Molecular regions controlling the activity of CNG channels

The alpha subunits of CNG channels of retinal photoreceptors (rod) and olfa ctory neurons (olf) are proteins that consist of a cytoplasmic NH2 terminus a transmembrane core region (including the segments S1-S6), and a cytoplas mic COOH terminus. The COOH terminus contains a cyclic nucleotide monophosp hate binding domain NBD) that is linked by the C-linker (CL) to the core re gion. The binding of cyclic nucleotides to the NBD promotes channel opening by an allosteric mechanism. We examined why the sensitivity to cGMP is 22 times higher in off than in rod by constructing chimeric channels and deter mining the [cGMP] causing half maximum channel activity (EC50). The charact eristic difference in the EC50 value between rod and olf was introduced by the NH2 terminus and the core-CL region, whereas the NBD showed a paradoxic al effect. The difference of the free energy difference Delta(DeltaG) was d etermined for each of these three regions with all possible combinations of the other two regions. For rod regions with respect to corresponding olf r egions, the open channel conformation was destabilized by the NH2 terminus (Delta (DeltaG) = - 1.0 to - 2.0 RT) and the core-CL region (Delta (DeltaG) = - 2.0 to - 2.9 RT), whereas it was stabilized by the NBD (Delta(DeltaG) = 0.3 to 1.1 RT). The NH2 terminus deletion mutants of rod and olf differed by Delta(DeltaG) of only 0.9 RT, whereas the wild-type channels differed b y the much larger value of 3.1 RT. The results show that in rod and olf, th e NH2 terminus, the core-CL region, and the NBD differ by characteristic De lta (DeltaG) values that do not depend on the specific composition of the o ther two regions and that the NH2, terminus generates the main portion of D elta(DeltaG) between the wild-type channels.