Structural insight into Ca²⁺ specificity in tetrameric cation channels

Apparent blockage of monovalent cation currents by the permeating blocker Ca²⁺ is a physiologically essential phenomenon relevant to cyclic nucleotide-gated (CNG) channels. The recently determined crystal structure of a bacterial homolog of CNG channel pores, the NaK channel, revealed a Ca ²⁺ binding site at the extracellular entrance to the selectivity filter. This site is not formed by the side-chain carboxylate groups from the conserved acidic residue, Asp-66 in NaK, conventionally thought to directly chelate Ca²⁺ in CNG channels, but rather by the backbone carbonyl groups of residue Gly-67. Here we present a detailed structural analysis of the NaK channel with a focus on Ca²⁺ permeability and blockage. Our results confirm that the Asp-66 residue, although not involved in direct chelation of Ca²⁺, plays an essential role in external Ca ²⁺ binding. Furthermore, we give evidence for the presence of a second Ca²⁺ binding site within the NaK selectivity filter where monovalent cations also bind, providing a structural basis for Ca²⁺ permeation through the NaK pore. Compared with other Ca²⁺-binding proteins, both sites in NaK present a novel mode of Ca²⁺ chelation, using only backbone carbonyl oxygen atoms from residues in the selectivity filter. The external site is under indirect control by an acidic residue (Asp-66), making it Ca²⁺-specific. These findings give us a glimpse of the possible underlying mechanisms allowing Ca²⁺ to act both as a permeating ion and blocker of CNG channels and raise the possibility of a similar chemistry governing Ca²⁺ chelation in Ca²⁺ channels.