Re-Introduction of Transmembrane Serine Residues Reduce the Minimum Pore Diameter of Channelrhodopsin-2

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.

Channelrhodopsin-2 Function is Modulated by Residual Hydrophobic Mismatch with the Surrounding Lipid Environment

“…This may be the first report describing the identification of particular residues and corresponding mutations central to the role of hydrophobic mismatch that was also followed by experimental validation for a multi-TM protein. Notably, our results provide a physical premise for previously observed effects on pore diameter by the V269S ChR2 mutation reported by us [32]. Analysis of our results highlights the importance of the lipid membrane in modulating the functional properties of ChR2 during optogenetic applications.…”

Characterization of a Highly Efficient Blue-shifted Channelrhodopsin from the Marine Alga Platymonas subcordiformis

“…S1), implicated in control of ion selectivity in CrChR2 (63)(64)(65), and Ser-136, which regulates the size of the channel pore (66). Of three positively charged residues that form a vestibule on the extracellular side of the channel pore in C1C2 (43), Lys-154 is substituted with Val in PsChR.…”

Transmembrane Domain Three Contributes to the Ion Conductance Pathway of Channelrhodopsin-2

“…Furthermore, we observe that although the permeability for all ions is reduced with the Q117E mutant (Table 3), the decrease in permeability is most dramatic for Na þ and K þ . Considering that it is well established that ions pass through ChR2 in a dehydrated state, we speculate that the extracellular pore entrance could be involved in cation dehydration before ion conductance and that small changes in H-bonding at the entrance of the pore have a variable effect on dehydrating cations as they are conducted by ChR2 (1,21). This suggests that H-bonding at the extracellular side of ChR2 is an important determinant of ion conductance where disruption of interhelical H-bonding results in a destabilization of helix-helix assembly and a subsequent decrease in ion permeability.…”

“…The cDNA of ChR2 (residues 1-309) with a hemagglutinin tag (YPYDVPDYA) on the C-terminus was subcloned into the vector pTLN as described (20)(21)(22). The ChR2 cysteine double replacement mutant (ChR2 C34A/C36A ) was created using the polymerase chain reaction-based QuikChange site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA) according to the manufacturer's instructions.…”