Möttig H et al. (2001), Molecular regions controlling the activity of C...

XB-ART-8636

J Gen Physiol 2001 Aug 01;1182:183-92.

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Molecular regions controlling the activity of CNG channels.

Möttig H , Kusch J , Zimmer T , Scholle A , Benndorf K .

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The alpha subunits of CNG channels of retinal photoreceptors (rod) and olfactory neurons (olf) are proteins that consist of a cytoplasmic NH(2) terminus, a transmembrane core region (including the segments S1-S6), and a cytoplasmic COOH terminus. The COOH terminus contains a cyclic nucleotide monophosphate binding domain NBD) that is linked by the C-linker (CL) to the core region. 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 olf than in rod by constructing chimeric channels and determining the [cGMP] causing half maximum channel activity (EC(50)). The characteristic difference in the EC(50) value between rod and olf was introduced by the NH(2) terminus and the core-CL region, whereas the NBD showed a paradoxical effect. The difference of the free energy difference Delta(DeltaG) was determined for each of these three regions with all possible combinations of the other two regions. For rod regions with respect to corresponding olf regions, the open channel conformation was destabilized by the NH(2) 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 NH(2) terminus deletion mutants of rod and olf differed by Delta(DeltaG) of only 0.9 RT, whereas the wild-type channels differed by the much larger value of 3.1 RT. The results show that in rod and olf, the NH(2) terminus, the core-CL region, and the NBD differ by characteristic Delta(DeltaG) values that do not depend on the specific composition of the other two regions and that the NH(2) terminus generates the main portion of Delta(DeltaG) between the wild-type channels.

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	Figure 1. Structure of the chimeras between rod and olf. As shown in the cartoons, each channel/chimera consists of an NH2 terminus (left), six transmembrane helices (S1–S6; vertical boxes) linked by extramembrane linkers including the pore region (lines), the cyclic nucleotide monophosphate binding domain (NBD; horizontal box plus adjacent line at the right), and the C-linker (CL; line between the sixth helix and the NBD). Deletion mutants (−1r, −1o) lack an NH2 terminus. Black boxes and fat lines indicate rod sequences, whereas white boxes and thin lines indicate olf sequences. At the right, the exact splice sites of the rod (r) and olf (o) sequences are given by the terminal amino acids. In the names, reading from the left roughly indicates the sequence of rod “r” or olf “o” regions in the direction from the NH2- to the COOH terminus. “1…6,” “L,” and “B” indicate the six transmembrane helices, the C-linker, and the NBD, respectively. For further explanation see text.
	Figure 2. Dose–response relationship for the activation of chimeric channels constructed by transferring rod segments into an olf environment. The names and the cartoons of the channels/chimeras are listed in the middle. Fat lines and black boxes indicate extramembrane and transmembrane segments of rod, respectively. Thin lines and white boxes indicate the respective segments of olf. The symbols in the left diagram correspond to those left to the names. The data points of the dose–response relationships stem from representative experiments. The curves are best fits to . The following parameters were obtained: for rod, EC50 = 45.0 μM, H = 2.3; for r3o, EC50 = 27.4 μM, H = 2.7; for o3r5o, EC50 = 2.3 μM, H = 2.1; for o5r, EC50 = 6.3 μM, H = 3.3; for r1o, EC50 = 7.5 μM, H = 3.0; for oLrBo, EC50 = 6.4 μM, H = 3.0; for oBr, EC50 = 1.54 μM, H = 3.6; and for olf, EC50 = 1.97 μM, H = 2.8. In the right diagram the values of EC50 are graphically illustrated by their mean ± SEM. The means were calculated from 4 to 16 patches. The systematic transfer of the rod segments into an olf background identifies the NH2 terminus, the S1–S2 region, and the C-linker as critical regions determining the higher EC50 value in rod compared with olf. The transfer of the rod NBD into an olf background caused a lower EC50 than in olf itself.
	Figure 3. EC50 for the activation of chimeric channels constructed by transferring single olf regions into a rod environment (top six chimeras), both termini together in the respective other environment (o1rBo, r1oBr; chimera seven and eight from top), and by deleting the NH2 terminus (bottom two chimeras). The EC50 values were obtained as described in Fig. 2. The systematic transfer of the olf segments into a rod background shows that the NH2 terminus and the C-linker are important regions determining the lower EC50 value in olf compared with rod. A small effect on the EC50 was also observed for the S5-P-S6 region. The means were calculated from 4 to 28 patches each.
	Figure 4. Differences in the free energy difference (Δ(ΔG)) caused by the three components NH2 terminus (NT), core plus C-linker (core-CL), and cyclic nucleotide monophosphate binding domain (NBD). In the channel schemes a black box indicates the rod origin whereas a white box indicates the olf origin. The eight chimeras and channels are ordered from top to bottom according to an increasing EC50 (for SEM see Fig. 1 and Fig. 2). The brackets (right of the channel schemes) indicate pairs of channels/chimeras in which only one of the three components differs. The corresponding Δ(ΔG) values (rod component with respect to olf component) were calculated according to . They are shown below each bracket in multiples of RT as bar graphs. A negative Δ(ΔG) value indicates that a rod region destabilizes the open channel conformation with respect to the corresponding olf region, and a positive Δ(ΔG) value indicates that a rod region stabilizes the open channel conformation with respect to the corresponding olf region.
	Figure 5. Differences in the free energy difference (Δ(ΔG)) of the NH2 terminus deletion mutants (−1o, −1r) and the respective channels/chimeras including an NH2 terminus. The comparisons are demonstrated in a fashion analogous to Fig. 4. The channels, chimeras, and deletion mutants are ordered from top to bottom according to an increasing EC50 (for SEM see Fig. 1 and Fig. 2). A negative Δ(ΔG) value indicates that a channel or channel region destabilizes the open channel conformation with respect to another channel or channel region, whereas a positive Δ(ΔG) value indicates that a channel or channel region stabilizes the open channel conformation with respect to another channel or channel region. Compared are the Δ(ΔG) values of rod with respect to olf (rod/olf), of −1r with respect to −1o (−1r/−1o), of both NH2 termini with respect to −1o (NT/−1o), and of both NH2 termini with respect to −1r (NT/−1r). Rod with respect to olf is energetically more different (Δ(ΔG) = −3.1 RT) than −1r with respect to −1o (Δ(ΔG) = −0.9 RT). The amount of Δ(ΔG) between channels with rod and olf NH2 terminus with a rod background is similar to the respective amount of Δ(ΔG) with an olf background (1.4 vs. 1.3 RT).

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