Abstract
Channelrhodopsin-2 (ChR2) is a light-activated, non-selective cation channel endogenous to the green algae Chlamydomonas reinhardtii. The unique properties of ChR2 have made it a useful tool in the field of optogenetics. However, the mechanism of ion conductance is not well resolved. Elucidation of the crystal structure of the channelrhodopsin chimera C1C2 has provided structural insight on the putative ChR2 ion conductance pathway. However, it is not clear how the chimeric structure correlates to ChR2 function. To directly examine the permeation pathway of ChR2, we have used a combination of molecular dynamics and two electrode voltage clamp. For our computational experiments, we modeled ChR2 using the C1C2 crystal structure as a template and performed steered molecular dynamics to pull a sodium ion through the proposed conductance pathway. Our results indicate that several binding sites are available for sodium passing through the channel. In addition, we used cysteine scanning mutagenesis and subsequent site-specific labeling to elucidate the permeation pathway of ChR2. Combined, analysis of our experimental results provides new insight into the mechanism of cation conductance by channelrhodopsin-2.
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