Cyclic nucleotide-gated (CNG) channels are composed of the tetramer of a-subunit alone or alpha- and beta-subunits. The alpha-subunits of these channels have a conserved glutamate (Glu) residue within the pore-forming region and the residue determines the selectivity as well as the affinity for the extracellular divalent cations. Using the high-affinity mutant (E363D) of bovine retinal CNG channel in which the Glu at position 363 was replaced to Asp, we constructed tandem dimers and investigated the binding characteristics of divalent cations to the site. The gating and permeation characteristics of individual homomeric tandem dimers are indistinguishable to those of homo-tetramers formed by parental monomers. The heteromeric tandem dimers showed the binding affinity for Sr2+ identical to the geometric mean of the affinities for two parent channels, indicating the energy additive and thus the simultaneous interaction. On the other hand, the binding affinity for Mg2+ followed the harmonic mean of those parent channels indicating that Mg2+ interacts more strongly with the subunit bearing Asp residue at the position. Thus the results strongly suggest that the Glu363 residues in the CNG channel pore be flexible enough to adapt different binding symmetries for different divalent cations. Moreover, the simultaneous interaction between the four Glu residues and Sr2+ provides an important structural constraint to the CNG channel outer vestibule of unknown structure. (C) 2002 Elsevier Science (USA). All rights reserved.