Transmembrane proton gradient collapse

Figure 1 Facing page) (A) Mechanism of remote loading of doxorubicin by transmembrane ammonium sulfate gradient. (B) Collapse of transmembrane ammonium ion gradient in SSL by nonactine induces doxorubicin release. (C) Collapse of transmembrane proton gradient in SSL by nigericin induces collapse of transmembrane ammonium ion gradient followed by release of DOX. Abbreviations. DOX, doxorubicin SSL, sterically stabilized liposome.

Collapse of Liposomal Transmembrane Ammonium Ion and Proton Gradient and Release of Encapsulated DOX by lonophores... 

The peptide subunit was easily incorporated into lipid bilayers of liposome, as confirmed by absorption and fluorescence spectroscopy. Formation of H-bonded transmembrane channel structure was confirmed by FT IR measurement, which suggests the formation of a tight H-bond network in phosphatidylcholine liposomes. Liposomes were first prepared to make the inside pH 6.5 and the outside pH 5.5. Then the addition of the peptide to such liposomal suspensions caused a rapid collapse of the pH gradient. The proton transport activity was comparable to that of antibiotics gramicidin A and amphotericin B. 

The fact that uncouplers are lipophilic weak acids (see above) explains their ability to collapse transmembrane pH gradients. Their lipophilic character allows uncouplers to diffuse relatively freely through the phospholipid bilayer. Because they are weak acids, uncouplers can release a proton to the solution on one side of the membrane and then diffuse across the membrane to fetch another proton. The chemiosmotic theory thus provides a simple explanation of the effects of uncouplers on oxidative phosphorylation. 

HPTS is a pH-sensitive fluorophore (pk, 7.3) [6]. The opposite pH sensitivity of the two excitation maxima permits the ratiometric (i.e. unambiguous) detection of pH changes in double-channel fluorescence measurements. The activity of synthetic ion channels is determined in the HPTS assay by following the collapse of an applied pH gradient. In response to an external base pulse, a synthetic ion channel can accelerate intravesicular pH increase by facilitating either proton efflux or OH influx (Fig. 11.5c). These transmembrane charge translocations require compensation by either cation influx for proton efflux or anion efflux for OH influx, i.e. cation or anion antiport (Fig. 11.5a). Unidirectional ion parr movement is osmotically disfavored (i.e. OH /M or X /H symport). HPTS efflux is possible with pores only (compare Fig. 11.5b/c). Modified HPTS assays to detect endovesiculation (Fig. 11.1c) [16], artificial photosynthesis [17] and catalysis by pores [18] exist. 

---