Voltage activation of reconstituted sodium channels: use of bacteriorhodopsin as a light-driven current source

We present a general method for the development and control of transmembrane potentials (delta psi) in reconstituted vesicles. The light-driven proton pump bacteriorhodopsin (bR) from Halobacterium halobium is the current source in the system, and the intensity of light controls the magnitude of delta psi at any given time. Transmembrane potentials were determined from the equilibrium distribution of hydrophobic ions, which was monitored by using either electron paramagnetic resonance spectroscopy and spin-labeled phosphonium ions or a tetraphenylphosphonium-selective electrode. A bias or holding potential was generated by using gradients of anions of limited permeability in the presence of an impermeable cation. Using n-methylglucamine or the polymer poly(ethylene imide) as the impermeable cation, the anions NO3- and SCN- were most effective in producing large (> -60 mV) negative diffusion potentials in egg phosphatidylcholine vesicles. In the presence of a NO(3-)-based negative holding potential (approx. -65 mV), bR is capable of depolarizing the membrane to at least the 0-mV level within a few seconds. More rapid depolarizations can be achieved by the application of a brief intense illumination preceding the preset illumination level (supercharging). The technique was successfully used to activate for the first time a population of unmodified voltage-dependent sodium channels purified from eel electroplax.