Arbuscular mycorrhiza and the rhizobia-legume symbiosis are two major root endosymbioses that facilitate plant nutrition. In Lotus japonicus, two symbiotic cation channels, CASTOR and POLLUX, are indispensable for the induction of nuclear calcium spiking, one of the earliest plant responses to symbiotic partner recognition. During recent evolution, a single amino acid substitution in DOES NOT MAKE INFECTIONS1 (DMI1), the POLLUX putative ortholog in the closely related Medicago truncatula, rendered the channel solo sufficient for symbiosis; castor, pollux, and castor pollux double mutants of L. japonicus were rescued by DMI1 alone, while both Lj-CASTOR and Lj-POLLUX were required for rescuing a dmi1 mutant of M. truncatula. Experimental replacement of the critical serine by an alanine in the selectivity filter of Lj-POLLUX conferred a symbiotic performance indistinguishable from DMI1. Electrophysiological characterization of DMI1 and Lj-CASTOR (wild-type and mutants) by planar lipid bilayer experiments combined with calcium imaging in Human Embryonic Kidney-293 cells expressing DMI1 (the wild type and mutants) suggest that the serine-to-alanine substitution conferred reduced conductance with a long open state to DMI1 and improved its efficiency in mediating calcium oscillations. We propose that this single amino acid replacement in the selectivity filter made DMI1 solo sufficient for symbiosis, thus explaining the selective advantage of this allele at the mechanistic level.