Transmembrane Serine Mutations reduce the Minimum Pore Diameter of Channelrhodopsin-2

Abstract

Channelrhodopsin-2 (ChR2) is a microbial-type rhodopsin that, together with channelrhodopsin-1, mediates phototactic behavior in the green algae Chlamydomonas reinhardtii. Like all other microbial-type rhodopsins, ChR2 has seven transmembrane domains with the chromophore all-trans retinal bound to a single lysine residue. However, unlike other microbial-type rhodopsins, ChR2 functions as a non-selective cation channel and not an ion pump. A sequence alignment of ChR2 with the proton pump bacteriorhodopsin (bR) reveals that ChR2 lacks specific motifs within the transmembrane domains that facilitate non-covalent interactions and contribute to protein stability. Specifically, there are eight TM serine residues, which have a high propensity for forming inter- and intrahelical hydrogen-bonds, present in bR that are absent in ChR2. We hypothesized that the re-introduction of serine residues at homologous positions in ChR2 would facilitate hydrogen-bonding, thus reducing the pore diameter of ChR2. We measured kinetics, reversal potentials, and calculated permeability ratios of alkali metal solutions using two-electrode voltage clamp. Applying excluded volume theory, we determined the minimum pore diameter of wild-type and mutant ChR2. Three mutants had either reduced functionality or low surface expression. Furthermore, four ChR2 mutants were determined to have smaller pore diameters than wild-type ChR2 while one mutant was similar to the phenotype, but had two-fold slower kinetics. Lastly, we replaced an endogenous serine residue with alanine and observed an increase in the pore diameter. An analysis of experimental results shows that re-introducing serine residues into the transmembrane domain of ChR2 can restrict the pore diameter while the removal of a transmembrane serine results in a larger pore. We suggest that the pore restriction is caused by the formation of an inter- or intrahelical hydrogen-bond and that multiple positions along the intracellular side of the transmembrane domains contribute to cation permeability.