Structural implications of weak Ca2+ block in Drosophila cyclic nucleotide-gated channels.
Bottom Line: The selectivity filter of the Drosophila CNG channel is similar to that of most other CNG channels except that it has a threonine at residue 318 instead of a proline.Moreover, mutating the corresponding threonine (T318) to proline in Drosophila CNG channels increased Ca(2+) 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 Ca(2+) block.
Affiliation: Department of Physiology and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390.Show MeSH
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Mentions: Because threonine 318 in Drosophila was found to be the key in gauging Ca2+ block, we asked whether the equivalent residue in mammalian CNG channels is important for the block. This was tested on bovine CNGA1 channel by replacing the equivalent proline (P366) with threonine, resulting in a selectivity filter sequence of TIGETPTP. The P366T mutation in bovine CNGA1 has no obvious effect on single-channel conductance and kinetics or cGMP-regulated gating (Fig. 6, A and B). However, contrary to our prediction based on the NaK2CNG mutants as well as the mutation on Drosophila CNGA, the Pro-to-Thr replacement in bovine CNGA1 has almost no effect on Ca2+ block. Outside-out patch recordings show that mutation only results in a subtle decrease of Ca2+ affinity (Ki = 3.5 ± 0.5 µM for wild type and Ki = 6.0 ± 0.7 µM for P366T; Fig. 6, C and D), suggesting that the equivalent Thr/Pro swap in CNGA1 does not compromise the structure integrity of the filter, and the channel thereby retains its strong Ca2+ block.
Affiliation: Department of Physiology and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390.