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(1) Background: After the discovery and application of <i>Chlamydomonas reinhardtii</i> channelrhodopsins, the optogenetic toolbox has been greatly expanded with engineered and newly discovered natural channelrhodopsins. However, channelrhodopsins of higher Ca²⁺ conductance or more specific ion permeability are in demand. (2) Methods: In this study, we mutated the conserved aspartate of the transmembrane helix 4 (TM4) within Chronos and <i>Ps</i>ChR and compared them with published ChR2 aspartate mutants. (3) Results: We found that the ChR2 D156H mutant (XXM) showed enhanced Na⁺ and Ca²⁺ conductance, which was not noticed before, while the D156C mutation (XXL) influenced the Na⁺ and Ca²⁺ conductance only slightly. The aspartate to histidine and cysteine mutations of Chronos and <i>Ps</i>ChR also influenced their photocurrent, ion permeability, kinetics, and light sensitivity. Most interestingly, <i>Ps</i>ChR D139H showed a much-improved photocurrent, compared to wild type, and even higher Na⁺ selectivity to H⁺ than XXM. <i>Ps</i>ChR D139H also showed a strongly enhanced Ca²⁺ conductance, more than two-fold that of the CatCh. (4) Conclusions: We found that mutating the aspartate of the TM4 influences the ion selectivity of channelrhodopsins. With the large photocurrent and enhanced Na⁺ selectivity and Ca²⁺ conductance, XXM and <i>Ps</i>ChR D139H are promising powerful optogenetic tools, especially for Ca²⁺ manipulation.