Effect of neurosteroids on a model lipid bilayer including cholesterol: An Atomic Force Microscopy study

Abstract

Amphiphilic molecules which have a biological effect on specific membrane proteins, could also affect lipid bilayer properties possibly resulting in a modulation of the overall membrane behavior. In light of this consideration, it is important to study the possible effects of amphiphilic molecule of pharmacological interest on model systems which recapitulate some of the main properties of the biological plasma membranes. In this work we studied the effect of a neurosteroid, Allopregnanolone (3α,5α-tetrahydroprogesterone or Allo), on a model bilayer composed by the ternary lipid mixture DOPC/bSM/chol. We chose ternary mixtures which present, at room temperature, a phase coexistence of liquid ordered (Lo) and liquid disordered (Ld) domains and which reside near to a critical point. We found that Allo, which is able to strongly partition in the lipid bilayer, induces a marked increase in the bilayer area and modifies the relative proportion of the two phases favoring the Ld phase. We also found that the neurosteroid shifts the miscibility temperature to higher values in a way similarly to what happens when the cholesterol concentration is decreased. Interestingly, an isoform of Allo, isoAllopregnanolone (3β,5α-tetrahydroprogesterone or isoAllo), known to inhibit the effects of Allo on GABAA receptors, has an opposite effect on the bilayer properties.

Effects of neurosteroids on a model membrane including cholesterol: A micropipette aspiration study

“…This result could be related to vesicles with slightly different lipid compositions as it is typically found for GUVs produced by electroformation and including lipid mixtures. Analogously, our results by AFM [30] show that some lipid bilayer patches produced after vesicle fusion on a solid support, even if from small unilamellar vesicles (SUVs), present phase separation whereas other patches show a homogeneous phase. In the specific case of the presence of phase separation, AFM data show also that the relative proportion of L o and L d domains is broadly distributed in the different lipid patches.…”

“…The mechanical property variations which are reported in the literature when exogenous molecules interact with lipid bilayers similar to DOPC are typically obtained for much higher concentrations than the ones used in our work [44]. Considering the comparison between DOPC and DOPC/bSM/chol GUVs, we can speculate that the variation in the stretching constant for the latter type of GUVs could be due to the variation of the relative proportion of L d and L o domains, as suggested by our AFM investigation [30]. Indeed, it has been demonstrated that the L o phase has different mechanical properties compared to the L d phase [53].…”

“…If the relative proportion of the two phases changes, the overall mechanical properties of the vesicles should also vary. In this sense, the above cited AFM investigation on this model system established that Allo favors a relative increase of the L d phase [30]. Other possible explanations for the effect of Allo on the mechanical properties of DOPC/bSM/chol GUVs could be related to the effect of the molecule on the order inside the lipid bilayer.…”

“…At variance with Allo, we found that isoAllo produced a decrease of the lipid bilayer area and a negligible variation of the mechanical properties. The obtained results are also interpreted on the basis of what is obtained when a supported lipid bilayer of the same lipid composition is studied by atomic force microscopy (AFM) as a function of Allo and isoAllo concentrations to which it is exposed [30]. Finally, we discuss the effects of the neurosteroid on the lipid bilayer properties in light of a general mechanism of action of lipophilic molecules on biological membranes [18].…”

“…A similar result is obtained by epifluorescence microscopy (see Supporting information). Moreover, Allo uptake shifts the relative distribution in favor of the L d phase [30]. It is interesting to note that other studies found that the insertion of exogenous molecules in lipid bilayers in the L o + L d coexistence region leads to a modification of the relative proportions of the two liquid phases [37,38].…”