of SPMVs lipid bilayers. The methanol increased the rotational and lateral mobilities of the outer monolayers more than of the inner monolayers. n-(9-Anthroyloxy)stearic acid (n-AS) were used to evaluate the effect of the methanol on the rotational mobility at the 16, 12, 9, 6, and 2 position of aliphatic chains present in phospholipids of the SPMVs outer monolayers. The methanol decreased the anisotropy of the 16-(9-anthroyloxy)palmitic acid (16-AP), 12-(9-anthroyloxy)stearic acid (12-AS), 9-(9-anthroyloxy)stearic acid (9-AS), and 6-(9-anthroyloxy)stearic acid (6-AS) in the SPMVs outer monolayer but it increased the anisotropy of 2-(9-anthroyloxy)stearic acid (2-AS) in the monolayers. The magnitude of the increased rotational mobility by the methanol was in the order at the position of 16, 12, 9, and 6 of aliphatic chains in phospholipids of the outer monolayers. Furthermore, the methanol increased annular lipid fluidity and also caused membrane proteins to cluster. The important finding is that was far greater increase by methanol in annular lipid fluidity than increase in lateral and rotational mobilities by the methanol. Methanol alters the stereo or dynamics of the proteins in the lipid bilayers by combining with lipids, especially with the annular lipids. In conclusion, the present data suggest that methanol, in additions to its direct interaction with proteins, concurrently interacts with membrane lipids, fluidizing the membrane, and thus inducing conformational changes of proteins known to be intimately associated with membranes lipids.

Keyword

Annular lipid fluidity; Membrane protein clustering; Methanol; Neuronal membranes; Transbilayer lateral and rotational mobility

MeSH Terms

Anisotropy
Cell Membrane
Cerebral Cortex
Diphenylhexatriene
Lipid Bilayers
Membrane Lipids
Membrane Proteins
Membranes
Methanol
Neurons
Palmitic Acids
Phospholipids
Proteins
Stearic Acids
Diphenylhexatriene
Lipid Bilayers
Membrane Lipids
Membrane Proteins
Methanol
Palmitic Acids
Phospholipids
Proteins
Stearic Acids

Figure

• 

  Fig. 1 The effect of methanol on excimer to monomer fluorescence intensity ratio (I'/I) of Py-3-Py in SPMVs. The excitation wavelength of Py-3-Py was 330 nm and the I'/I values were calculated from the 480 nm to 379 nm signal ratio. SPMVs was treated ±4 mM TNBS, pH 8.5, at 4℃ for 80 min. Py-3-Py was incorporated into SPMVs and fluorescence measurements were performed at 37℃ (pH 7.4). Untreated (inner and outer monolayers, ▪); TNBS treated (inner monolayer, •); calculated for outer monolayer (▴) by eq. 3 as described in Materials and Methods. Each point represents the mean±SEM of 5 determinations. An asterisk and double asterisks signify p<0.05 and p<0.01, respectively, compared to control by Student's t-test.

• 

  Fig. 2 The effect of methanol on annular lipid fluidity in SPMVs. Py-3-Py was excited through RET from tryptophan (excitation wavelength, 286 nm) and the excimer to monomer fluorescence intensity ratio (I'/I) was calculated from the 480 nm to 379 nm signal ratio. Fluorescence measurements were performed at 37℃ (pH 7.4). Each point represents the mean±SEM of 5 determinations. An asterisk and double asterisks signify p<0.05 and p<0.01, respectively, compared to control by Student's t-test.

• 

  Fig. 3 The effect of methanol on protein clustering in SPMVs. Efficiency of RET from tryptophan to Py-3-Py was taken as a measure of protein clustering and calculated by eq. 4. Fluorescence measurements were performed at 37℃ (pH 7.4). Each point represents the mean±SEM of 5 determinations. An asterisk and double asterisks signify p<0.05 and p<0.01, respectively, compared to control by Student's t-test.

• 

  Fig. 4 The effect of methanol on the anisotropy (r) of DPH in SPMVs. SPMVs were treated ±2 mM TNBS, pH 8.5, at 4℃ for 40 min. DPH was incorporated and fluorescence measurements were performed at 37℃ (pH 7.4). Values from untreated membranes represent inner+outer monolayer; Untreated (inner and outer monolayers, ▪); TNBS treated (inner monolayer, •); calculated for outer monolayer (▴) by eq. 7 as described in Materials and Methods. Each point represents the mean±SEM of 5 determinations. An asterisk and double asterisks signify p<0.05 and p<0.01, respectively, compared to control by Student's t-test.

• 

  Fig. 5 Effects of methanol on the anisotropy (r) of 16-(9-anthroyloxy) palmitic acid (16-AP), 12-(9-anthroyloxy)stearic acid (12-AS), 9-(9-anthroyloxy)stearic acid (9-AS), 6-(9-anthroyloxy)stearic acid (6-AS) and 2-(9-anthroyloxy)stearic acid (2-AS) in SPMVs. Fluorescence measurements were performed at 37℃ (pH 7.4). Each point represents the mean±SEM of 5 sample determinations. The asterisk and double asterisk denote p<0.05 and p<0.01, respectively, compared to the control according to a Student's t-test.

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