Membrane Binding of Hydrophobic Ions: Application of New Kinetic Techniques

Understanding membrane transport processes such as ion occlusion reactions of ion pumps and transporters and the ion gating of channels requires knowledge of lipid bilayer electrostatics. A simple example of the effect of membrane electrostatics on ion transport is the much higher permeability of the membrane to hydrophobic anions, such as tetraphenylborate (TPB-), compared to hydrophobic cations, such as tetraphenylphosphonium (TPP+) or tetraphenylarsonium (TPA+). This has been attributed to the membrane dipole potential, of which a major contributor has been determined to be oriented water dipoles in the lipid headgroup region of the membrane. From the ratio of the TPB- to TPP+ or TPA+ conductances, the magnitude and polarity of the dipole potential can be estimated. Using the voltage-sensitive dye RH421 in conjunction with the stopped-flow technique and solid-supported membrane electrophysiology here we show that the transport of these ions is not simply a diffusion through the membrane but rather occurs in jumps between discrete binding sites within the membrane. The hydrophobic anion TPB- causes much greater RH421 spectral changes than TPA+. This could be explained by a combination of a stronger interaction of TPB- with RH421 and a deeper binding of TPB- within the membrane compared to TPA+. The experimental methods, used here for the first time to study the kinetics of ion transport across membranes, are potentially applicable to investigations of the membrane permeability of charged drug molecules, in particular anticancer agents.