Encoding the Light-Sensitivity of Channelrhodopsin-2

Abstract

Microbial rhodopsins are routinely used as light-controlled switches in neurobiology. Their versatile applicability relies on the simplicity of the optogenetic approach. Light-sensitivity is promoted to the host cell encoded in genetic information. However, the signal output is limited by the expression level and the molecular properties of the rhodopsins. The light-sensitivity of the system can set a limit for its usage. Different strategies might be envisaged how to tune and increase the responsiveness on a molecular level in the case of channelrhodopsin-2 (ChR2), a light-gated cation channel from Chlamydomonas reinhardtii: 1) increased absorption cross-section and quantum efficiency, 2) increased single channel conductance, 3) increased lifetime of the open state and 4) an amplification system. Here, we follow up the different strategies in a combined biophysical and neurobiological approach. As a first step we have developed the tools to study the different properties to have experimental access to the molecular properties from a spectroscopic and electrophysiological side. Especially, the development of fusing different rhodopsins into a single entity allows the discrimination between effects on the expression level and an increased single channel current by using one of the rhodopsins as a molecular ruler. In a next step we looked into the light-induced dynamical changes that accompany the photocycle of ChR2. This strategy allows us to map the conformational changes connected to the open state of the channel. We further generated a more calcium permeable mutant (CatCh, L132C) whose action on tuning the light sensitivity is different. Here, we propose a model that a local calcium concentration increase at the cytoplasmic membrane screens negative surface charges leading to a higher probability of opening the voltage-dependent sodium channels responsible for the onset of action potentials in neurons.