FIGURE 2.
Lack of exchange between vesicles and bilayer lipids and bulk effect of DOTAP on DPhPC vesicles. A, lipids from the bilayer do not over time mix with lipids from the vesicles. DPhPC and DOTAP vesicles containing KvAP fused to a DOTAP:DPhPC, 2:1, bilayer (left) and to a DPhPC bilayer (right), respectively, are incubated for 15 min (black) or 1 h (red) at a holding potential of −100 mV. In both cases, no significant change of the GV was observed. B, increase of the temperature does not promote mixing between the lipids of the bilayer and the vesicles. DPhPC and DOTAP vesicles containing KvAP were fused to a DOTAP:DPhPC, 2:1, bilayer (left) and a DPhPC bilayer, respectively (right). The bilayers were kept at room temperature (black) or between 35 and 40 °C (red). No significant changes indicated lipid mixing between DOTAP and DPhPC at higher temperature. V½ and VS values are presented in Table 2 (n = 2–4). C, the voltage dependence of KvAP reconstituted in DPhPC vesicles was shallower when fused to a DOTAP:DPhPC, 2:1, bilayer (red) than to a DPhPC bilayer (black). Current traces show one normalized pulse at a saturating voltage (+60 mV) and one less depolarizing pulse where the difference of steepness of the GV curves creates a difference in the open probability (−60 mV, dotted lines). The data are fit with a single Boltzmann function (solid lines, Table 1), n = 3 and 7, error bars are S.D.