Vectorial Ion Transport by Channelrhodopsin-2

Abstract

Channelrhodopsins represent a third member of microbial-type rhodopsins and have gained considerable attention in neurobiology as a tool to control the excitability of neurons. The other photoreceptors act either as light-driven ion pumps like bacteriorhodopsin (BR) or as part of a relay system like the sensory rhodopsins (SR). In contrast, channelrhodopsins are light-gated ion channels that allow the passive permeation of cations over the membrane barrier after light activation. Previously, we could follow the spectral characteristics of different photointermediates and relate them to the functional states of the channel. In the gating process, the Schiff-base undergoes a deprotonation reaction before it relaxes into a red-shifted species. The red-shifted photointermediate is characteristic for the open state with a lifetime of 10 ms.So far, the role of the protonation reactions is not fully understood. Channelrhodopsins possess a glutamate residue at the homologue position of the proton acceptor D85 in BR. The main difference among the microbial-type rhodopsins is found in the nature of the proton donor at the opposite side of the retinal moiety towards the cytoplasm: In BR, an aspartic acid reprotonates the Schiff-base in a fast manner as expected for an efficient proton pump, while in SR an aromatic residue leads to a long-lived deprotonated state of the Schiff-base. In channelrhodopsins, the homologue residue is a histidine that could allow a reprotonation of the observed deprotonated Schiff-base from the cytoplasmic side. As a net result, one proton would be transported per photocycle. Here we show that one can indeed observe such a vectorial ion translocation under illumination of purified and proteoliposome reconstituted channelrhodopsin-2. Therefore, the mechanism of channelrhodopsin-2 shows similar features as other rhodopsins, i.e. control of the accessibility to the Schiff-base by light-induced isomerization of the retinal and vectorial ion transport.