Measuring Proton Depletion in the Vicinity of Proton Channels

Abstract

Proton channels have evolved to provide a pH regulatory mechanism, affording the extrusion of protons from the cytoplasm at all membrane potentials. Previous evidence has suggested that channel-mediated acid extrusion could significantly change the local concentration of protons in the vicinity of the channel. In this experiments we directly measure the proton depletion caused by activation of Hv1 proton channels, using patch-clamp fluorometry recordings from channels labeled with the Venus fluorescent protein at intracellular domains. The fluorescence of the Venus protein is very sensitive to pH, thus behaving as a genetically encoded sensor of local pH. Eliciting outward proton currents increases the fluorescence intensity of Venus. This dequenching is related to the magnitude of the current and not to channel gating and is dependent on the pH gradient. Increasing or decreasing the distance of Venus to the membrane or placing the fluorescent protein at the amino or carboxy terminus did no produce different fluorescence behavior, indicating that the depletion microdomain is larger than 90 A, which is an estimate of the distance of the Venus protein from the inner face of the membrane. Changing the pH-dependent properties of Venus through mutations allows tuning of the pH sensitivity of the Venus-Hv1 construct. Our results provide direct evidence of local proton depletion due to flux through the proton-selective channel.