Scentsational Ion Channels
Authors: Stuart Firestein
Journal: Neuron (1996)

Ion channels gated by cyclic nucleotide second messenger (CNG channels) occupy a pivotal position in the transduction cascade in both vision and olfaction. Their activation by either cAMP or cGMP is the last step in the intracellular biochemical cascade and the first step in the generation of the electrical response to a stimulus. The discovery of CNG channels, first in photoreceptors and then in olfactory neurons, and subsequent cloning (reviewed recently by19Zagotta W.N. Siegelbaum S.A. Annu. Rev. Neurosci. 1996; 19: 235-263Crossref PubMed Scopus (403) Google Scholar) introduced them as a new ion channel family, but one that was curiously related to voltage-gated channels in both gene and protein structure. In the last few years, significant biophysical advances have been made in appreciating the mechanisms of gating, ion selectivity, and ligand sensitivity in this class of channels. More recently their presence in other neuronal and non-neuronal cell types has been shown, although their role in these nonsensory contexts remains largely unknown. In addition to the biophysical interest in CNG channels, their direct activation by cyclic nucleotides has made them useful as detectors of the activity of the enzymatic cascades that generate cAMP or cGMP as intracellular messengers. That is, instead of activating a kinase (PKA for example) cascade that eventually modulates the behavior of a cell, in visual and olfactory receptors the production of cyclic nucleotide results immediately in electrical activity that can be measured easily and with high fidelity. Thus, the existence of these channels is often taken to indicate the identity of the underlying signal transduction scheme used by a particular cell for a particular signal. If it is a cGMP-sensitive channel, then one expects photoreceptor like mechanisms, e.g., a Gi GTPase and a cGMP-dependent phosphodiesterase; if cAMP activates the channel, then a Gs and adenylyl cyclase are the prime suspects. It is in this context that three recent contributions involving the role of cyclic nucleotide-gated channels in chemosensory systems are especially relevant (Brunet et al., 1996; Coburn and Bargmann, 1996; Komatsu et al., 1996). Adding to their interest as a group is that two of the papers utilize the powerful genetics of the nematode Caenorhabditis elegans, while the other exploits the equally powerful, if more expensive, genetics of the knockout mouse. The importance of cAMP in vertebrate olfactory transduction was established by biochemical, molecular, and physiological experiments carried out by several laboratories during the 1980s (recently reviewed by16Shepherd G.M. Neuron. 1994; 13: 771-790Abstract Full Text PDF PubMed Scopus (250) Google Scholar). The final common step in the vertebrate response to many, if not all, odors is the generation of cAMP and the activation of a cAMP-gated channel (11Nakamura T. Gold G.H. Nature. 1987; 325: 442-444Crossref PubMed Scopus (823) Google Scholar, 6Firestein S. Darrow B. Shepherd G.M. Neuron. 1991; 6: 825-835Abstract Full Text PDF PubMed Scopus (134) Google Scholar), leading to depolarization and action potential generation. In frogs, it was shown that out of a panel of some 65 odors about half generated a biochemically significant increase in cAMP (17Sklar P.B. Anholt R.R.H. Snyder S.H. J. Biol. Chem. 1986; 261: 15538-15543Abstract Full Text PDF PubMed Google Scholar). This was initially taken as evidence that a second pathway might exist for these odors. However, a simple but elegant experiment by Gold and colleagues (10Lowe G. Nakamura T. Gold G.H. Proc. Natl. Acad. Sci. USA. 1989; 86: 5641-5645Crossref PubMed Scopus (106) Google Scholar) showed that this result might be explained rather by the fact that frogs simply have a reduced sensitivity (or even none at all) to those odors for which no cAMP could be detected and that a single pathway was still sufficient to account for chemotransduction in this species. Nonetheless, in the late 1980s, biochemical evidence for an inositol 1,4,5-trisphosphate (IP3) pathway was put forward, primarily by the laboratory of Heinz Breer in Stuttgart (1Boekhoff I. Raming K. Breer H. J. Comp. Physiol. B. 1990; 160: 99-103Crossref Scopus (48) Google Scholar). Breer and his colleagues showed that in response to a small subset of odors they could detect the transient generation of IP3 in a preparation consisting of enriched ciliary membranes from olfactory neurons of rats. Additionally, it was found that odors that increased cAMP did not increase IP3 and vice versa: in other words, the two pathways operated separately and were selective for different odors. Similar evidence supporting an IP3 pathway was developed in catfish and other species (reviewed by3Breer H. Semin. Cell Biol. 1994; 5: 25-32Crossref PubMed Scopus (56) Google Scholar). Unfortunately, the physiological evidence for an IP3-mediated olfactory response has never been as strong as the biochemical. cAMP analogs applied to single cells under patch clamp recording conditions cause an inward depolarizing current in virtually every olfactory neuron tested in vertebrates ranging from newt to rat, whereas IP3 or its analogs injected into a cell through a patch pipette rarely if ever induce electrical activity (7Firestein S. Zufall F. Shepherd G.M. J. Neurosci. 1991; 11: 3565-3572Crossref PubMed Google Scholar). An exception to this is a report by 12Okada Y. Teeter J.H. Restrepo D. J. Neurophys. 1994; 71: 595-602Crossref PubMed Scopus (49) Google Scholar of IP3-induced currents in isolated rat olfactory neurons. However, in this case, injection of IP3 produced at least two different currents, and neither was shown to be able to suppress an odorant-induced current, as was the case for cAMP-dependent currents. A further difficulty for the IP3 hypothesis is that IP3 channels are not normally found on the plasma membrane of cells, but are restricted to endoplasmic reticulum or other intracellular organelles. Olfactory cilia have no endoplasmic reticulum or other known internal Ca2+ storage organelle, and therefore an IP3-gated channel would, of necessity, have to be in the plasma membrane. This has yet to be demonstrated conclusively in a vertebrate. (The story is different for invertebrates, as will be discussed below.) A novel approach to this controversy is reported in the recent paper by Brunet et al. (1996) in which a knockout mouse devoid of a functional cAMP-gated channel was tested for its ability to respond to odors. The gene mutation deleted a large region from the middle of the coding region, producing a protein that fails to form a functional channel subunit. Nonetheless, the knockout animals appeared to posses an otherwise normal olfactory epithelium showing high levels of expression of olfactory-specific genes encoding proteins in the transduction pathway. Only the functional α subunit of the CNG channel was absent in the knockout animals, indicating that the targeted channel mutation was specific and caused no generalized effects in olfactory neurons. One curious early result with these mice was that a large number of the male mice hemizygous for the mutant channel (the α subunit gene apparently resides on the X chromosome) died within 1 day of birth. Although these animals appeared normal at birth, they were unable to locate nipples and failed to suckle even if placed near a nipple. This is consistent with well-established behavioral data showing the importance of “nipple odors” in promoting suckling behavior (8Greer C.A. Stewart W.B. Teicher M.H. Shepherd G.M. J. Neurosci. 1982; 2: 1744-1759PubMed Google Scholar). The mutant mice, as might be expected, suffered from severe dehydration. This early indication of anosmia (the absence of an olfactory sense) was borne out in electrophysiological experiments. While individual olfactory neurons had normal resting potentials and were capable of generating action potentials, indicating that they possessed a normal complement of functioning voltage-gated channels, the electrical component of the odor response appeared to be compromised. To assess the odor responsiveness, Brunet et al. utilized a field potential recording technique known as the electro-olfactogram (EOG). The EOG is an extracellular recording method that sums the activity of cells in a local region of intact olfactory epithelium. Although there are some artifacts associated with the placement of the recording electrode (i.e., the depth at which the electrode is placed can significantly alter the amplitude of the responses), it is a generally reliable method for making a global measurement of odor sensitivity. Normally, the EOG is a negative transepithelial potential of a few millivolts amplitude that arises due to the extracellular current flow generated by the inward sensory current across the membrane of the olfactory cilia. In these experiments, EOG measurements were made from the olfactory epithelia of 1-day-old pups exposed to brief pulses of a panel of nine odorants. Four of these odorants had been reported to elicit increases in cAMP, while five led to IP3 generation in biochemical assays. Additionally, three “natural” odor stimuli known to be behaviorally significant were tested. These included mouse urine (conspecific recognition), coyote urine (predator recognition), and peanut butter (an attractive food source used as bait in mouse traps). The results were unequivocal. Wild-type mice responded to all of the odor stimuli with a negative EOG wave; hemizygous mutant mice responded to none of the odors. The knockouts were completely anosmic. Without a cAMP-gated channel, and therefore without the intact cAMP cascade, there appeared to be no other alternatives for producing an excitatory response to odors. Although this says nothing about inhibitory (hyperpolarizing) responses, there is currently only slight evidence that inhibition plays an important role in olfactory signaling in vertebrates (reviewed by16Shepherd G.M. Neuron. 1994; 13: 771-790Abstract Full Text PDF PubMed Scopus (250) Google Scholar). How can these results be reconciled with the biochemical evidence for odor-induced IP3 production? According to Brunet et al., they cannot, and indeed, it is difficult to see a way out. There are several caveats, as with virtually any knockout results, but none seem to be particularly serious in this instance. Are there more far-reaching effects of the knockout than are documented? Does the absence of a CNG channel during development somehow inhibit the expression of one or more IP3 related enzymes (i.e., is the IP3 pathway intact in the knockout animal)? Are there unexpected compensatory effects due to the absence of the CNG channel? (An excellent recent review by Lem and Makino [1996] details such unexpected results in the phototransduction cascade of transgenic mice.) In spite of these concerns, the evidence here seems quite compelling: the CNG channel is clearly an integral part of the transduction machinery and the resultant anosmia is so thorough that ascribing the results to secondary deficits lacks parsimony. One might imagine that, since all of these experiments were performed on 1-day-old mice, the IP3 pathway appears and functions later in development, although this too seems unlikely since the olfactory epithelium is fully developed at birth in mammals. Finally, there is the possibility that IP3 is not directly involved in olfactory transduction but plays a modulatory role. In recent biochemical experiments (13Ronnett G.V. Cho H. Hester L.D. Wood S.F. Snyder S.H. J. Neurosci. 1993; 13: 1751-1758Crossref PubMed Google Scholar), most odorant stimuli were shown to produce both IP3 and cAMP, suggesting that IP3 may indeed play a such a secondary role. It now seems imperative to replicate the original biochemical and physiological experiments in which IP3 generation was observed in the CNG channel knockout mice. At the same time that the IP3 pathway is coming under attack in vertebrates, the evidence for an alternative to the cAMP cascade is mounting in invertebrates. In biochemical assays similar to those utilized in rat, Breer and his colleagues also showed an increase in IP3 to pheromonal odors in insects (2Boekhoff I. Tareilus E. Strotmann J. Breer H. EMBO J. 1990; 9: 2453-2458Crossref PubMed Scopus (252) Google Scholar). Unlike the vertebrate situation, there is also strong physiological evidence to support an odor-dependent IP3 response in invertebrates. In the lobster, an IP3-gated channel has been identified in the plasma membrane, and in a particularly striking experiment, a patch of membrane containing this IP3 channel was inserted into a lobster olfactory neuron, where it could be activated by applying odors to the cell (5Fadool D.A. Ache B.W. Neuron. 1992; 9: 907-918Abstract Full Text PDF PubMed Scopus (204) Google Scholar). This provided two crucial pieces of evidence lacking in vertebrates. An IP3 channel clearly existed on the plasma membrane (this is still the only definitive evidence anywhere for a plasma membrane IP3 channel) and the level of IP3 in an intact, living olfactory neuron increased with odor stimulation. Finally, there is some preliminary evidence from genetic screens that odor transduction in Drosophila is mediated in part by enzymes generally found in the IP3 pathway (18Smith D. Curr. Opin. Neurobiol. 1996; 6: 500-505Crossref PubMed Scopus (21) Google Scholar), although this work is not yet conclusive. Interestingly the results of investigations in C. elegans aimed at identifying a CNG channel in the chemosensory pathway have provided not only strong evidence for a cAMP transduction cascade, but also a further indication that IP3 may play a role in these animals as well. Two laboratories (Cori Bargmann at the University of California, San Francisco, and Yasumi Oshima at Kyushu University) have identified subunits of a CNG channel in the nematode by characterizing and cloning the genes responsible for previously identified chemosensory and thermosensory mutations. However, the C. elegans mutants are still able to detect distinct subsets of odors, suggesting that in C. elegans CNG channels might not be the whole story. CNG channels are now known to exist as heteromultimers of two subunits called OCNC1 and OCNC2 (or alternatively α and β; 19Zagotta W.N. Siegelbaum S.A. Annu. Rev. Neurosci. 1996; 19: 235-263Crossref PubMed Scopus (403) Google Scholar). Four subunits are required to form a functional channel, but the stoichiometry is unknown. The α subunit can form a homomultimeric channel when expressed in a heterologous system. The β subunit when expressed alone cannot be activated by cyclic nucleotides, but when coexpressed with α subunits forms a heteromeric channel whose properties more closely resemble those of the native channel. In the C. elegans papers, Komatsu et al. have identified the α subunit and Coburn and Bargmann the β subunit of a CNG channel. In both instances, the investigators proceeded along similar experimental lines. Mutant animals were tested for chemosensory deficits, the mutant genes were localized, cloned, and in the case of the α subunit, functionally expressed. Proteins of 733 amino acids (α) and 800 amino acids (β) were predicted. These displayed significant sequence similarity to the vertebrate CNG channel subunits. Both C. elegans subunits would be predicted to be sensitive to cGMP, based on sequence similarities to the retinal rod type channel in the cyclic nucleotide-binding region. The α subunit, encoded by the gene tax-4, demonstrates some peculiar biophysical properties when expressed as a homomeric protein. The Kd for cGMP was 1.4 × 10−7, significantly lower than the vertebrate channel, which possesses a Kd around 3 × 10−5. In addition, the Hill coefficient was less than 1, suggesting that the channel could be activated by a single molecule of cGMP. In the vertebrate at least 2, and most likely 3 or 4, molecules of cyclic nucleotide are required for activation. A future collaboration between these two laboratories should enable the coexpression of the two subunits, providing important information on the characteristics of the wild-type channel. What about the behavior of the mutant animals? In contrast with the knockout mouse, both the tax-4 and tax-2 mutants were not completely anosmic. Both animals displayed specific anosmia to salts and the odorants benzaldehyde, 2-butanone, and isoamyl alcohol. However, they responded normally to the odorants diacetyl, pyrazine, and trimethylthiazole. C. elegans use several pairs of identified neurons for chemosensory processing of different odors, and this behavior is consistent with defects in the functioning of the AWC olfactory neurons, but normal functioning in the AWA olfactory neurons. Using green fluorescent protein (GFP) as a marker for tax-4 or tax-2 expression, both laboratories were able to demonstrate that the channel was expressed in AWC neurons and not in the AWA neurons. Thus, it appears that the AWA olfactory neurons utilize an alternative signal transduction cascade. Even a brain surgeon would be thinking about looking for IP3-associated enzymes. The possible existence of an IP3 pathway in C. elegans is relevant to the identification of the family of odor receptors as well. Bargmann's laboratory recently cloned the first odor receptor gene in an invertebrateodr-10 (15Sengupta P. Chou J.H. Bargmann C.I. Cell. 1996; 84: 899-909Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar), which is expressed exclusively in the AWA chemosensory neurons, the neurons that do not posses the CNG channel. Interestingly these receptors are only mildly similar to the vertebrate odor receptor family, and may therefore represent a class of receptors that could be coupled to the IP3 pathway rather than to cAMP. Could there be an additional family of receptors coupled to the cAMP pathway that might share more homology with the vertebrate family? As an added bonustax-2 and tax-4 expression were also detected in neurons known to be important in temperature sensitivity, and both mutants were indeed defective in thermosensory abilities, hinting that thermosensation is also mediated by a G protein–coupled receptor and cyclic nucleotide–based second messenger system. This could certainly provide an important breakthrough in understanding a still largely mysterious sensory system. Finally, but perhaps most enticing, Coburn and Bargmann also observed striking defects in the growth of sensory axons in tax-2 and tax-4 mutants. The defects were primarily expressed as an overgrowth of axonal processes, with subsets of sensory neurons displaying either inappropriate additional axonal branches or a failure to terminate upon reaching their target. Coburn and Bargmann used a Tax-2/GFP fusion protein and observed strong expression of the CNG channel β subunit in both the cilia and the axons, in particular at the synaptic terminals of chemosensory neurons. A possible role for CNG channels in development has been quietly gaining currency. CNG channels can be activated by nitric oxide (NO) in the absence of cyclic nucleotide (4Broillet M.-C. Firestein S. Neuron. 1996; 16: 377-385Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), and NO synthase is known to be expressed transiently in the mammalian olfactory epithelium during development and neuronal regeneration (14Roskams A.J. Bredt D.S. Dawson T.M. Ronnett G.V. Neuron. 1994; 13: 289-299Abstract Full Text PDF PubMed Scopus (219) Google Scholar). It was recently proposed that this might be a mechanism for providing activity during neuronal growth in the absence of a stimulus (4Broillet M.-C. Firestein S. Neuron. 1996; 16: 377-385Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Now it appears that the effect may be even more direct, especially if CNG channels can also be localized to axons of vertebrate chemosensory neurons. Kai Zinn and his associates have recently found high levels of expression of β subunit in the growth cones of hippocampal neurons in culture (as discussed by Coburn et al.), and the β subunit alone, while unresponsive to cyclic nucleotides, can be activated by NO (4Broillet M.-C. Firestein S. Neuron. 1996; 16: 377-385Abstract Full Text Full Text PDF PubMed Scopus (133) Google ScholarSoc. Neurosci., abstract). This may also help to explain the striking finding in the knockout mouse that the olfactory epithelium appeared to have developed normally, in spite of the complete anosmia owing to the loss of the α subunit. Perhaps β subunits are still expressed in the neuronal axons, permitting normal growth and development. It appears that CNG channels may be for more than just smelling the roses. Errata et al.NeuronDecember, 1996In Brief Full-Text PDF Open Archive