Abstract
The activation of cyclic nucleotide-gated (CNG) channels is the final step in olfactory and visual transduction. Previously we have shown that, in addition to their activation by cyclic nucleotides, nitric oxide (NO)-generating compounds can directly open olfactory CNG channels through a redox reaction that results in the S-nitrosylation of a free SH group on a cysteine residue. To identify the target site(s) of NO, we have now mutated the four candidate intracellular cysteine residues Cys-460, Cys-484, Cys-520, and Cys-552 of the rat olfactory rCNG2 (alpha) channel into serine residues. All mutant channels continue to be activated by cyclic nucleotides, but only one of them, the C460S mutant channel, exhibited a total loss of NO sensitivity. This result was further supported by a similar lack of NO sensitivity that we found for a natural mutant of this precise cysteine residue, the Drosophila melanogaster CNG channel. Cys-460 is located in the C-linker region of the channel known to be important in channel gating. Kinetic analyses suggested that at least two of these Cys-460 residues on different channel subunits were involved in the activation by NO. Our results show that one single cysteine residue is responsible for NO sensitivity but that several channel subunits need to be activated for channel opening by NO.
Highlights
Activation of cyclic nucleotide-gated (CNG)1 channels is the final step in the transduction pathways in both vision and olfaction [1]
Of the 8 cysteine residues distributed throughout the rat olfactory rCNG2 (␣) channel (Fig. 1A), our previous biochemical evidence identified 1 residue located on the intracellular face of the channel as the putative target site for S-nitrosylation [21]
In order to determine the possible contribution of each of these residues to the activation of the channel by nitric oxide (NO), we prepared a series of mutant rCNG2 (␣) channel subunits sequentially replacing the intracellular cysteine residues in positions 460, 484, 520, and 552 (Fig. 1, A and B) with serine residues by site-directed mutagenesis [28, 34]
Summary
Activation of cyclic nucleotide-gated (CNG) channels is the final step in the transduction pathways in both vision and olfaction [1]. The opening of olfactory and photoreceptor CNG channels relies on the binding of at least two molecules of cAMP or cGMP These ubiquitous cyclic nucleotide second messengers bind at intracellular sites on the channel protein to activate a nonspecific cation conductance. The gaseous messenger nitric oxide (NO) has been proposed to exert its gating effects on the CNG channel by a redox modulation of at least one of these intracellular cysteines by reactive nitrogen species that are downstream of NO itself (i.e. the nitrosonium ion, NOϩ) [21] This direct regulation of protein by NO, so-called “S-nitrosylation,” has been proposed to play a critical role in many processes such as blood pressure regulation, host defense, and neurotransmission [22, 23]. We found that the cysteine in position 460, within the C-linker region just N-terminal to the CN binding region, is the critical residue in the reaction that leads to channel activation by NO
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