Structural implications of weak Ca2+ block in Drosophila cyclic nucleotide-gated channels.

Calcium permeability and the concomitant calcium block of monovalent ion current ("Ca2+ block") are properties of cyclic nucleotide gated (CNG) channel fundamental to visual and olfactory signal transduction. Although most CNG channels bear a conserved glutamate residue crucial for Ca2' block, the degree of block displayed by different CNG channels varies greatly. For instance, the Drosophila melanogaster CNG channel shows only weak Ca2+ block despite the presence of this glutamate. We previously constructed a series of chimeric channels in which we replaced the selectivity -filter of the bacterial nonselective cation channel NaK with a set of CNG channel filter sequences and determined that the resulting NaK2CNG chimeras displayed the ion selectivity and Ca2+ block properties of the parent CNC channels. Here, we used the same strategy to determine the structural basis of the weak Ca2"' block observed in the Drosophila GNG channel. The selectivity filter of the Drosophila CNG channel is similar to that of most other CNC, channels except that it has a threonine at residue 318 instead of a proline. We constructed a NaK chimera, which we called NaK2CNG:Dm, which contained the Drosophila, selectivity filter sequence. The high resolution structure of NaK2CNG-Din revealed a filter structure different from those of NaK and all other previously investigated NaK2CNG chimeric channels. Consistent with this structural difference, functional studies of the NaK2CNG-Dm chimeric channel demonstrated a loss of Ca2+ block compared with other NaK2CNG chimeras. Moreover, mutating the corresponding threonine (T318) to proline in Drosophila CNG channels increased Ca' block by 16 times. These results imply that a simple replacement of a threonine for a proline in Drosophila CNG channels has likely given rise to a distinct selectivity filter conformation that results in weak Ca2+ block.