Voltage-dependent Ca2+ channels (Cavs) are indispensable for coupling action potentials with Ca2+ signaling in living organisms. The structure of Cavs is similar to that of voltage-dependent Na+ channels (Navs). It is known that prokaryotic Navs can obtain Ca2+ selectivity by negative charge mutations of the selectivity filter, but native prokaryotic Cavs had not yet been identified. We report the first identification of a native prokaryotic Cav, CavMr, whose selectivity filter contains a smaller number of negatively charged residues than that of artificial prokaryotic Cavs. A relative mutant whose selectivity filter was replaced with that of CavMr exhibits high Ca2+ selectivity. Mutational analyses revealed that the glycine residue of the CavMr selectivity filter is a determinant for Ca2+ selectivity. This glycine residue is well conserved among subdomains I and III of eukaryotic Cavs. These findings provide new insight into the Ca2+ selectivity mechanism that is conserved from prokaryotes to eukaryotes.,Electrical signals in the brain and muscles allow animals – including humans – to think, make memories and move around. Cells generate these signals by enabling charged particles known as ions to pass through the physical barrier that surrounds all cells, the cell membrane, at certain times and in certain locations. The ions pass through pores made by various channel proteins, which generally have so-called “selectivity filters” that only allow particular types of ions to fit through. For example, the selectivity filters of a family of channels in mammals known as the Cavs only allow calcium ions to pass through. Another family of ion channels in mammals are similar in structure to the Cavs but their selectivity filters only allow sodium ions to pass through instead of calcium ions. Ion channels are found in all living cells including in bacteria. It is thought that the Cavs and sodium-selective channels may have both evolved from Cav-like channels in an ancient lifeform that was the common ancestor of modern bacteria and animals. Previous studies in bacteria found that modifying the selectivity filters of some sodium-selective channels known as BacNavs allowed calcium ions to pass through the mutant channels instead of sodium ions. However, no Cav channels had been identified in bacteria so far, representing a missing link in the evolutionary history of ion channels. Shimomura et al. have now found a Cav-like channel in a bacterium known as Meiothermus ruber. Like all proteins, ion channels are made from amino acids and comparing the selectivity filter of the M. ruber Cav with those of mammalian Cavs and the calcium-selective BacNav mutants from previous studies revealed one amino acid that plays a particularly important role. This amino acid is a glycine that helps select which ions may pass through the pore and is also present in the selectivity filters of many Cavs in mammals. Together these findings suggest that the Cav channel from M. ruber is similar to the mammal Cav channels and may more closely resemble the Cav-like channels thought to have existed in the common ancestor of bacteria and animals. Since other channel proteins from bacteria are useful genetic tools for studies in human and other animal cells, the Cav channel from M. ruber has the potential to be used to stimulate calcium signaling in experiments.,© 2020, Shimomura et al.