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PLoS One
2012 Jan 01;711:e50018. doi: 10.1371/journal.pone.0050018.
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Re-introduction of transmembrane serine residues reduce the minimum pore diameter of channelrhodopsin-2.
Richards R
,
Dempski RE
.
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.
Figure 2. Representative ChR2 photocurrent traces recorded at â120 mV.Black bars indicate illumination with 473 nm light. (A) Normalized photocurrent traces. Mutant photocurrents were normalized to WT ChR2 on the same day in 115 mM Na+ solution at pH 9. Values are reported as mean±S.E.M (nâ=â3â9). Statistically significant values are denoted by a *(*P<0.001; **P<0.04). (B) Reduced photocurrent for I197S compared to WT ChR2 on the same day. M255S and P234S had similar results. (C) Western blot of I197S, P234S, and M255S ChR2 mutants. ChR2-HA tagged band appears at â¼35 kDa. (D) Typical electrophysiological recordings for single serine mutants. Mutants retained inward rectification of cations, but with altered permeability, kinetics, and/or inactivation ratios.
Figure 3. Shifts in reversal potential and permeability ratio comparison of single serine ChR2 constructs.Changes in reversal potential were calculated by subtracting Erev in cation X+ from Erev in Na+ (mean±S.E.M; nâ=â7â25). Permeability ratios were calculated using changes in reversal potential. Values are reported as mean±S.E.M (nâ=â7â25). For significance testing, mutant ChR2 values were compared to WT in the same solution. Statistically significant values are denoted by a *(P<0.001).
Figure 4. Kinetic parameters for single serine ChR2 constructs.Kinetic values were obtained by fitting ChR2 photocurrents to a standard biexponential equation. All parameters were calculated in 115 mM Na+ solution at pH 9. For on and decay times, traces were fit at â120 mV to ensure ChR2 was in the dark adapted state. Values reported as mean±S.E.M (nâ=â8â23). Significant differences are marked with a *(P<0.05). (A) Summary of photocurrent exponential fits. On time was calculated from baseline current to peak current, decay time was calculated from peak to stationary current, and off time was calculated from stationary to baseline current when the light is switched off. (B) Off rate comparison of the slow G224S mutant versus WT at different membrane potentials. The off rate of ChR2 had a strong dependence on the holding potential of the cell. Diamond, WT; crossed X, G224S. All values were statistically significant (as described in C) (C) Summary of kinetic parameters and inactivation ratios for single serine ChR2 mutants. Values are reported as mean±S.E.M (nâ=â8â23). Statistically significant values are denoted by a *(P<0.05).
Figure 5. Steady-state to peak current ratios for WT and G181S ChR2.Ratios were calculated after normalization to â1 µA at â120 mV in cationic solutions (115 mM XCl, 2 mM BaCl2, 1 mM MgCl2, Tris; pH 9; nâ=â14â18). Significant differences are marked with a *(P<0.05). (A) Comparison of WT and G181S photocurrents in Na+ solution. (B) Iss/Ip comparison for WT and G181S in different cationic solutions. Wild-type ChR2 is colored white while G181S is colored black.
Figure 6. Relationship of relative permeability and alkali ionic radii for ChR2 serine mutations.Relative permeability ratios were calculated using reversal potentials for each mutant. (A) Plotting the square root of relative permeability ratios vs. ionic radius yielded a linear fit for each data set. Solid line/diamond, WT; dashed line/square, V269S; broken line/X S136A. For all mutants, the larger alkali metal ions (K+, Rb+, and Cs+) were less permeable than the smaller alkali metal ions (Li+, Na+). These results were consistent with excluded volume theory [36]. (B) Minimum pore fitting parameters and pore size calcualtions for single serine ChR2 mutants and S136A. The coefficients a and b correspond to the y-intercept and slope of the fitted data, respectively.
Figure 1. Simplified photocycle and ChR2 single serine mutation model.(A) Simple four-state model of the channelrhodopsin-2 photocycle. The two nonconducting states consist of a dark-adapted (C1) and desensitized (C2) state. The conducting states represent an early conducting state (O1) and a late conducting state (O2). Blue arrows represent illumination with blue light. (B) Structural model of ChR2 based on the channelrhodopsin chimera C1C2 (PDB entry: 3UG9) highlighting the locations of serine mutations. Three residues (black) had highly reduced photocurrents, while 5 mutations showed changes in pore size, permeability, and/or kinetics (orange). Residue S136 is shown in purple. The retinal chromophore, which is covalently bound to K257, is shown in yellow. This figure was prepared using Visual Molecular Dynamics [37]. (C) Sequence comparison of transmembrane domains between bacteriorhodopsin and channelrhodopsin-2. Residues that correspond to serine mutations are highlighted in orange. Adapted from [9].
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