T4pA

20 ms

Fig. 4. Cys-460 is the nitric oxide target site. A comparison is shown of single-channel recordings from channels composed of either wild-type CNG subunits (CNGA2) or C460S mutant or C484S mutant subunits expressed in HEK 293 cells. For the wild-type and C484S mutant channels, cAMP (50 fiM) and SNC (100 fiM) elicited high-frequency channel activity. In the case of the C460S mutant channel, no activation could be observed after SNC treatment and a decrease in open probability is observed after cAMP treatment compared with the wild-type channel. All recordings were performed at —60 mV, holding potential. Adapted with permission from M.-C. Broillet, J. Biol. Chem. 275, 15135 (2000).

activity at particular cysteines. Different degrees of accessibility to NO due to protein conformation, different reaction rates with NO at different cysteines due to redox status of the immediate environment, or cysteines in positions that may have no functional consequences on nitrosylation, could also account for the observation that in most proteins relatively few free thiols are in fact involved in nitrosylation-induced activity.28 In the CNG channel, our concentration-response data indicate a Hill coefficient of less than 2, suggesting that as few as two of the four target cysteines may actually interact with NO. It does not, however, preclude activity at all four sites.

An important role for cysteine residues in CNG channel activation has been proposed by Gordon et al?x These authors have shown, by work on the rod CNGA1 subunit of the CNG channel, that the N- and C-terminal regions of each subunit interact in the tetrameric channel. This interaction involves particular cysteine residues with the formation of a disulfide bond between Cys-35 (N terminal) and Cys-481 (C terminal). Brown et al?2 have also found that Cys-481 is necessary for the potentiation of the rod CNGA1 channel response to cAMP and cGMP. This cysteine residue, located in the C-terminal region, corresponds exactly to the NO target site in the olfactory CNG channel (Cys-460), confirming that this amino acid, which is highly conserved among the different cloned CNG channels, likely plays a critical role in channel gating and in the potentiation of cyclic nucleotide action.

The fact that Cys-460 is a potent activator of channel gating by NO and also affects channel gating by cAMP indicates the importance of this residue for channel function.

Nitric Oxide and Olfaction

Nitric oxide synthase has been shown to be expressed in olfactory neurons during development and possibly during neuronal regeneration in adult life.33'34 Developing or regenerating olfactory neurons are dependent on outside stimuli, that is, odors, for their activity, and these odors may not always be present in the environment during critical growth periods. Thus, NO production may be important in maintaining activity in developing neurons independent of the stimulus environment. Because the CNG channels are highly permeable to Ca2+ ions35 their activation could also provide a pathway for increasing intracellular Ca2+ concentration, a first step in many cellular processes.

Acknowledgments

I sincerely thank O. Randin for preparing the figures. This work was supported by grants from the Fonds National Suisse de la Recherche (31-51061.97 and 3130-051920.97).

31 S. E. Gordon, M. D. Varnum, and W. N. Zagotta, Neuron 19,431 (1997).

32 l. A. Brown, S. D. Snow, and T. L. Haley, Biophys. J. 75, 825 (1998).

33 A. J. Roskams, D. S. Bredt, T. M. Dawson, and G. V. Ronnett, Neuron 13, 289 (1994).

34 H. Zhao, S. Firestein, and C. A. Greer, NeuroReport 6, 149 (1994).

35 F. Zufall and S. Firestein, J. Neurophysiol. 69, 1758 (1993).

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