All-trans-retinal is a closed-state inhibitor of rod cyclic nucleotide-gated ion channels

Rod vision begins when 11-cis-retinal absorbs a photon and isomerizes to all-trans-retinal (ATR) within the photopigment, rhodopsin. Photoactivated rhodopsin triggers an enzyme cascade that lowers the concentration of cGMP, thereby closing cyclic nucleotide-gated (CNG) ion channels. After isomerization, ATR dissociates from rhodopsin, and after a bright light, this release is expected to produce a large surge of ATR near the CNG channels. Using excised patches from Xenopus oocytes, we recently showed that ATR shuts down cloned rod CNG channels, and that this inhibition occurs in the nanomolar range (aqueous concentration) at near-physiological concentrations of cGMP. Here we further characterize the ATR effect and present mechanistic information. ATR was found to decrease the apparent cGMP affinity, as well as the maximum current at saturating cGMP. When ATR was applied to outside-out patches, inhibition was much slower and less effective than when it was applied to inside-out patches, suggesting that ATR requires access to the intracellular surface of the channel or membrane. The apparent ATR affinity and maximal inhibition of heteromeric (CNGA1/CNGB1) channels was similar to that of homomeric (CNGA1) channels. Single-channel and multichannel data suggest that channel inhibition by ATR is reversible. Inhibition by ATR was not voltage dependent, and the form of its dose-response relation suggested multiple ATR molecules interacting per channel. Modeling of the data obtained with cAMP and cGMP suggests that ATR acts by interfering with the allosteric opening transition of the channel and that it prefers closed, unliganded channels. It remains to be determined whether ATR acts directly on the channel protein or instead alters channel-bilayer interactions.     

Movement of gating machinery during the activation of rod cyclic nucleotide-gated channels.

In the visual and olfactory systems, cyclic nucleotide-gated (CNG) ion channels convert stimulus-induced changes in the internal concentrations of cGMP and cAMP into changes in membrane potential. Although it is known that significant activation of these channels requires the binding of three or more molecules of ligand, the detailed molecular mechanism remains obscure. We have probed the structural changes that occur during channel activation by using sulfhydryl-reactive methanethiosulfonate (MTS) reagents and N-ethylmaleimide (NEM). When expressed in Xenopus oocytes, the alpha-subunit of the bovine retinal channel forms homomultimeric channels that are activated by cGMP with a K1/2 of approximately 100 microM. Cyclic AMP, on the other hand, is a very poor activator; a saturating concentration elicits only 1% of the maximum current produced by cGMP. Treatment of excised patches with MTS-ethyltrimethylamine (MTSET) or NEM dramatically potentiated the channel's response to both cyclic nucleotides. After MTSET treatment, the dose-response relation for cGMP was shifted by over two orders of magnitude to lower concentrations. The effect on channel activation by cAMP was even more striking. After modification, the channels were fully activated by cAMP with a K1/2 of approximately 60 microM. This potentiation was abolished by conversion of Cys481 to a nonreactive alanine residue. Potentiation occurred more rapidly in the presence of saturating cGMP, indicating that this region of the channel is more accessible when the channel is open. Cys481 is located in a linker region between the transmembrane and cGMP-binding domains of the channel. These results suggest that this region of the channel undergoes significant movement during the activation process and is critical for coupling ligand binding to pore opening. Potentiation, however, is not mediated by the recently reported interaction between the amino- and carboxy-terminal regions of the alpha-subunit. Deletion of the entire amino-terminal domain had little effect on potentiation by MTSET.     

Mechanisms of modulation by internal protons of cyclic nucleotide-gated channels cloned from sensory receptor cells.

We have examined the modulation by internal protons of cyclic nucleotide-gated (CNG) channels cloned from bovine olfactory receptor cells and retinal rods. CNG channels were studied in excised inside-out membrane patches from Xenopus laevis oocytes previously injected with the mRNA encoding for the subunit 1 of olfactory or rod channels. Channels were activated by cGMP or cAMP, and currents as a function of cyclic nucleotide concentrations were measured as pHi varied between 7.6 and 5.0. Increasing internal proton concentrations caused a partial blockage of the single-channel current, consistent with protonation of a single acidic site with a pK1 of 4.5-4.7, both in rod and in olfactory CNG channels. Channel gating properties were also affected by internal protons. The open probability at low cyclic nucleotide concentrations was greatly increased by lowering pHi, and the increase was larger when channels were activated by cAMP than by cGMP. Therefore, internal protons affected both channel permeation and gating properties, causing a reduction in single-channel current and an increase in open probability. These effects are likely to be caused by different titratable groups on the channel.     

Functional activity of photoreceptor cyclic nucleotide-gated channels is dependent on the integrity of cholesterol- and sphingolipid-enriched membrane domains

Rod and cone photoreceptor cyclic nucleotide-gated (CNG) channels play pivotal roles in phototransduction. This work investigates the functional significance of photoreceptor CNG channel association with membrane microdomains enriched in raft lipids, cholesterol and sphingolipids. The primary subunits of cone and rod CNG channels, CNGA3 and CNGA1, respectively, were heterologously expressed in HEK 293 cells, and channel activity was determined by ratiometric measurement of [Ca (2+)] i in response to cyclic guanosine monophosphate (cGMP) stimulation. CNGA3 was found to be largely insoluble following Triton X-100 extraction and cofractionationed with biochemically isolated membrane domains enriched in caveolin-1. Cofractionation of both natively expressed CNGA3 and CNGB1 (the modulatory subunit of the rod CNG channel) with the low buoyant density, caveolin-1-enriched membranes was also confirmed in mouse retinas. The functional significance of this association was established by the observed negative effects of depletion of raft lipids on the channel activity. Treatment with the cholesterol depleting agent, methyl-beta-cyclodextrin (MCD), significantly inhibited CNGA3 and CNGA1 activation in response to cGMP stimulation. MCD treatment lowered cellular cholesterol levels by approximately 45% without altering fatty acid composition, suggesting that the inhibition of channel activity by MCD treatment is not due to perturbation of other membrane lipids. Treatment with the sphingolipid biosynthesis inhibitor myriocin resulted in impaired activation and cytosolic redistribution of CNGA3, suggesting that the integrity of the membrane domains is critical for the channel cellular processing and plasma membrane localization. This study demonstrates the association of photoreceptor CNG channels with membrane domains enriched in raft lipids and indicates, for the first time, that raft lipids modulate the plasma membrane localization and functional activity of photoreceptor CNG channels.     

Mutating three residues in the bovine rod cyclic nucleotide-activated channel can switch a nucleotide from inactive to active

Cyclic nucleotide-gated (CNG) channels, which were initially studied in retina and olfactory neurons, are activated by cytoplasmic cGMP or cAMP. Detailed comparisons of nucleotide-activated currents using nucleotide analogs and mutagenesis revealed channel-specific residues in the nucleotide-binding domain that regulate the binding and channel-activation properties. Of particular interest are N(1)-oxide cAMP, which does not activate bovine rod channels, and Rp-cGMPS, which activates bovine rod, but not catfish, olfactory channels. Previously, we showed that four residues coordinate the purine interactions in the binding domain and that three of these residues vary in the alpha subunits of the bovine rod, catfish, and rat olfactory channels. Here we show that both N(1)-oxide cAMP and Rp-cGMPS activate rat olfactory channels. A mutant of the bovine rod alpha subunit, substituted with residues from the rat olfactory channel at the three variable positions, was weakly activated by N(1)-oxide cAMP, and a catfish olfactory-like bovine rod mutant lost activation by Rp-cGMPS. These experiments underscore the functional importance of purine contacts with three residues in the cyclic nucleotide-binding domain. Molecular models of nucleotide analogs in the binding domains, constructed with AMMP, showed differences in the purine contacts among the channels that might account for activation differences.     

Mechanism of inhibition of cyclic nucleotide-gated ion channels by diacylglycerol

Cyclic nucleotide-gated (CNG) channels are critical components in the visual and olfactory signal transduction pathways, and they primarily gate in response to changes in the cytoplasmic concentration of cyclic nucleotides. We previously found that the ability of the native rod CNG channel to be opened by cGMP was markedly inhibited by analogues of diacylglycerol (DAG) without a phosphorylation reaction (Gordon, S.E., J. Downing-Park, B. Tam, and A.L. Zimmerman. 1995. Biophys. J. 69:409-417). Here, we have studied cloned bovine rod and rat olfactory CNG channels expressed in Xenopus oocytes, and have determined that they are differentially inhibited by DAG. At saturating [cGMP], DAG inhibition of homomultimeric (alpha subunit only) rod channels was similar to that of the native rod CNG channel, but DAG was much less effective at inhibiting the homomultimeric olfactory channel, producing only partial inhibition even at high [DAG]. However, at low open probability (P(o)), both channels were more sensitive to DAG, suggesting that DAG is a closed state inhibitor. The Hill coefficients for DAG inhibition were often greater than one, suggesting that more than one DAG molecule is required for effective inhibition of a channel. In single-channel recordings, DAG decreased the P(o) but not the single-channel conductance. Results with chimeras of rod and olfactory channels suggest that the differences in DAG inhibition correlate more with differences in the transmembrane segments and their attached loops than with differences in the amino and carboxyl termini. Our results are consistent with a model in which multiple DAG molecules stabilize the closed state(s) of a CNG channel by binding directly to the channel and/or by altering bilayer-channel interactions. We speculate that if DAG interacts directly with the channel, it may insert into a putative hydrophobic crevice among the transmembrane domains of each subunit or at the hydrophobic interface between the channel and the bilayer.     

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 (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.     

Conformational changes in S6 coupled to the opening of cyclic nucleotide-gated channels.

In cyclic nucleotide-gated channels (CNG), direct binding of cyclic nucleotides in the carboxy-terminal region is allosterically coupled to opening of the pore. A CNG1 channel pore was probed using site-directed cysteine substitution to elucidate conformational changes associated with channel opening. The effects of cysteine modification on permeation suggest a structural homology between CNG and KcsA pores. We found that intersubunit disulfide bonds form spontaneously between S399C residues in the helix bundle when channels are in the closed but not in the open state. While MTSET modification of pore-lining residues was state dependent, Ag(+) modification of V391C, in the inner vestibule, occurred at the same diffusion-limited rate in both open and closed states. Our results suggest that the helix bundle undergoes a conformational change associated with gating but is not the activation gate for CNG channels.     

Subunit interactions in the activation of cyclic nucleotide-gated ion channels.

Cyclic nucleotide-gated (CNG) ion channels of retinal photoreceptors and olfactory neurons are multimeric proteins of unknown stoichiometry. To investigate the subunit interactions that occur during CNG channel activation, we have used tandem cDNA constructs of the rod CNG channel to generate heteromultimeric channels composed of wild-type and mutant subunits. We introduced point mutations that affect channel activation: 1) D604M, which alters the relative ability of agonists to promote the allosteric conformational change(s) associated with channel opening, and 2) T560A, which primarily affects the initial binding affinity for cGMP, and to a lesser extent, the allosteric transition. At saturating concentrations of agonist, heteromultimeric channels were intermediate between wild-type and mutant homomultimers in agonist efficacy and apparent affinity for cGMP, cIMP, and cAMP, consistent with a model for the allosteric transition involving a concerted conformational change in all of the channel subunits. Results were also consistent with a model involving independent transitions in two or three, but not one or four, of the channel subunits. The behavior of the heterodimers implies that the channel stoichiometry is some multiple of 2 and is consistent with a tetrameric quaternary structure for the functional channel complex. Steady-state dose-response relations for homomultimeric and heteromultimeric channels were well fit by a Monod, Wyman, and Changeux model with a concerted allosteric opening transition stabilized by binding of agonist.     

Movement of the C-helix during the gating of cyclic nucleotide-gated channels

Movements within the cyclic nucleotide-binding domain of cyclic nucleotide-gated channels are thought to underlie the initial phase of channel gating (Tibbs, G. R., D. T. Liu, B. G. Leypold, and S. A. Siegelbaum. 1998. J. Biol. Chem. 273:4497-4505; Zong, X., H. Zucker, F. Hofmann, and M. Biel. 1998. EMBO J. 17:353-362; Matulef, K., G. E. Flynn, and W. N. Zagotta. 1999. Neuron. 24:443-452; Paoletti, P., E. C. Young, and S. A. Siegelbaum. 1999. J. Gen. Physiol. 113:17-33; Johnson, J. P., and W. N. Zagotta. 2001. Nature. 412:917-921). To investigate these movements, cysteine mutation was performed on each of the 28 residues (Leu-583 to Asn-610), which span the agonist-binding domain of the alpha-subunit of the bovine rod cyclic nucleotide-gated channel. The effects of Cd(2+) ions, 2-trimethylammonioethylmethane thiosulfonate (MTSET) and copper phenanthroline (CuP) on channel activity were examined, in excised inside-out patches in the presence and in the absence of a saturating concentration of cGMP. The application of 100 microM Cd(2+) in the presence of saturating concentration of cGMP caused an irreversible and almost complete reduction of the current in mutant channels E594C, I600C, and L601C. In the absence of cGMP, the presence of 100 microM Cd(2+) caused a strong current reduction in all cysteine mutants from Asp-588 to Leu-607, with the exception of mutant channels A589C, M592C, M602C, K603C, and L606C. The selective effect of Cd(2+) ions was very similar to that observed when adding the oxidizing agent CuP to the bath medium, except for mutant channel G597C, where CuP caused a stronger current decrease (67 +/- 7%) than Cd(2+) (23 +/- 4%). In the absence of cGMP, MTSET caused a reduction of the current by >40% in mutant channels L607C, L601C, I600C, G597C, and E594C, whereas in the presence of cGMP only mutant channel L601C was affected. The application of MTSET protected many mutant channels from the effects of Cd(2+) and CuP. These results suggest that, when CNG channels are in the open state, residues from Asp-588 to Leu-607 are in an alpha-helical structure, homologous to the C-helix of the catabolite gene activator protein (Weber, I. T., and T. A. Steitz. 1987. J. Mol. Biol. 198:311-326). Furthermore, residues Glu-594, Gly-597, Ile-600, and Leu-601 of these helices belonging to two different subunits must be in close proximity. In the closed state the C-helices are in a different configuration and undergo significant fluctuations.     

Matrix metalloproteinase-9 and -2 enhance the ligand sensitivity of photoreceptor cyclic nucleotide-gated channels

Photoreceptor cyclic nucleotide-gated (CNG) channels are the principal ion channels responsible for transduction of the light-induced change in cGMP concentration into an electrical signal. The ligand sensitivity of photoreceptor CNG channels is subject to regulation by intracellular signaling effectors, including calcium-calmodulin, tyrosine kinases and phosphoinositides. Little is known, however, about regulation of channel activity by modification to extracellular regions of CNG channel subunits. Extracellular proteases MMP9 and -2 are present in the interphotoreceptor matrix adjacent to photoreceptor outer segments. Given that MMPs have been implicated in retinal dysfunction and degeneration, we hypothesized that MMP activity may alter the functional properties of photoreceptor CNG channels. For heterologously expressed rod and cone CNG channels, extracellular exposure to MMPs dramatically increased the apparent affinity for cGMP and the efficacy of cAMP. These changes to ligand sensitivity were not prevented by destabilization of the actin cytoskeleton or by disruption of integrin mediated cell adhesion, but could be attenuated by inhibition of MMP catalytic activity. MMP-mediated gating changes exhibited saturable kinetic properties consistent with enzymatic processing of the CNG channels. In addition, exposure to MMPs decreased the abundance of full-length expressed CNGA3 subunits, with a concomitant increase in putative degradation products. Similar gating effects and apparent proteolysis were observed also for native rod photoreceptor CNG channels. Furthermore, constitutive apparent proteolysis of retinal CNGA1 and retinal MMP9 levels were both elevated in aged mice compared with young mice. Together, these results provide evidence that MMP-mediated proteolysis can regulate the ligand sensitivity of CNG channels.     

Matrix metalloproteinase-9 and -2 enhance the ligand sensitivity of photoreceptor cyclic nucleotide-gated channels

Photoreceptor cyclic nucleotide-gated (CNG) channels are the principal ion channels responsible for transduction of the light-induced change in cGMP concentration into an electrical signal. The ligand sensitivity of photoreceptor CNG channels is subject to regulation by intracellular signaling effectors, including calcium-calmodulin, tyrosine kinases and phosphoinositides. Little is known, however, about regulation of channel activity by modification to extracellular regions of CNG channel subunits. Extracellular proteases MMP9 and -2 are present in the interphotoreceptor matrix adjacent to photoreceptor outer segments. Given that MMPs have been implicated in retinal dysfunction and degeneration, we hypothesized that MMP activity may alter the functional properties of photoreceptor CNG channels. For heterologously expressed rod and cone CNG channels, extracellular exposure to MMPs dramatically increased the apparent affinity for cGMP and the efficacy of cAMP. These changes to ligand sensitivity were not prevented by destabilization of the actin cytoskeleton or by disruption of integrin mediated cell adhesion, but could be attenuated by inhibition of MMP catalytic activity. MMP-mediated gating changes exhibited saturable kinetic properties consistent with enzymatic processing of the CNG channels. In addition, exposure to MMPs decreased the abundance of full-length expressed CNGA3 subunits, with a concomitant increase in putative degradation products. Similar gating effects and apparent proteolysis were observed also for native rod photoreceptor CNG channels. Furthermore, constitutive apparent proteolysis of retinal CNGA1 and retinal MMP9 levels were both elevated in aged mice compared with young mice. Together, these results provide evidence that MMP-mediated proteolysis can regulate the ligand sensitivity of CNG channels.     

Primary structure and functional expression of a Drosophila cyclic nucleotide-gated channel present in eyes and antennae.

Cyclic nucleotide-gated (CNG) ion channels serve as downstream targets of signalling pathways in vertebrate photoreceptors and olfactory sensory neurons. Whether CNG channels subserve similar functions in invertebrate photoreception and olfaction is unknown. We have cloned genomic DNA and cDNA encoding a cGMP-gated channel from Drosophila. The gene contains at least seven exons. Heterologous expression of cloned cDNA in both Xenopus oocytes and HEK 293 cells gives rise to functional ion channels. The Drosophila CNG channel is approximately 50-fold more sensitive to cGMP than to cAMP. The voltage dependence of blockage by divalent cations is different compared with the CNG channel of rod photoreceptors, and the Ca2+ permeability is much larger. The channel mRNA is expressed in antennae and the visual system of Drosophila. It is proposed that CNG channels are involved in transduction cascades of both invertebrate photoreceptors and olfactory sensillae.     

Mechanism of inhibition of cyclic nucleotide-gated channel by protein tyrosine kinase probed with genistein

Rod cyclic nucleotide-gated (CNG) channels are modulated by changes in tyrosine phosphorylation catalyzed by protein tyrosine kinases (PTKs) and phosphatases (PTPs). We used genistein, a PTK inhibitor, to probe the interaction between the channel and PTKs. Previously, we found that in addition to inhibiting tyrosine phosphorylation of the rod CNG channel alpha-subunit (RETalpha), genistein triggers a noncatalytic inhibitory interaction between the PTK and the channel. These studies suggest that PTKs affects RETalpha channels in two ways: (1) by catalyzing phosphorylation of the channel protein, and (2) by allosterically regulating channel activation. Here, we study the mechanism of noncatalytic inhibition. We find that noncatalytic inhibition follows the same activity dependence pattern as catalytic modulation (phosphorylation): the efficacy and apparent affinity of genistein inhibition are much higher for closed than for fully activated channels. Association rates with the genistein-PTK complex were similar for closed and fully activated channels and independent of genistein concentration. Dissociation rates were 100 times slower for closed channels, which is consistent with a much higher affinity for genistein-PTK. Genistein-PTK affects channel gating, but not single channel conductance or the number of active channels. By analyzing single channel gating during genistein-PTK dissociation, we determined the maximal open probability for normal and genistein-PTK-bound channels. genistein-PTK decreases open probability by increasing the free energy required for opening, making opening dramatically less favorable. Ni(2+), which potentiates RETalpha channel gating, partially relieves genistein inhibition, possibly by disrupting the association between the genistein-PTK and the channel. Studies on chimeric channels containing portions of RETalpha, which exhibits genistein inhibition, and the rat olfactory CNG channel alpha-subunit, which does not, reveals that a domain containing S6 and flanking regions is the crucial for genistein inhibition and may constitute the genistein-PTK binding site. Thus, genistein-PTK stabilizes the closed state of the channel by interacting with portions of the channel that participate in gating.     

A cysteine scan of the inner vestibule of cyclic nucleotide-gated channels reveals architecture and rearrangement of the pore

Cyclic nucleotide-gated (CNG) channels belong to the P-loop-containing family of ion channels that also includes KcsA, MthK, and Shaker channels. In this study, we investigated the structure and rearrangement of the CNGA1 channel pore using cysteine mutations and cysteine-specific modification. We constructed 16 mutant channels, each one containing a cysteine mutation at one of the positions between 384 and 399 in the S6 region of the pore. By measuring currents activated by saturating concentrations of the full agonist cGMP and the partial agonists cIMP and cAMP, we show that mutating S6 residues to cysteine caused both favorable and unfavorable changes in the free energy of channel opening. The time course of cysteine modification with 2-aminoethylmethane thiosulfonate hydrochloride (MTSEA) was complex. For many positions we observed decreases in current activated by cGMP and concomitant increases in current activated by cIMP and cAMP. A model where modification affected both gating and permeation successfully reproduced the complex time course of modification for most of the mutant channels. From the model fits to the time course of modification for each mutant channel, we quantified the following: (a) the bimolecular rate constant of modification in the open state, (b) the change in conductance, and (c) the change in the free energy of channel opening for modification of each cysteine. At many S6 cysteines, modification by MTSEA caused a decrease in conductance and a favorable change in the free energy of channel opening. Our results are interpreted within the structural framework of the known structures of KcsA and MthK. We conclude that: (a) MTSEA modification affects both gating and permeation, (b) the open configuration of the pore of CNGA1 channels is consistent with the structure of MthK, and (c) the modification of S6 residues disrupts the helical packing of the closed channel, making it easier for channels to open.     

Mechanism of allosteric modulation of rod cyclic nucleotide-gated channels

The cyclic nucleotide-gated (CNG) channel of retinal rod photoreceptor cells is an allosteric protein whose activation is coupled to a conformational change in the ligand-binding site. The bovine rod CNG channel can be activated by a number of different agonists, including cGMP, cIMP, and cAMP. These agonists span three orders of magnitude in their equilibrium constants for the allosteric transition. We recorded single-channel currents at saturating cyclic nucleotide concentrations from the bovine rod CNG channel expressed in Xenopus oocytes as homomultimers of alpha subunits. The median open probability was 0.93 for cGMP, 0.47 for cIMP, and 0.01 for cAMP. The channels opened to a single conductance level of 26-30 pS at +80 mV. Using signal processing methods based on hidden Markov models, we determined that two closed and one open states are required to explain the gating at saturating ligand concentrations. We determined the maximum likelihood rate constants for two gating schemes containing two closed (denoted C) and one open (denoted O) states. For the C left and right arrow C left and right arrow O scheme, all rate constants were dependent on cyclic nucleotide. For the C left and right arrow O left and right arrow C scheme, the rate constants for only one of the transitions were cyclic nucleotide dependent. The opening rate constant was fastest for cGMP, intermediate for cIMP, and slowest for cAMP, while the closing rate constant was fastest for cAMP, intermediate for cIMP, and slowest for cGMP. We propose that interactions between the purine ring of the cyclic nucleotide and the binding domain are partially formed at the time of the transition state for the allosteric transition and serve to reduce the transition state energy and stabilize the activated conformation of the channel. When 1 microM Ni2+ was applied in addition to cyclic nucleotide, the open time increased markedly, and the closed time decreased slightly. The interactions between H420 and Ni2+ occur primarily after the transition state for the allosteric transition.     

A point mutation in the pore region alters gating, Ca(2+) blockage, and permeation of olfactory cyclic nucleotide-gated channels

Upon stimulation by odorants, Ca(2+) and Na(+) enter the cilia of olfactory sensory neurons through channels directly gated by cAMP. Cyclic nucleotide-gated channels have been found in a variety of cells and extensively investigated in the past few years. Glutamate residues at position 363 of the alpha subunit of the bovine retinal rod channel have previously been shown to constitute a cation-binding site important for blockage by external divalent cations and to control single-channel properties. It has therefore been assumed, but not proven, that glutamate residues at the corresponding position of the other cyclic nucleotide-gated channels play a similar role. We studied the corresponding glutamate (E340) of the alpha subunit of the bovine olfactory channel to determine its role in channel gating and in permeation and blockage by Ca(2+) and Mg(2+). E340 was mutated into either an aspartate, glycine, glutamine, or asparagine residue and properties of mutant channels expressed in Xenopus laevis oocytes were measured in excised patches. By single-channel recordings, we demonstrated that the open probabilities in the presence of cGMP or cAMP were decreased by the mutations, with a larger decrease observed on gating by cAMP. Moreover, we observed that the mutant E340N presented two conductance levels. We found that both external Ca(2+) and Mg(2+) powerfully blocked the current in wild-type and E340D mutants, whereas their blockage efficacy was drastically reduced when the glutamate charge was neutralized. The inward current carried by external Ca(2+) relative to Na(+) was larger in the E340G mutant compared with wild-type channels. In conclusion, we have confirmed that the residue at position E340 of the bovine olfactory CNG channel is in the pore region, controls permeation and blockage by external Ca(2+) and Mg(2+), and affects channel gating by cAMP more than by cGMP.     

Single-channel properties of ionic channels gated by cyclic nucleotides.

This paper presents an extensive analysis of single-channel properties of cyclic nucleotide gated (CNG) channels, obtained by injecting into Xenopus laevis oocytes the mRNA encoding for the alpha and beta subunits from bovine rods. When the alpha and beta subunits of the CNG channel are coexpressed, at least three types of channels with different properties are observed. One type of channel has well-resolved, multiple conductive levels at negative voltages, but not at positive voltages. The other two types of channel are characterized by flickering openings, but are distinguished because they have a low and a high conductance. The alpha subunit of CNG channels has a well-defined conductance of about 28 pS, but multiple conductive levels are observed in mutant channels E363D and T364M. The conductance of these open states is modulated by protons and the membrane voltage, and has an activation energy around 44 kJ/mol. The relative probability of occupying any of these open states is independent of the cGMP concentration, but depends on extracellular protons. The open probability in the presence of saturating cGMP was 0.78, 0.47, 0.5, and 0.007 in the w.t. and mutants E363D, T364M, and E363G, and its dependence on temperature indicates that the thermodynamics of the transition between the closed and open state is also affected by mutations in the pore region. These results suggest that CNG channels have different conductive levels, leading to the existence of multiple open states in homomeric channels and to the flickering behavior in heteromeric channels, and that the pore is an essential part of the gating of CNG channels.     

Nitric Oxide Inhibition of the Rat Olfactory Cyclic Nucleotide-Gated Cation Channel

The effects of nitric oxide (NO) and other cysteine modifying agents were examined on cyclic nucleotide-gated (CNG) cation channels from rat olfactory receptor neurons. The NO compounds, S-nitroso-cysteine (SNC) and 3-morpholino-sydnonomine (SIN-1), did not activate the channels when applied for up to 10 min. The cysteine alkylating agent, N-ethylmaleimide (NEM), and the oxidising agent, dithionitrobensoate (DTNB), were also without agonist efficacy. Neither SNC nor DTNB altered the cAMP sensitivity of the channels. However, 2-min applications of SIN-1, SNC and DTNB inhibited the cAMP-gated current to approximately 50% of the control current level. This inhibition showed no spontaneous reversal for 5 min but was completely reversed by a 2-min exposure to DTT. The presence of cAMP protected the channels against NO-induced inhibition. These results indicate that inhibition is caused by S-nitrosylation of neighboring sulfhydryl groups leading to sulfhydryl bond formation. This reaction is favored in the closed channel state. Since recombinantly expressed rat olfactory α and β CNG channel homomers and α/β heteromers are activated and not inhibited by cysteine modification, the results of this study imply the existence of a novel subunit or tightly bound factor which dominates the effect of cysteine modification in the native channels. As CNG channels provide a pathway for calcum influx, the results may also have important implications for the physiological role of NO in mammalian olfactory receptor neurons. Received: 30 March 1998/Revised: 17 June 1998