Structural insight into Ca 2+ specificity in tetrameric cation channels

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Abstract
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 Ca2+ 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 Ca2+ 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.