Abstract
Channelrhodopsin-2 (ChR2) is a microbial-type rhodopsin found in the green algae Chlamydomonas reinhardtii. Under physiological conditions, ChR2 is an inwardly rectifying cation channel that permeates a wide range of mono- and divalent cations. Although this protein shares a high sequence homology with other microbial-type rhodopsins, which are ion pumps, ChR2 is an ion channel. A sequence alignment of ChR2 with bacteriorhodopsin, a proton pump, reveals that ChR2 lacks specific motifs and residues, such as serine and threonine, known to contribute to non-covalent interactions within transmembrane domains. We hypothesized that reintroduction of the eight transmembrane serine residues present in bacteriorhodopsin, but not in ChR2, will restrict the conformational flexibility and reduce the pore diameter of ChR2. In this work, eight single serine mutations were created at homologous positions in ChR2. Additionally, an endogenous transmembrane serine was replaced with alanine. We measured kinetics, changes in reversal potential, and permeability ratios in different alkali metal solutions using two-electrode voltage clamp. Applying excluded volume theory, we calculated the minimum pore diameter of ChR2 constructs. An analysis of the results from our experiments show that reintroducing serine residues into the transmembrane domain of ChR2 can restrict the minimum pore diameter through inter- and intrahelical hydrogen bonds while the removal of a transmembrane serine results in a larger pore diameter. Therefore, multiple positions along the intracellular side of the transmembrane domains contribute to the cation permeability of ChR2.
Highlights
Channelrhodopsin-2 (ChR2) is a light-activated cation channel found in the eyespot region of the green algae Chlamydomonas reinhardtii [1]
An analysis of our experiments demonstrates that the size selectivity filter for ion conductance is not located at a single region within the transmembrane domains
Mutations were located in various areas of the transmembrane domain; C87S, G181S, G224S, and M255S were located near the retinal binding pocket, I197S, Q210S, and V269S were located at the cytoplasmic end of the transmembrane domain, and P234S was located at the extracellular end of the transmembrane domain (Figure 1B)
Summary
Channelrhodopsin-2 (ChR2) is a light-activated cation channel found in the eyespot region of the green algae Chlamydomonas reinhardtii [1]. Unlike other rhodopsins, ChR2 is able to conduct a large array of mono- and divalent cations with a high selectivity for protons [4]. Large cations such as guanidinium (ionic radius ,2.4 A ) [10] and dimethyl-amine (ionic radius ,2.6 A ) [10] can pass through the channel which suggests a large pore diameter [4,11]. ChR2 current decays from an initial peak current to stationary current. The stationary current decays biexponentially to a desensitized closed state once the light is turned off [4,12,13]
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