Chloride Transport Across Planar Lipid Bilayers and Cell Membranes by Steriod-Based Synthetic Anion Transporters

Abstract

Cholapods are steroid-based synthetic anion transporters derived from cholic acid that bind anions with high affinity and promote their efflux from liposomes. To understand better cholapod-mediated anion transport, we studied the cholapods AS09, LJ09 and TL145 in planar lipid bilayers, single cells and polarised epithelia. When compared with AS09, LJ09 has higher anion affinity, whereas TL145 has greater lipid solubility. In planar lipid bilayers, cholapod-mediated currents relaxed to a steady-state after an initial peak when membrane voltage was stepped. Anion transport by cholapods was concentration-dependent with anion conductance decreasing with increasing anionic radius. Evaluation of the rate constants for anion transport by cholapods using the method of Lauger (Science 1972;178:24-30) suggested that movement of the anion-transporter complex across the membrane is the rate-limiting step of cholapod-mediated anion transport. To investigate cholapod-mediated anion transport in single cells, we used Fischer rat thyroid epithelial cells stably expressing the halide-sensitive yellow fluorescent protein (FRT-YFP) (gift from A Verkman, UCSF) to monitor anion influx. In the absence of cholapod, addition of iodide (10 mM) to FRT-YFP cells had little or no effect on fluorescence. However, in the presence of cholapod, YFP fluorescence decreased following iodide influx into FRT-YFP cells and when iodide was washed from the external solution fluorescence quenching reversed. Finally, to evaluate cholapod-mediated transport at the tissue level, we grew FRT-YFP cells as polarised epithelia and measured transepithelial Cl- movement in Ussing chambers. Treatment of FRT-YFP epithelia with cholapods led to the development of transepithelial Cl- current that was absent in untreated epithelia. Because TL145 was the most effective transporter in all assays, we conclude that lipid solubility is an important determinant of transmembrane anion transport by the cholapod family of synthetic anion transporters. [Supported by the EPSRC].