Membranes with Charged Lipids

As discussed in Section 7.2, strong electric pulses applied to single-component giant vesicles made of zwitterionic lipids like PCs induce the formahon of pores, which reseal within tens of milliseconds. When negatively charged lipids, like phosphatidylglycerol (PC) or phosphatidylserine, are present in a membrane a very different response of the vesicles can be observed, partially influenced by the medium conditions [142]. 

In this sechon some applicahon aspects of giant vesicle electroporation are considered. In parhcular, it will be demonstrated that creahng macropores in GUVs and observing their closing dynamics can be successfully apphed to the evaluation of material properties of membranes. While in Section 7.4.2 we saw that such experiments can be used to characterize membrane stability in terms of the crihcal porahon potenhal f c, here we will find out how one can also evaluate the edge tension of porated membranes. In addition, another apphcation based on electro-porahon, namely vesicle electrofusion, is introduced whereby the use of GUVs as microreactors suitable for the synthesis of nanoparhcles is demonstrated. 

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Early investigations of peptides in membrane model systems included studies of mel-letin 124,125 220 221 spectra and polarization properties. This water-soluble peptide is found to be structureless in solution at neutral pH but was sensitive to environmental change. The undecapeptide hormone, substance P, a member of the tackykinin family, was also found by Choo et a].1222 to be unstructured in solution at physiological pH and to aggregate at high pH or on interaction with charged lipids. These data were used as counter-evidence to a hypothesis that the membrane surface structured the peptide to facilitate interaction with the receptor. 

Conveniently, one monitors the increase of the fluorescence intensity of an encapsulated self-quenched dye, eg 50 mM 6-carboxyfluorescein (6-CF) (160) or calcein (161), as it permeates the vesicles. pHJGlucose is more generally applicable because it does not interact with charged lipid membrane surfaces and can be used at low ionic strength. Alternatively, a dilute solution of 6-CF (10 /am) is encapsulated and Co + (500 /am) is added to the external solution which quenches the fluorescence. Permeation of either Co + ions or the dye across the membranes causes a reduction in fluorescence intensity. 

FIGURE 6-15 Schematic representation of the ion permeability modulation for cation-responsive voltammetric sensors based on negatively charged lipid membranes. Complexation of the guest cation to the phospholipid receptors causes an increase of the permeability for the anionic marker ion. (Reproduced with permission from reference 49.)... 

Figure 3.6 compares iso-pH permeabilities of ketoprofen at various pH values in a 2% DOPC-dodecane model (open circles) and the 20% soy lecithin with SLS in the acceptor compartment (filled circles, data in Table 3.5). In the presence of the latter negatively charged lipids (with the make-up similar to that of BBM in Table 3.1), ketoprofen is intrinsically more permeable, by a factor of 17. The UWL limit, indicated by the solid curves in low-pH solutions, and consistent with the permeability Pu 19.8 x 10-6 cm s 1 (log Pu —4.7), masks the true intrinsic permeability of the membranes, P0. However, it is possible to deduce the membrane permeability if the pKa is known. In Fig. 3.6, the bending in the dashed (calculated) curves at pH 4 corresponds to the pKa of the molecule. Due to the UWL, the point of bending is shifted to higher pH values in the solid (measured) curves. The difference between the apparent pKa (pK 5.3 for DOPC and 6.3 for soy) and the true pKa (4.12) is the same as the difference between log P0 and log Pu [23],... 

P-gp substrates are in general either neutral or cationic at physiological pH (weak bases). Weak bases can cross the lipid membrane in the uncharged form and reprotonate in the negatively charged cytosolic leaflet of the membrane. With a few exceptions (e.g., the tetraphenyl phosphonium ion, which can reach the cytosolic membrane leaflet due to charge delocalization [70]), permanently charged cations do not cross the cell membrane and therefore cannot interact with P-gp in intact cells. They can, however, insert into the cytosolic leaflet in inside-out cellular vesicles and are then transported by P-gp [42, 71]. 

The organization of lipids around the plasma membrane Ca2+-transport ATPase of erythrocytes has been also determined by FRET. Taking advantage of the intrinsic fluorescence of the ATPase due to tryptophan residues and labeling different types of lipids with pyrene, it was demonstrated that the transporter is preferentially surrounded by negatively charged lipids such as phosphoinositides [167],... 

As detailed in chapter 17, biological membranes are basically lipid—think fat or oil—in nature with some attached proteins. As such, these thin sheets of phospholipids and proteins are nearly impermeable to charged particles such as sodium, potassium, or chloride ions. While the isolation of the cell interior from the exterior ionic environment is critical in many ways, it is also true that controlled permeability to ions may be critical. In fact, it is the near-impermeability of biological membranes to ions that permits control of ion transport across them by certain, specific proteins. 

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