## 4 Discussion
The measured reversal potentials for this mutant indicate severely reduced progressive proton selectivity, lower inactivation and accelerated kinetics. Previously, we had observed a similar effect at this position with the V269S mutant as this mutation caused a reduction in channel permeability and pore size [32]. Interestingly, TM7 undergoes reorientation and movement upon photoisomerization [59] and recent MD simulations comparing all-trans retinal and 13-cis retinal bound C1C2 structures revealed outward cytoplasmic bending of TM7 [59]. This is a common movement in microbial-rhodopsins that leads to ion transport [60; 61]. Previously, we had attributed the reduction in pore size and function of the V269S mutation to a water-mediated hydrogen bond network that prevents large movements of TM7. However, our experimental results here indicate the importance of lowering RHM at this position and the observed effects on channel permeability for both V269S and V269N in addition to the reduction in pore diameter observed for V269S. This suggests that lowering RHM of ground state ChR2 leads to an unfavorable positioning of TM7 in the open state which restricts the minimum pore diameter. This is further supported by analysis of our kinetic modeling of V269N, which exhibits destabilization between O1 and O2 compared to WT [V269N R\({}_{\text{f}}\) = 5.08; WT R\({}_{\text{f}}\) = 6.67 where R\({}_{\text{f}}\) is the reaction coefficient (e12/e21)] and reduced maximal current. We propose that once the pore is formed, V269N becomes exposed to an unfavorable environment or allows for further water penetration exposing proximal hydrophobic residues resulting in the loss of function. This correlates with the conformational movement of TM7 during pore formation and the influx in water prior to ion conductance [15; 16; 62]. Functionally similar was the effect of Q210 mutation to alanine. Our computational results showed a large reduction in the RHM energy penalty with Q210A. This large reduction is a result of positioning the hydrophobic alanine residue in a favorable environment and also a consequence of the elimination of the Q210 mismatch which decreases the water penetration to the neighboring V269, thereby further lowering the RHM. As functional correlates, we observed a reduction in photocurrent, lower inactivation and slightly accelerated decay kinetics. Although there was no change in selectivity under our experimental conditions, this is unsurprising as TM4 has not been shown to contribute to the permeation pathway. Notably, Q210 is also highly conserved among channelrhodopsins and has a clear structural impact on ChR2 function as suggested by previous serine substitution [32]. The kinetic parameters also indicated a similar destabilization of the open state preference to O2 and significantly reduced quantum efficiency of the C2 to O2 transition as in V269N. To test whether or not our results were a direct consequence of RHM, we created an expected RHM null mutant at this position (Q210N), which we expected to have limited functional impact. Indeed, we found that reversal potentials and kinetics of Q210N are similar to those of WT ChR2. While Q210N did have increased photocurrent, the permeability and kinetics remained similar to WT. Therefore, the increased photocurrent of Q210N is likely not related to RHM. Our modeling also revealed limited impact on photocycle kinetic parameters for this mutant. It remains unclear how decreasing RHM at Q210 affects structural rearrangements of TM4 as little is known about the movement of this TM. However, because mutation of Q210 to alanine also changes the environment at V269 on TM7, it is possible that there is a secondary effect sensed through TM7, which is critical for channel activation. Our results have documented the clear and quantitatively substantiated effects of residual hydrophobic mismatch at the cytoplasmic interface in ChR2 on channel function.