A Single Intracellular Cysteine Residue Is Responsible for the Activation of the Olfactory Cyclic Nucleotide-gated Channel by NO

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 (α) 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 melanogasterCNG 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. 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 (α) 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 melanogasterCNG 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. cyclic nucleotide-gated cyclic nucleotide nitric oxide human embryonic kidney green fluorescent protein base pair S-nitrosocysteine Activation of cyclic nucleotide-gated (CNG)1 channels is the final step in the transduction pathways in both vision and olfaction (1.Zagotta W.N. Siegelbaum S.A. Annu. Rev. Neurosci. 1996; 19: 235-263Crossref PubMed Scopus (421) Google Scholar). 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. This conductance has a significant permeability to calcium ions (1.Zagotta W.N. Siegelbaum S.A. Annu. Rev. Neurosci. 1996; 19: 235-263Crossref PubMed Scopus (421) Google Scholar), and thus, CNG channel activation can lead not only to depolarization but also to Ca2+ influx. Native olfactory CNG channels are constructed from at least three different but highly homologous subunits, variously called CNG2 or α (original designation OCNC1 (2.Dhallan R.S. Yau K.W. Schrader K.A. Reed R.R. Nature. 1990; 347: 184-187Crossref PubMed Scopus (515) Google Scholar)), CNG5 or β (original designation OCNC2 (3.Bradley J. Li J. Davidson N. Lester H.A. Zinn K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8890-8894Crossref PubMed Scopus (210) Google Scholar, 4.Liman E.R. Buck L.B. Neuron. 1994; 13: 611-621Abstract Full Text PDF PubMed Scopus (215) Google Scholar)), and CNG4.3 (5.Sautter A. Zong X. Hofmann F. Biel M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4696-4701Crossref PubMed Scopus (106) Google Scholar) or CNCβ1b (6.Bonigk W. Bradley J. Muller F. Sesti F. Boekhoff I. Ronnett G.V. Kaupp U.B. Frings S. J. Neurosci. 1999; 19: 5332-5347Crossref PubMed Google Scholar) (for nomenclature of CNG channel subunits see Ref. 7.Biel M. Zong X. Hofmann F. Trends Cardiovasc. Med. 1996; 6: 274-280Crossref PubMed Scopus (20) Google Scholar). The different subunits of the native channel are thought to assemble in a heterotetrameric structure (8.Liu D.T. Tibbs G.R. Siegelbaum S.A. Neuron. 1996; 16: 983-990Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 9.Liu D.T. Tibbs G.R. Paoletti P. Siegelbaum S.A. Neuron. 1998; 21: 235-248Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The gating of CNG channels following cyclic nucleotide binding has been the subject of intensive research (1.Zagotta W.N. Siegelbaum S.A. Annu. Rev. Neurosci. 1996; 19: 235-263Crossref PubMed Scopus (421) Google Scholar, 8.Liu D.T. Tibbs G.R. Siegelbaum S.A. Neuron. 1996; 16: 983-990Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 9.Liu D.T. Tibbs G.R. Paoletti P. 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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, NO+) (21.Broillet M.-C. Firestein S. Neuron. 1996; 16: 377-385Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). 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.Moncada S. Higgs A. N. Engl. J. Med. 1993; 2002–2012Google Scholar, 23.Broillet M.-C. Cell. Mol. Life Sci. 1999; 55: 1036-1042Crossref PubMed Scopus (176) Google Scholar). Ion channel regulation has also been postulated to occur by S-nitrosylation (24.Lei S.Z. Pan Z.H. Aggarwal S.K. Chen H.S.V. Hartman J. Sucher N.J. Lipton S.A. Neuron. 1992; 8: 1087-1099Abstract Full Text PDF PubMed Scopus (686) Google Scholar, 25.Bolotina V.M. Najibi S. Palacino J. Pagano P. Cohen R.A. Nature. 1994; 368: 850-853Crossref PubMed Scopus (1510) Google Scholar, 26.Li Z. Chapleau M.W. Bates J.N. Bielefeldt K. Lee H.-C. Abboud F.M. Neuron. 1998; 20: 1039-1049Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 27.Xu L. Eu J.P. Meissner G. Stamler J.S. Science. 1998; 279: 234-236Crossref PubMed Scopus (857) Google Scholar). Among ion channels, only the CNG channel has been shown to be directly activated by NO (21.Broillet M.-C. Firestein S. Neuron. 1996; 16: 377-385Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Of the 8 cysteine residues distributed throughout the rat olfactory rCNG2 (α) channel (Fig. 1 A), our previous biochemical evidence identified 1 residue located on the intracellular face of the channel as the putative target site for S-nitrosylation (21.Broillet M.-C. Firestein S. Neuron. 1996; 16: 377-385Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). We have therefore focused on the cysteines located on the intracellular face of the channel, and we generated a series of mutant subunit constructs in which each of these cysteines was changed to a serine residue. After expression in HEK 293 cells, we were able to test each of these channels for activation by cAMP and/or NO. 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. Cysteine to serine mutants of rCNG2 (α) were generated by substituting the specific cysteine residues with serines using the polymerase chain reaction-based mutagenesis described by Nelson and Long (28.Nelson R.M. Long G.L. Anal. Biochem. 1989; 180: 147-151Crossref PubMed Scopus (294) Google Scholar). Pfu polymerase (Stratagene, CA) was used to reduce the rate of contaminating mutations. The point mutants were designated as C460S, C484S, C520S, and C552S. All constructs were verified by sequencing. Human embryonic kidney (HEK) 293 cells were grown at 37 °C in minimal essential medium supplemented with 10% horse serum and 1% gentamicin. A pCIS expression vector (Genentech, CA) containing either the wild type rat olfactory subunit rCNG2 (α) (29.Chen T.-Y. Peng Y.-W. Dhallan R.S. Ahamed B. Reed R.R. Yau K.-W. Nature. 1993; 362: 764-767Crossref PubMed Scopus (274) Google Scholar) or one of the four different types of mutant rCNG2 (α) channel was used to perform transient transfections using a standard calcium phosphate protocol (30.Gorman C.M. Gies D.R. McCray G. DNA Protein Eng. Techniques. 1990; 2: 3-10Google Scholar). The cells were co-transfected with a vector containing the gene for the green fluorescent protein (GFP) (kind gift of M. Chalfie) at a 1:1 molar ratio. Patch clamp recordings were made 2–3 days after transfection. GFP was used as an indicator of transfection success (31.Cubitt, A. B., Heim, R., Adams, S. R., Boyd, A. E., Gross, L. A., and Tsien, R. Y. (1995) Trends Biochem. Sci. 448–455Google Scholar, 32.Broillet M.-C. Firestein S. Neuron. 1997; 18: 951-958Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), efficiency, and probable expression of rCNG2 (α) wild type or GFP was in cells with a using that the HEK 293 cells were days after transfection with rCNG2 (α) wild type or mutant The total was by of total was on and The was at °C with located at wild type rCNG2 (α) region The was by clamp recordings were made using the A. B. PubMed Scopus Google Scholar). were from and to of to The was the as the (see that both of the were in The channel were using an clamp CA) and on were and on a using the The was and the was The have been using the and was by the of the of after with a vector containing the gene for the GFP to for cells with a of either single channel or of in the The were using an clamp CA) and on a using the and with the was on from the of of and (24.Lei S.Z. Pan Z.H. Aggarwal S.K. Chen H.S.V. Hartman J. Sucher N.J. Lipton S.A. Neuron. 1992; 8: 1087-1099Abstract Full Text PDF PubMed Scopus (686) Google Scholar). This for All were in the for a to the This was by the and a in to a of All recordings were made at The are as S.E. were using the test for to activate olfactory CNG channels in we identified the probable NO target site as one of four intracellular cysteine residues (21.Broillet M.-C. Firestein S. Neuron. 1996; 16: 377-385Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). shown in Fig. and the four intracellular cysteines of the rat olfactory CNG channel rCNG2 (α) occur to the of Cys-520, and are within the cyclic nucleotide binding and the is in a region of the that the to the CN binding known as the C-linker to the of each of these residues to the activation of the channel by NO, we a series of mutant rCNG2 (α) channel subunits the intracellular cysteine residues in 460, and (Fig. and with serine residues by mutagenesis (28.Nelson R.M. Long G.L. Anal. Biochem. 1989; 180: 147-151Crossref PubMed Scopus (294) Google Scholar, Anal. Biochem. 1992; PubMed Scopus Google Scholar). This is a in which the SH group of the thought to be the redox site of NO is by an of each mutant of expression in HEK 293 cells (Fig. 1 channels were as by in cells (see using with the green fluorescent protein (GFP) as an indicator of transfection M.-C. Firestein S. Neuron. 1997; 18: 951-958Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), we found of protein expression in different This to containing from 1 to several the three channels with in the CN binding region C520S, and we were to that each of these mutants a channel with single channel not different from of wild type rCNG2 (α) channels (Fig. and a of cAMP which the of the wild type rCNG2 (α) channel, the open open and single channel and not were similar in three we not test this cAMP binding not appear to be by these single cysteine channel of wild type and mutant rCNG2 channels after activation by cAMP wild type and C460S and wild type and C460S and wild type and C460S and in a S.E. of the NO S-nitrosocysteine at channel in these three mutants that was similar to that in the rCNG2 (α) wild type channel. the were by the of NO activation (Fig. these we that the cysteine residues located in the site are not involved in the activation of the channel by NO. to the results the cysteine residue in the C-linker region a channel that was to NO (Fig. of the NO to to channel after of These single channel be with recordings in with of channels not This result the Cys-460 as the only intracellular cysteine residue involved in the of channel activation by NO. a further we made of the Drosophila CNG channel (kind gift of U. B. which is the only known CNG channel that this highly cysteine residue, a to the CNG channels and the three cysteine residues in the site A. Frings S. M. R. Kaupp U.B. J. 1994; 13: PubMed Scopus Google Scholar). with A. Frings S. M. R. Kaupp U.B. J. 1994; 13: PubMed Scopus Google Scholar), we found that activated the D. channel in HEK 293 as we from our results with C460S, this channel was also found to be to NO (Fig. This further verified the of this single cysteine residue in the NO activation and the results with the rat olfactory C460S mutant channel. activation by cAMP at the single channel also to be by the C460S The cAMP was that this mutant channel was but a significant in the open and an in the of the channel were with the wild type rCNG2 (α) channel the cAMP sensitivity was not we used with of channels and in the of of cAMP Fig. we similar for cAMP for the wild type rCNG2 (α) channel and for the mutant C460S The of the to the a of a of for the rCNG2 (α) channel, of and of for the C460S These were not further that this cysteine residue not affect nucleotide binding but only the channel gating shown NO to with a single cysteine residue channel A CNG channel is to be a of or different types of subunits (8.Liu D.T. Tibbs G.R. Siegelbaum S.A. Neuron. 1996; 16: 983-990Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 9.Liu D.T. Tibbs G.R. Paoletti P. Siegelbaum S.A. Neuron. 1998; 21: 235-248Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). of the subunits the C-linker cysteine for for and for the subunit four cysteine residues channel that can This the as to four subunits be for channel activation. we of the that the rCNG2 (α) subunit a channel of four subunits, to the for these we used with of channels and of in at from 1 to of 1 to 1 (Fig. The activated with a that was (see and to base of were by the in each to the by cAMP in that was by the activation of rCNG2 (α) at Activation by NO a with a of that the opening of a single channel on the binding of at least two NO The for are in Fig. on a of expression in of with of channels, and this to the of the of Cys-460 by NO using a A the of at different on such an The to the activation of channels, as from the single channel conductance to the of the NO is only to a but the to these effects through a on the single channel We focused on the rate of of the after a in and the rate of the to the in To the shown in Fig. the of the by the of was with A single was to the of the (for see Fig. from 1 to from to Fig. the by the were as a of The rate for was not into this not only NO binding but was also by the see and The of this an of the rate and the an of the rate to We a on of and of This of is to the by an of the after the of these two we of the as We a in with the one at by the This of to a free NO of 1 in which is to the NO that has been in the F. M. J. 1993; 13: PubMed Scopus Google Scholar). modulation of by NO has now been in a of Trends Sci. 1995; 16: Full Text PDF PubMed Scopus Google Scholar, S. L.A. J. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) and an for protein modulation to of these is that a redox reaction at free SH on cysteine residues. in only a have the specific cysteine residues been J. 1997; 11: PubMed Scopus Google Scholar, J.S. L. Eu J.P. J. K. Science. 1997; PubMed Scopus Google Scholar). Among ion channels the has been but specific cysteine have not been These (24.Lei S.Z. Pan Z.H. Aggarwal S.K. Chen H.S.V. Hartman J. Sucher N.J. Lipton S.A. Neuron. 1992; 8: 1087-1099Abstract Full Text PDF PubMed Scopus (686) Google Scholar), channels V.M. Najibi S. Palacino J. Pagano P. Cohen R.A. Nature. 1994; 368: 850-853Crossref PubMed Scopus (1510) Google Scholar), channels in Z. Chapleau M.W. Bates J.N. Bielefeldt K. Lee H.-C. Abboud F.M. 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Sci. 448–455Google Scholar). the we have to the of a cysteine by olfactory CNG channels in which specific cysteine residues have been mutated to The result of our is that a single cysteine residue within the intracellular C-linker region in the rCNG2 (α) channel subunit is shown to be the NO target essential and of the channel, in the of cyclic intracellularly located cysteines appear not to in the gating of the channel. is not for several cysteine residues on a protein to be for the of a total of free SH but only are thought to L. Eu J.P. Meissner G. Stamler J.S. Science. 1998; 279: 234-236Crossref PubMed Scopus (857) Google Scholar). the precise by NO are the of a has been postulated on base J.S. Lipton S.A. Sucher N.J. Neuron. 1997; 18: Full Text Full Text PDF PubMed Scopus Google Scholar). The proposed is can be of or can be or and can be or The important of the is to be the residues following the this in the CNG channel only the Cys-460, identified by our biochemical and as the NO target the (i.e. and the channel is made of four subunits (8.Liu D.T. Tibbs G.R. Siegelbaum S.A. Neuron. 1996; 16: 983-990Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 9.Liu D.T. Tibbs G.R. Paoletti P. Siegelbaum S.A. Neuron. 1998; 21: 235-248Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), are four sites channel. also the of NO at of to NO to protein different reaction with NO at different cysteines to redox of the or cysteines in that have also for the that in a free are in involved in J.S. Lipton S.A. Sucher N.J. Neuron. 1997; 18: Full Text Full Text PDF PubMed Scopus Google Scholar). the CNG channel, our a of that as as two of the four target cysteines with NO. at four The activation of a CNG channel is a binding and from a to an open studies have that the the E.H. Tibbs G.R. Siegelbaum S.A. Nature. 1994; 372: 369-374Crossref PubMed Scopus (179) Google Scholar, 15.Gordon S.E. Zagotta W.N. Neuron. 1995; 14: 857-864Abstract Full Text PDF PubMed Scopus (141) Google Scholar) and the of that the site to the also channel activation. A residue within this region that with and ions has been in modulation of both and olfactory CNG channel gating S.E. Zagotta W.N. Neuron. 1995; 14: 177-183Abstract Full Text PDF PubMed Scopus (133) Google Scholar). Zong X. Hofmann F. Biel M. 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