I. INTRODUCTION

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Ion channels that are directly activated by cyclic nucleotides [cyclic nucleotide-gated (CNG) channels] are relatively recent arrivals in the world of ion channels. Their discovery was intimately tied with the quest for the intracellular messenger that mediates the photoresponse in retinal photoreceptors. Since the late 1960s two messenger molecules, Ca²⁺ and cGMP, were the most likely candidates to control the "light-sensitive conductance" in the outer segement envelope from rod photoreceptors (for review, see Ref. 431). Because the prevailing dogma was that cyclic nucleotides control the activity of proteins through phosphorylation mediated by cyclic nucleotide-dependent kinases, it came as a surprise when in 1985 Fesenko et al. (103) reported that cGMP can directly activate the light-dependent channel of rods.

Within a relatively short time, similar channels were identified in cone photoreceptors (153), chemosensitive cilia of olfactory sensory neurons (OSNs) (293), and the pineal gland (94). Molecular cloning of CNG channels became possible when the channel protein was purified and unequivocally identified by functional reconstitution into artificial liposomes and lipid bilayers (79, 151). Partial amino acid information derived from the purified protein allowed the successful cloning and functional expression of the first CNG channel gene (188). The molecular identification of a CNG channel has sparked much progress over the past several years. It is now obvious that CNG channels are not unique to photoreceptors and OSNs, but are expressed in other neurons and nonneuronal tissues alike. CNG channels belong to a heterogeneous gene superfamily of ion channels that share a common transmembrane topology and pore structure and that harbor in their COOH-terminal region a binding domain for nucleoside 3',5'-cyclic monophosphates (cNMPs). Other members of this superfamily are the so-called hyperpolarization-activated and cyclic nucleotide-gated (HCN) pacemaker channels (for review, Ref. 189), the ether-a-gogo (EAG) and human eag-related gene (HERG) family of voltage-activated K⁺ channels (for review, see Ref. 117), and several plant K⁺ channels commonly referred to as KAT, AKT, and KST channels (for review, see Ref. 359). The focus of this review is on CNG channels.

	II. BASIC FUNCTIONAL PROPERTIES: AN OVERVIEW

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Cyclic nucleotides directly activate CNG channels by binding to a site on the channel protein. The dependence of channel activation on the ligand concentration is steep, indicating that several, most probably four, molecules of the ligand are required to fully open the channels. All CNG channels respond to some extent to both cAMP and cGMP. In rods and cones, CNG channels sharply discriminate between cAMP and cGMP, whereas channels in chemosensitive cilia of OSNs by and large respond equally well to both ligands. The ability to discriminate between ligands is commonly referred to as ligand selectivity. Selectivity can be achieved either by differential control of ligand affinity or efficacy or a combination of both. Ligand affinity is a measure of how tightly cyclic nucleotides bind to the channel. Efficacy refers to the ability to open the channel once the ligand has been seated in the binding cavity. The molecular basis of ligand affinity, efficacy, and selectivity is discussed in section VII.

CNG channels are nonselective cation channels that poorly discriminate between alkali ions and even allow the passage of divalent cations, in particular Ca²⁺. Therefore, at rest (60 mV) and when bathed in a physiological ion milieu, CNG channels conduct mixed inward currents carried by Na⁺ and Ca²⁺. To permeate, the ions bind to a site inside the channel pore. The dwell time at this binding site is significantly longer for Ca²⁺ than for monovalent cations. As an important result, Ca²⁺ blocks the current of the more permeant Na⁺. The pronounced Ca²⁺ permeability and the concomitant blockage of Na⁺ current by divalent cations is crucially important for the channel's function and underlies, for example, the ability of rod photoreceptors to detect single photons and to adapt to steady illumination. The interaction of Ca²⁺ with the channel pore and the consequences for the physiology of cells is discussed in section VIII.

Unlike ligand-gated neurotransmitter receptors, CNG channels do not desensitize in the continuous presence of the ligand. Both the cooperative and sustained activation predestines CNG channels to serve as a molecular switch that faithfully tracks the cAMP or cGMP concentration in a cell. Although the channels do not desensitize, their activity is nonetheless modulated, notably by the Ca²⁺-binding protein calmodulin and by phosphorylation. The potential mechanisms of this modulation and its physiological significance are presented in section IX.

	III. EXCURSION ON CYCLIC NUCLEOTIDES

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Studies on cellular cAMP and cGMP signaling frequently relied on the use of chemical derivatives of cAMP and cGMP. Ever since the first substances have been deployed for studies of cAMP- and cGMP-dependent protein kinases (PKA and PKG, respectively) and phosphodiesterases (PDE), the list of new derivatives has grown long. More importantly for the subject of this review, derivatives of cyclic nucleotides were also instrumental for functional studies of CNG channels in various cellular systems. cAMP and cGMP have been modified at the cyclic phosphodiester group, the 2'- and 3'-hydroxyls of the ribofuranose moiety, and at the adenine and guanine ring systems (see Fig. 12). Phosphorothioate derivatives of cAMP and cGMP have been employed to study the activation properties of native CNG channels in photoreceptors (217, 442) and olfactory neurons (217) and the heterologously expressed A1 and A2 channels (217; see sect. VII). Cyclic nucleotides substituted at the C-8 position have proven particularly valuable for cellular studies. For one reason, a number of substituents at C-8 render these molecules more membrane permeant than cAMP and cGMP itself. For example, 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) and 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (8-pCPT-cGMP) are 6- and 90-fold, respectively, more lipophilic than cGMP (63). Therefore, these compounds readily penetrate membranes providing a simple route for delivery to the intracellular binding sites. Another favorable feature of most but not all C-8-substituted derivatives is their high potency in activating CNG channels (54, 66, 203, 390, 442). For example, in rod photoreceptors and OSNs, 8-BrcGMP and 8-pCPT-cGMP activate CNG channels at ~10- and 80-fold, respectively, lower concentrations than cGMP (112, 411, 442). Finally, 8-BrcGMP and 8-pCPT-cGMP are poor substrates for several PDE isoforms and resist hydrolysis. 8-pCPT-cGMP is not measurably hydrolyzed by three different PDEs, and the rate of 8-BrcGMP hydrolysis is 4- to 40-fold lower than that for cGMP (63). The PDE6 of rods and cones hydrolyzes 8-BrcGMP 170- to 500-fold more slowly than cGMP (19, 442). Thus the triad of lipophilicity, potency of activation, and resistance to hydrolysis makes these C-8-substituted cyclic nucleotides attractive agents for the study of CNG channels.

Owing to the multiple actions of cyclic nucleotides inside intact cells, however, results obtained with these derivatives may sometimes be difficult to interpret. Delivering, for example, a relatively high concentration of cyclic nucleotides to intact cells will eventually activate all cAMP- and cGMP-dependent processes including phosphorylation by PKA and PKG, activation or inhibition of some PDE isoforms, and eventually cAMP-dependent regulation of gene expression. In addition, hydrolysis-resistant analogs may behave as competitive antagonists that bind to the catalytic site of PDE and thereby hinder hydrolysis of both cAMP and cGMP. As a result, endogenous cyclic nucleotides may accumulate during the course of the experiment. Finally, the infusion of cells with cyclic nucleotides either from a pipette in the whole cell configuration or by bathing in a medium containing membrane-permeable analogs is inherently slow in relation to the speed of action on kinases, PDEs, and CNG channels. Therefore, it will be exceedingly difficult to experimentally dissect the action of cyclic nucleotides on CNG channels from other cellular effects.

This kind of experiment can be ameliorated using chemical derivatives "dubbed" caged cyclic nucleotides. Caged compounds are molecules whose biological activity has been disabled by chemical modification. Photolysis cleaves the modifying group ("uncaging"), thereby rapidly releasing the active molecule. (For a collection of reviews on caged compounds, see Reference 261.) Four different classes of caging groups have been used: 4,5-dimethoxy-2-nitrobenzyl (DMNB), 1-(2-nitrophenyl)ethyl (NPE), desoxybenzoinyl (Desyl), and derivatives of (7-methoxy-coumarin-4-yl)methyl (MCM) (Fig. 1). Synthesis of caged cyclic nucleotides by esterification of the phosphodiester group produces mixtures of axial and equatorial forms. The diastereomers differ significantly in solubility and solvolytic stability (150). We recommend that the pure isomeric forms are used for experiments. To be useful, caged cyclic nucleotides must meet specific requirements.

View larger version (14K): [in this window] [in a new window]   Fig. 1. Structure of caged cGMP and caging groups. Top two panels: axial and equatorial forms of cGMP. R, caging groups. Bottom panels: structure of four different caging groups (see text).

First, they should dissolve well in aqueous solution (~100 µM-10 mM). The higher the concentration of the caged compound inside the cell, the more cAMP or cGMP is released per flash. Second, caged compounds must be resistant toward solvolysis (the caging group is attached to the cyclic nucleotide through an ester group that can undergo hydrolysis in an aqueous medium). Otherwise, the free cyclic nucleotide is produced during the course of an experiment. Third, caged cyclic nucleotides should display high photoefficiencies, i.e., high molar absorptivities and high quantum yields. Finally, the photochemical reaction that releases the cyclic nucleotide should be fast (1 ms) compared with the physiological reaction under study.

The NPE- and DMNB-caged compounds either photolyze relatively slowly (NPE) or display rather low photoefficiencies (DMNB) (Table 1). Desyl-caged cAMP is very sensitive to solvolysis in aqueous buffer solution (Table 1), and the MCM-caged cyclic nucleotides are poorly soluble (148). However, caged derivatives of MCM combine a set of favorable properties rendering them ideal tools for intracellular studies (Table 1). For example, [6,7-bis(carboxymethoxy)coumarin-4-yl]methyl (BCMCM) esters of cAMP and cGMP are highly soluble (>1 mM) (147) and display a high quantum yield (0.1-0.15, Ref. 147; Table 1). The (7-diethylaminocoumarin-4-yl)methyl (DEACM) derivatives have an even higher quantum yield and absorptivity than the BCMCM-caged congeners. Moreover, MCM-based caged cyclic nucleotides are extremely stable in aqueous solution and react quickly within a few nanoseconds. The CMCM and BCMCM compounds are negatively charged, therefore, their high solubility; consequently, they are poorly membrane permeable and must be introduced into the cell by means of the recording pipette. Recently, bismethoxy and bisethoxy esters of BCMCM have been synthesized. These compounds are neutral, penetrate cell membranes, and accumulate inside the cell due to hydrolysis of the ester groups at the coumaryl moiety (V. Hagen and U. B. Kaupp, unpublished data). They are compounds of choice for studies on cell suspensions.

Caged cyclic nucleotides have been used to study 1) the rate of activation of the rod CNG channel in excised patches (185) and in the whole cell configuration (150, 339); 2) the Ca²⁺ permeability of CNG channels in intact rods and cones, OSNs, and cell lines (97, 311); 3) the desensitization of the olfactory CNG channel by Ca²⁺/calmodulin (CaM) (45); and 4) cyclic nucleotide-stimulated Ca²⁺ entry in mammalian spermatozoa (417).

	IV. CELLULAR FUNCTION

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The function of CNG channels has been firmly established in rod and cone photoreceptors, in extraretinal photoreceptors, and in sensory neurons of the olfactory epithelium. Electrophysiological studies of CNG channels in these sensory neurons provided a wealth of information regarding their ligand sensitivity, mechanism(s) of activation, modulation, and ion selectivity. The underlying channel polypeptides have been identified by molecular cloning; coexpression of the respective subunits in cell lines produces channels that recapitulate many, if not all, properties of the CNG channels in their native membranes. Although CNG channels also exist in other neurons and nonneuronal tissues, their specific functions are yet to be determined rigorously. Several other ion channels, whose molecular identity is uncertain or not known, have been speculated to be directly controlled by cyclic nucleotides.

Photoreceptors in invertebrates fall into two morphologically distinct subtypes: rhabdomeric photoreceptors with a microvilli-derived photosensitive structure and ciliary photoreceptors, whereas vertebrate photoreceptors are of the ciliary type. The ciliary-type photoreceptors, whether vertebrate or invertebrate, respond to light either with a depolarization or hyperpolarization, whereas the rhabdomere-type invertebrate photoreceptors respond exclusively with a depolarization. Ciliary photoreceptors, whether depolarizing or hyperpolarizing, vertebrate or invertebrate, use cGMP-signaling pathways. The targets of these pathways are CNG channels that either open or close in response to light. Although the phototransduction mechanism in depolarizing rhabdomeric photoreceptors is not entirely clear, the light-dependent channels belong to the family of trp/trpl channels that are targeted by a phosphoinositide pathway (365). Notwithstanding this well-established pathway, the presence of CNG channels in rhabdomeric photoreceptors has been considered. We will discuss the function of CNG channels in 1) the hyperpolarizing rods and cones, 2) the pinealocytes, 3) the depolarizing photoreceptor in the parietal eye of some lizards, 4) the hyperpolarizing ciliary photoreceptor of an invertebrate, and 5) rhabdomeric photoreceptors of invertebrates.

Rods respond to a light stimulus with a brief hyperpolarization by closing CNG channels in the surface membrane of the outer segment (for review, see Ref. 431). In the dark, channels are activated by the binding of cGMP, allowing a steady cation current ("dark current") to flow into the outer segment. Light triggers a sequence of enzymatic reactions that leads to the hydrolysis of cGMP. When CNG channels close, the inward current ceases and the cell hyperpolarizes. The enzyme cascade comprises the photopigment rhodopsin (R), the G protein transducin (T), and a PDE. Light stimulation decreases the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i), which 1) initiates the recovery from the light response by enhancing the synthesis of new cGMP molecules and 2) adjusts the sensitivity of the transduction machinery, a process known as light adaptation (for review, see Ref. 330). The CNG channel is crucially important for the control of [Ca²⁺]_i, because it provides the only source for Ca²⁺ influx into the outer segment. In rods, between 10 and 18% of the dark current (30 pA) is carried by Ca²⁺ (141, 229, 296, 435). Ca²⁺ entry through open CNG channels is balanced by Ca²⁺ extrusion through a Na⁺/Ca²⁺-K⁺ exchange mechanism (reviewed in Refs. 268, 329; Fig. 2). In light, when CNG channels close but the exchanger continues to clear Ca²⁺ from the cytosol, the balance is disturbed between Ca²⁺ entry and Ca²⁺ extrusion. The resulting decline in [Ca²⁺]_i provides a negative feedback mechanism that controls at least three biochemical processes. First, the activity of the guanylyl cyclase (GC) that synthesizes cGMP is stimulated as Ca²⁺ levels decrease. The Ca²⁺ sensitivity of the GC is relayed by two small Ca²⁺-binding proteins, designated GC-activating proteins (GCAP1 and GCAP2). At rest, when [Ca²⁺]_i is ~300-500 nM, the GCAPs prevail in the inactive form with Ca²⁺ bound. In light, when [Ca²⁺]_i is lowered to 50-100 nM, Ca²⁺ dissociates from GCAPs; the Ca²⁺-free form then stimulates GC activity (for review, see Refs. 202, 313). Second, the lifetime of active PDE is shortened through the phosphorylation of light-activated rhodopsin (R*) by the rhodopsin kinase. This reaction is mediated by another small Ca²⁺-binding protein, recoverin (for review, see Ref. 202). Finally, the ligand sensitivity of the CNG channel increases as [Ca²⁺]_i decreases. The regulation of ligand sensitivity by Ca²⁺ is mediated by a third Ca²⁺-dependent protein, CaM (169, 280). All three reactions by various degrees help to restore the dark state and to adjust the light sensitivity of the cell (reviewed in Ref. 330).

View larger version (36K): [in this window] [in a new window]   Fig. 2. Ca2+ feedback mechanism in rod and cone photoreceptors involving cyclic nucleotide-gated channels. PDE, phosphodiesterase; GCAP, guanylyl cyclase-activating protein.

A similar transduction scheme exists in cones, the photoreceptors responsible for vision in bright light. The fundamentally same events underlie phototransduction in rods and cones, and the two photoreceptor types utilize similar protein isoforms of the enzyme cascade. However, the light sensitivity of cones is 30- to 100-fold lower than that of rods, and cones adapt over a wider range of light intensities than rods (reviewed in Ref. 330). It has been suggested that differences in the Ca²⁺ homeostasis underlie the distinct light sensitivity and adaptation range of the two photoreceptor types. Important elements that control the dynamics and size of the changes in [Ca²⁺]_i are cell volume, the rate of Ca²⁺ clearance by the Na⁺/Ca²⁺-K⁺ exchanger, the Ca²⁺-buffering capacity of the cytoplasm, and Ca²⁺ entry through CNG channels (see Ref. 274 for a thorough discussion). Several observations demonstrate that the CNG channels in rods and cones differ in ion permeation, ligand sensitivity, and modulation by Ca²⁺. The relative ion permeability P_Ca/P_Na of CNG channels is more than three times larger in cones than in rods (21.7 and 6.5, respectively; Refs. 322, 414), and under physiological ionic conditions, the fraction of the dark current carried by Ca²⁺ is about twofold larger in cones than in rods (311, 318). The Na⁺/Ca²⁺-K⁺ exchange current in cones is at least one order of magnitude larger than that in rods. From these observations it has been inferred that the light-stimulated changes in [Ca²⁺]_i are far larger and faster in cones compared with rods.

The cGMP sensitivity of the CNG channel and its modulation by Ca²⁺ is also different in intact outer segments of rods and cones. At elevated [Ca²⁺]_i (i.e., in the dark state), the K_1/2 for cGMP can be as large as 550 µM in cones (mean K_1/2 = 335.5 µM; Ref. 333) compared with rods (37.8-40 µM; Refs. 295, 352). In truncated or electropermeabilized rods, the Ca²⁺-dependent modulation of the ligand sensitivity is only 1.5- to 2-fold, similar to the effect of Ca²⁺/CaM on detached membrane patches (295, 333, 352). In contrast, the range of the CNG channel modulation in intact cones is much wider than in rods and is not well mimicked by Ca²⁺/CaM in detached patches from the outer segment (145). This has led to the hypothesis that an unknown factor, which is lost upon patch excision, is responsible for the larger range of modulation of the ligand sensitivity in cones (333). In summary, the cGMP sensitivity, its modulation by [Ca²⁺]_i, and the Ca²⁺ permeation are profoundly different in CNG channels of rods and cones, supporting the notion that the CNG channel is a pivotal determinant of the dynamics of Ca²⁺ homeostasis in vertebrate photoreceptor cells.

CNG channels in cones serve a second function that is absent in rods. Light produces a graded hyperpolarization in rods and cones that is up to 35 mV in amplitude. Not all of this response range is effectively transmitted to the postsynaptic bipolar and horizontal cells. The highly nonlinear input-output relation of the rod synapse is largely accounted for by the voltage dependence of presynaptic Ca²⁺ channels. At the dark resting voltage of 35 mV, a fraction of the Ca²⁺ channel is open, and the continuous Ca²⁺ entry sustains a tonic release of the neurotransmitter glutamate from the synaptic terminal. The Ca²⁺ channels are characterized by an activation threshold of approximately 45 mV (14). Therefore, when a rod is hyperpolarized to values more negative than approximately 45 mV, the Ca²⁺ channels close and synaptic transmission ceases (12, 27). In contrast to rods, synaptic transmission in cone photoreceptors continues as the light-induced voltage response grows to 70 mV (23, 114, 308). Whereas the small overlap of the voltage range of Ca²⁺ channel activation and the voltage range produced by light can explain signal clipping at the rod synapse, it fails to explain the broader voltage range over which synaptic transmission operates in cones. This conundrum has been partially solved by the discovery of CNG channels in the inner segment and synaptic terminal of cones (341, 358). The density of CNG channels in the inner segment is low, whereas in the cone terminal these channels appear to come in clusters (358). If the clusters were located near release sites, CNG channels would be ideally suited to control the Ca²⁺-dependent release of glutamate. In fact, experimental maneuvers that activate CNG channels also trigger exocytotic events and release glutamate from the cone terminal (341, 358). The cGMP sensitivities measured in patches of membrane excised either from the outer segment or the axon terminal are indistinguishable, suggesting that CNG channels from both locales are built from identical or similar subunits. The cGMP sensitivity of the CNG channels in the synapse is as unusually low as that of CNG channels in the cone outer segment of the fish retina (K_1/2 = 206 and 335.5 µM, respectively; Refs. 333, 358). We note, however, that the high K_1/2 value in fish cones required an intact cone photoreceptor, whereas the K_1/2 of synaptic channels was determined in excised patches.

CNG channels could serve two different functions in the cone synapse. First, these channels might extend the voltage range over which synaptic transmission operates by providing a sustained Ca²⁺ influx even at very negative voltages. Second, nitric oxide (NO) is a good candidate to serve as retrograde neurotransmitter that is released onto cone terminals from other retinal cells (358). An NO synthase (NOS) is predominantly found in the inner segment of rods and cones and in processes of bipolar cells in the outer plexiform layer of the retina (204, 227, 240). Furthermore, a soluble form of guanylate cyclase (sGC) is found in the inner segment of cones and is stimulated by NO (204). Thus CNG channels may play an important role in the modulation of synaptic transmission by NO in the axon terminals of cones.

The pineal regulates various physiological functions by nocturnal secretion of the hormone melatonin. Light sensitivity of the pineal has been retained in most vertebrates, except mammals. Pinealocytes, the light-sensitive cells, display hyperpolarizing responses to brief pulses of light (328, 398) and express several retinal proteins including arrestin, recoverin, rhodopsin kinase, phosducin, GC, and a cGMP-specific PDE (for review, see Refs. 212, 250). Dryer and Henderson (94) recorded CNG channel activity from excised inside-out patches of dissociated photoreceptors from the chick pineal. These CNG channels in extraretinal photoreceptors feature all the hallmarks of CNG channels in retinal photoreceptors (94, 95). Activation is half-maximal between 10 and 50 µM cGMP. Even fully activated channels display frequent brief transitions to the closed state. For this reason, the open probability (P_o) becomes not unity at saturating cGMP concentrations. The brief closing events are more frequent at negative than at positive membrane potentials. Similar properties have been reported for CNG channels from retinal photoreceptors (158, 262, 305). Moreover, expression of several CNG channel subunits in the pineal has been confirmed by in situ hybridization and immunohistochemistry (see sect. VI). These results collectively show that the light response in pinealocytes of lower vertebrates is produced by activation of a cGMP-signaling pathway, which leads to the closure of cGMP-selective ion channels. Chick pineal cells display a circadian rhythm in cGMP concentration (152, 388). It is therefore conceivable that CNG channels are involved in regulating the output of the intrinsic circadian oscillator.

Some lizards do have a parietal-eye, or third eye, on top of their head. The parietal eye seems likely to convey information about changes in light intensity and spectral composition during dusk and dawn. The parietal-eye photoreceptors resemble in their morphology rod and cones of the vertebrate retina, yet they depolarize in response to a flash of light (377). This suggested that parietal-eye photoreceptors, like rhabdomeric photoreceptors of the invertebrate eye, might utilize a phosphoinositide-signaling cascade rather than the cGMP-signaling pathway of retinal rods and cones. It came as a surprise when Finn et al. (105) convincingly demonstrated that the outer segment membrane of the parietal-eye photoreceptors harbors a high density of CNG channels with all the hallmarks of CNG channels from rods and cones: the channels are selectively activated by cGMP, cAMP is much less effective, the channels are nonselective among monovalent cations and are permeable to Ca²⁺ ions, channels are blocked by L-cis-diltiazem, and Ca²⁺/CaM reduces the cGMP-activated current by reducing the ligand sensitivity.

What type of CNG channel is expressed in the parietal-eye? CNG channels of retinal cones are significantly more Ca²⁺ permeable than those of rods (113, 146, 155, 322). The relative selectivity for Ca²⁺ over alkali cations has been determined from reversal potentials (V_rev) under well-defined ionic conditions in excised patches from rod and cone (322) and parietal-eye photoreceptors (105). In cones of striped bass, P_Ca/P_Na = 21.7; in rods of tiger salamander, P_Ca/P_Na = 5.9 (146); and in the parietal-eye, P_Ca/P_Na = 8.1-10.3 (105). Thus, at least with respect to the Ca²⁺ permeability, the CNG channel in parietal-eye photoreceptors behaves more like the CNG channel of rods than that of cones. However, permeability ratios of native CNG channels are not invariant but depend on the cGMP concentrations (146; see sect. VIII). When comparing relative ion permeabilities, this complication must be kept in mind.

The depolarizing light response is produced by an increase in the cytosolic cGMP concentration that is controlled by an unusual cGMP-signaling pathway (426). In the dark, cGMP is synthesized continuously by GC activity and rapidly degraded by PDE activity. The elevated PDE activity in the dark seems to rest on a constitutively active G protein, whereas the mechanism that keeps GC active in the dark is not known. Light acts by inhibiting the PDE through another G protein, permitting the cGMP concentration to rise and CNG channels to open.

The retina of some molluscan eyes is composed of two layers of photoreceptors: depolarizing rhabdomeric-type cells, similar to those found in most other invertebrates, and ciliary photoreceptors that hyperpolarize in light (269). The mechanism underlying the hyperpolarizing light responses has been studied in two scallop species, Pecten and Lima. Light stimulation under voltage clamp activates an outward current that is accompanied by a decrease in the cellular input resistance. The V_rev of the light-stimulated current lies near the equilibrium potential for K⁺ (E_K) (126), demonstrating that the light-dependent channel is highly K⁺ selective and that the hyperpolarizing light response is brought about by opening K⁺ channels rather than by closing nonselective cation channels, as in retinal rods and cones. In a series of incisive experiments, Gomez and Nasi (85) convincingly demonstrated that 1) the inositol trisphosphate (IP₃)/Ca²⁺-signaling pathway is not crucial for phototransduction, 2) the photoreceptors rely on cGMP as the internal messenger of the transduction cascade, and 3) the light-dependent channel is opened by cGMP. The latter two observations imply that light elevates cGMP, although it is unknown whether this involves the inhibition of a PDE or the stimulation of a GC.

In contrast to the Ca²⁺-permeable CNG channels of retinal photoreceptors and OSNs, the Pecten channel is virtually impermeable to Ca²⁺, and the K_1/2 values for blockage by extracellular Ca²⁺ and Mg²⁺ are 1-2 orders of magnitude higher (299). The significant K⁺ selectivity, the lack of Ca²⁺ permeability, and the weak divalent block suggest that the pore architecture is more like that of K⁺ channels than that of CNG channels. It is interesting to note that a cGMP-sensitive K⁺ channel also seems to underlie the light response of a photosensitive neuron in the abdominal ganglion of a marine mollusc (136). This cell generates slow, depolarizing light responses due to the closure of K⁺ channels that are kept open in the dark by cGMP. The protein(s) forming the cGMP-dependent K⁺ channel is unknown. Its molecular identification is eagerly awaited, as it will certainly further our understanding of the molecular mechanisms that govern ion selectivity in CNG channels.

During the 1970s and 1980s, when the cGMP-signaling pathway in vertebrate photoreceptors was elucidated, several groups examined the possibility that light also regulates the cGMP (or cAMP) concentration in depolarizing rhabdomeric photoreceptors of invertebrates and that cGMP mediates the light response by opening ion channels in the microvilli membrane. Injection of cGMP into Limulus ventral photoreceptors produced a depolarization that mimics the receptor potential (176). Superfusion with cGMP of membrane patches excised from the light-sensitive lobe of the ventral photoreceptor activated channels that closely resembled the channels activated by light in cell-attached patches (13). In contrast, perfusion of photoreceptors from Drosophila eyes with various cGMP analogs was without effect (73). The cDNAs of CNG channel subunits have been cloned from Drosophila melanogaster and Limulus polyphemus (22, 69, 278). The precise sites of expression of the two channel subunits are not known. Further advances with respect to the function of CNG channels in invertebrate brain will require precise cellular and subcellular localization of the channel proteins.

OSNs are embedded in the olfactory epithelium lining the cavity of the nose. OSNs are bipolar neurons; a single dendrite extends to the apical surface of the neuroepithelium, and a single axon projects to the olfactory bulb. From the tip of the dendrite ~20-50 thin cilia extend into the layer of mucus that covers the epithelium. The cilium is the site where the chemoelectrical transduction takes place. It is the functional equivalent of the outer segment of retinal photoreceptors. It houses all the molecular components to register odorants, to amplify the signal by a series of enzymatic reactions, and to generate the electrical response. Like rods, OSNs are exquisitely sensitive; they can respond to stimulation by a few odorant molecules. This high sensitivity is accompanied by a rich selectivity. Humans are probably able to discriminate between more than 10,000 or so different odorous compounds. This enormous achievement is endowed by an array of several hundreds up to a thousand odorant receptors with overlapping specificity for a few odorants (for review, see Ref. 327).

Quite remarkably, a cousin of the retinal CNG channels takes center stage in odorant signaling; the vast majority of OSNs respond to brief pulses of odorants with a transient receptor current by opening cAMP-gated channels in the ciliary membrane (107, 293). In contrast to the outer segment of photoreceptors, where the light-sensitive current is solely carried by CNG channels, the receptor current originating in chemosensitive cilia has two ionic components: an inward cationic component mediated by CNG channels, followed by an inward anionic component mediated by Ca²⁺-activated Cl channels. In addition to cAMP, other signaling molecules have been implicated in odorant transduction, especially the two gaseous messengers NO and carbon monoxide (CO). We will critically examine the experimental foundation for these hypotheses.

Finally, a cGMP-signaling pathway that is targeting a highly cGMP-selective CNG channel has been identified in a small subset of OSNs, whereas components that furnish the prototypical cAMP-signaling pathway are absent in those cells. These observations provide compelling evidence that cGMP serves as the principal messenger for chemosensory signaling in some OSNs.

A) THE PROTOTYPICAL CAMP-SIGNALING PATHWAY. A milestone in olfactory research was the cloning of a family of odorant receptors by Buck and Axel (61). Since then a vast number of putative odorant receptors have been cloned. Although functional expression of these receptors in heterologous systems has been accomplished in only a few cases (219, 415), it is taken for granted that these receptors feed into a common cAMP-signaling pathway. The principal players of this signaling pathway are shown in Figure 3, top. The binding of odorants to their cognate receptors in the membrane of chemosensitive cilia first activates a G protein (G_olf) and then an adenylyl cyclase (ACIII). The ensuing rise in the concentration of cAMP opens CNG channels and thereby produces a depolarization of the cell membrane. Like its retinal cousins, the CNG channel in OSNs is highly Ca²⁺ permeable (97, 113, 294), and channel activation causes a rapid increase of [Ca²⁺]_i (230, 232). The odorant-stimulated rise of [Ca²⁺]_i plays an important role in both excitation and odor adaptation. It serves as a feedforward signal that enhances the depolarizing response by activating Ca²⁺-dependent Cl channels, which, in fact, carry a large fraction of the receptor current (197, 226). In rat OSNs, as much as 85% of the olfactory response can be mediated by Cl channels (251). The increase of [Ca²⁺]_i serves also as a delayed negative feedback signal that reduces the cAMP sensitivity of the CNG channels and stimulates cAMP hydrolysis by a Ca²⁺-dependent ciliary form of PDE (PDE1C2) (42). Both processes, channel desensitization and PDE activation, are controlled by Ca²⁺/CaM (42, 72, 248).

View larger version (44K): [in this window] [in a new window]   Fig. 3. The cAMP-signaling pathway and the cGMP-signaling pathway in olfactory sensory neurons (OSNs). OR, odorant receptor; Golf, G protein expressed in OSNs; CaM, calmodulin; ClC, Ca2+-activated Cl channel; PDE1C2, CaM-dependent phosphodiesterase; PDE2, cGMP-regulated phosphodiesterase; GC-D, guanylyl cyclase type D; ACIII, Ca2+-dependent adenylyl cyclase type III. The two pathways do not exist in one and the same OSN.

In a series of elegant double-pulse experiments using short puffs of odorants or flashes of ultraviolet light that rapidly release cAMP from a caged compound, Kurahashi and Menini (224) show that the principal mechanism underlying odorant adaptation acts at the CNG channel and that regulatory mechanisms upstream of the channel contribute little to the odorant-induced reduction of the cell's sensitivity, at least on a time scale of several tens of seconds. Additional mechanisms of adaptation that appear to operate on a longer time regime include phosphorylation of odorant receptors (38) and adenylyl cyclase (412).

B) NO AND CO: GASEOUS MESSENGERS INVOLVED IN ODORANT SIGNALING? NO is a short-lived molecule capable of diffusing across membranes and reacting with a variety of targets. It is produced by various isoforms of NOS, some of which are activated by Ca²⁺/CaM. The physiological concentrations of NO are in the picomolar range (8). The most common action of NO involves the activation of sGC, i.e., NO stimulates the synthesis of cGMP. However, NO can also regulate the activity of various proteins by reacting with cysteine sulfhydryls, a covalent modification known as S-nitrosylation (for review, see Ref. 50). The direct activation by NO was also reported for the olfactory CNG channel (52). Perfusion of membrane patches excised from the soma or dendrite of OSNs of tiger salamander with NO donors like S-nitrosocysteine (SNC) produced single-channel events similar to those observed in the presence of cAMP. NO-stimulated channel activity persisted for up to 30 min in the absence of NO donors and was mimicked by sulfhydryl-modifying reagents like N-ethylmaleimide (NEM), iodoacetamide (IAA), and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) in a reversible manner. SNC was also a potent agonist of the heterologously expressed rat A2 and A2/A4 channels (53). When C460 in the A2 subunit was mutated to a serine residue, the activation of the homomeric channel by NO was completely abolished (51). Mutation of other intracellularly located cysteine residues was without effect. Moreover, a CNG channel subunit from Drosophila that is lacking this particular cysteine residue (22) was not NO sensitive (51). These observations collectively support the notion that NO works through S-nitrosylation. However, it is noted that CNG channels in rod and cone photoreceptors and from C. elegans do not become activated by covalent modification with NO (209, 358, 400), although their respective A subunits carry a cysteine residue at a homologous position.

Stimulation of OSNs with a short pulse of 500 µM SNC in the whole cell recording configuration elicits a sizable inward current that, in contrast to the current in excised patches, returns to baseline within 1.5 s (52). The authors reasoned that this current was due to covalent modification by NO, because the recording pipette contained no GTP, which is required for cGMP synthesis. This observation then indicates that the S-nitrosylation product inside the cell is 100-1,000 times less stable than in the excised membrane patch. Although redox reactions inside the cell might rapidly reverse the S-nitrosylation, it is conceivable that some endogenous GTP was still available to support cGMP synthesis either because equilibration between the pipette and the cell interior was not complete and/or because GTP was generated from ATP by transphosphorylation reactions involving guanylate kinase and nucleotide diphosphate kinase activity (see Ref. 422 and references therein). The chemical nature of the modification in native channels from OSNs and heterologously expressed A2 subunits seems to be different as well. The NO-induced channel activity persisted for many minutes in excised patches of OSNs, but rapidly faded with a rate constant of 3.8 s¹ ( = 260 ms) in patches with the A2 homomeric channel (51). Such a rapid decay would seem incompatible with the formation of high-molecular-weight nitrosothiol species, which are stable on a time scale of several minutes to hours (378). This raises the intriguing possibility that reversible binding of either SNC or NO itself gates the channel open.

Lynch (256), working with rat OSNs, was unable to reproduce the actions of NO donors and sulfhydryl-modifying reagents on the native olfactory CNG channel. In fact, the NO donors SIN-1 and SNC at moderate concentrations inhibit the cAMP-stimulated currents rather than activating the unliganded channels. Moreover, the oxidizing agent DTNB caused a permanent inhibition. Inhibition brought about by either agent was reversed by a 2-min wash with dithiothreitol. The similar inhibitory effects of NO and DTNB suggest that two neighboring S-nitrosothiols combine to form a relatively stable disulfide bond. Because NEM displays no inhibitory action, it can be concluded that alkylation of either one or both of the paired cysteines does not replicate the effect of the disulfide formation.

Future work must establish the physiological function of covalent NO modification of the olfactory CNG channel. Two questions are particularly pertinent. The concentration of NO in the olfactory epithelium has not been determined; therefore, it is not known whether the concentrations of NO in the ciliary layer reach the high levels of NO needed to activate the olfactory channel (125). Second, NO potently activates sGC in cells (EC₅₀ ~20-250 nM; Refs. 28, 380). The high sensitivity raises the question what fraction of CNG channels becomes activated by S-nitrosylation at NO concentrations that fully activate sGC and thereby cGMP synthesis.

An alternative mechanism of the action of NO in odorant signaling involving sGC and cGMP was proposed by other groups. These investigations were stimulated by the fact that the CNG channel of chemosensitive cilia is exquisitely sensitive to both cAMP and cGMP. In fact, the K_1/2 of half-maximal activation is twofold lower for cGMP than for cAMP (1.0-2.4 µM and 2-4 µM, respectively; Refs. 40, 58, 72, 112, 208, 293). The slightly higher sensitivity for cGMP spurred several authors to explore a potential physiological role for channel regulation by cGMP.

Breer and Shepherd (49) proposed that the NO/cGMP system is involved in both excitation and some form of olfactory adaptation. What is the experimental evidence for these hypotheses? Hefty doses of odorant stimulate a retracted elevation of cGMP concentration in isolated olfactory cilia or cultured OSNs (48, 220, 404). The response is abolished by N^G-nitro-L-arginine, a selective inhibitor of NO formation by NOS. These observations have been interpreted to indicate that an odorant-induced Ca²⁺ influx activates NOS via Ca²⁺/CaM and, thereby, initiates NO production. The highly membrane-permeable and diffusible NO might activate sGC in this and in neighboring cells. Specifically, Breer and Shepherd (49) proposed that recruitment of adjacent neurons via the NO/cGMP system could serve as a mechanism to encode very intense stimuli. The NO/cGMP system, however, is unlikely to play such a role in the adult epithelium, because developing and regenerating OSNs, but not mature OSNs, contain NOS activity (47, 196, 221, 345).

A variation of this theme has been proposed by Zufall and collaborators (234, 235, 444). These authors provide evidence that the CO/cGMP system might be responsible for a long-lasting form of odor adaptation. Variants of this hypothesis can be traced to previous reports showing that pretreatment of olfactory preparations with membrane-permeable cGMP derivatives attenuates the second messenger response to odorant stimuli in rat cilia and the receptor current in the olfactory epithelium of the bullfrog (319). CO, like NO, stimulates the synthesis of cGMP by sGC (59, 115, 193); therefore, CO has been proposed to serve as an endogenous gaseous messenger in the nervous system (260). Indeed, OSNs contain high levels of the CO-producing enzyme heme oxygenase-2 and produce CO upon stimulation with odorants (173, 174, 404).

How might CO engender odor adaptation? Odor stimulation elicits two kinetically distinct inward currents: a large and fast transient current mediated by the cAMP-signaling system, followed by a small and persistent current of only a few picoampères in amplitude (444). It is this "background" current that appears to depend on cGMP and is underlying long-term adaptation. The authors specifically proposed that the small background current is flowing through CNG channels and that cGMP-dependent Ca²⁺ entry is a crucial step in the development of long-lasting adaptation. The results are based on an experimental protocol involving successive puffs of odorant. The first conditioning pulse of odorant provides the reference cellular response and sets into motion the adapting processes. The second test pulse probes the change in odorant sensitivity caused by the first pulse. Using this experimental design, Kurahashi and Menini (224) and Reisert and Matthews (335) identified a form of odorant adaptation that is virtually instantaneous and operates on a time scale of a few seconds. The amplitude of the adapted response depends on the time of the delivery of the test pulse. For short interpulse times (~1-5 s), the response amplitude is significantly reduced; amplitudes gradually increase with the interpulse time span (224, 230, 335). The time constant for complete recovery of the sensitivity is of the order of 3-10 s and depends on the odorant concentration of the conditioning pulse (224); intense stimuli require longer times for complete recovery than weaker stimuli. The recovery time critically depends on the rate of Ca²⁺ clearance from the cell by a Na⁺/Ca²⁺ exchange mechanism (230, 335), underpinning the idea that Ca²⁺/CaM modulation of the CNG channel accounts for most odorant adaptation.

The understanding of long-term adaptation is incomplete. For example, does a stimulated cell enter a state of short-term adaptation first, then recover from stimulation, and finally transit to a state of long-lasting adaptation? For obvious reasons, the paired-pulse protocol (pulse every 30 s) designed to study long-term adaptation does not cover the time regime of short-term adaptation (<10 s) (444). Therefore, it is not clear whether the lower sensitivity revealed by the second pulse reflects a delayed or incomplete recovery from the first pulse rather than a slowly progressing adaptation. How does long-term adaptation relate to short-term adaptation if a Ca²⁺-dependent shift of CNG channel sensitivity is underlying both forms of adaptation? It is intriguing that cells recover from the massive Ca²⁺ influx during the cAMP-mediated odorant response within 5-10 s or so (224, 230, 335), whereas it takes several minutes to recover from the much smaller cGMP-mediated response. Does this finding imply that the feedback mechanisms, i.e., hydrolysis of the cyclic nucleotide and extrusion of Ca²⁺, are different for the cAMP and cGMP response? Because the rate of clearance from the Ca²⁺ load by the Na⁺/Ca²⁺ exchanger sets the recovery time (335), the long-term effects in some cells might result from an altered Ca²⁺ homeostasis that is characterized by a slower rate of Ca²⁺ clearance. This interpretation is supported by the observation that only a subpopulation of cells displays long-term adaptation.

C) A CGMP-SELECTIVE CNG CHANNEL IN MAMMALIAN CHEMOSENSORY NEURONS. A small population of OSNs that project to a group of atypical glomeruli in the main olfactory bulb, the so-called necklace glomeruli, houses a different repertoire of signaling molecules (178, 273). This subgroup of OSNs expresses an olfactory-specific guanylyl cyclase (GC-D), a cGMP-stimulated isoform of PDE (PDE2), and a cGMP-selective CNG channel (splice variant of the cone A3). These three proteins are highly enriched in the chemosensitive cilia. Most intriguingly, the characteristic markers for the enzymatic makeup of the prototypical cAMP pathway in OSNs, i.e., G_olf, ACIII, PDE1C2, and three distinct CNG channel subunits (A2, A4, and B1b), are absent from this subset of neurons (see Fig. 3, bottom). These findings rule out the coexistence of the known cAMP-signaling pathway and this novel cGMP-dependent pathway and argue for cGMP as the principal messenger in this subset of OSNs. GC-D is a member of the family of receptor-type GCs that become activated by binding of peptide hormones to the extracellular domain (111). Although no ligand has yet been identified for GC-D, this subgroup of OSNs may not respond to normal volatile odorants, but, possibly, to ligands that control some aspects of reproductive behavior. Because very few OSNs use this cGMP-signaling pathway, it has not been feasible to electrically record from these cells. It is, however, anticipated that stimulation of this unique cell type with the cognate ligand of GC-D will produce a depolarizing receptor current and an increase in [Ca²⁺]_i by opening cGMP-selective channels.

The vomeronasal organ (VNO) or Jacobson's organ is a chemosensitive organ present in most vertebrates. It is important for the detection of pheromones that convey information between individuals of the same species. The sensory neurons of the VNO have a bipolar organization, and their axons terminate in a specialized brain region, the accessory olfactory bulb. In situ hybridization studies revealed that a modulatory CNG channel subunit that is also found in OSNs (A4, see nomenclature in sect. V) is reportedly expressed in these neurons, whereas the principal A2 subunit of OSNs is lacking (30). The A4 subunit itself does not form functional CNG channels (44, 242), raising the intriguing possibility that it might coassemble with members of other channel families.

Alternatively, the A4 subunit might become activated by another ligand of unknown nature. Indeed, Broillet and Firestein (53) reported that the A4 subunit, when heterologously expressed, produced NO-activated Ca²⁺-selective channels and that single NO-activated channels from VNO neurons have some properties in common with the heterologously expressed channels. The significance of these findings is unclear, because recent evidence suggests that signaling in the VNO is mediated by the phospholipase C/IP₃ pathway and TRP channels (231, 243, 270, 445).

A cyclic nucleotide-sensitive conductance with quite unique properties has been described in a subset of taste cells in the frog. Superfusion of excised inside-out patches from the apical end of frog taste receptor cells suppressed a current (207) that reversed at about 50 mV under symmetrical biionic conditions (110 mM intracellular K⁺/110 mM extracellular Na⁺), arguing that the channel is K⁺ selective. Maneuvers intended to raise the intracellular cGMP concentration (perfusion with IBMX or 8-BrcGMP) reduced whole cell inward currents that reverse at ~0 mV. The discrepancy between results acquired in the excised-patch and whole cell configuration raises questions as to the nature of the ionic conductance. The underlying channels appear to have a ligand sensitivity and selectivity that are both distinctively different from those of other CNG channels. The action of cAMP and cGMP on excised patches was exquisitely sensitive (K_1/2 = 77-160 nM and K_1/2 = 16-36 nM, respectively). Moreover, cAMP and cGMP are significantly more potent than their 8-bromo-substituted analogs, whereas for the photoreceptor and olfactory CNG channels the opposite is true. The dependence of current suppression on the concentration of cAMP, cGMP, and 8-bromo-substituted congeners is described by a simple binding isotherm (Hill coefficient n is unity). From the indirect modulation by cyclic nucleotides of Ca²⁺-activated K⁺ channels in the same patch, it has been inferred that the cNMP-suppressible conductance is also Ca²⁺ permeable, although this interpretation has not been substantiated by direct demonstration of Ca²⁺ permeation. The authors propose that tastants, by binding to G protein-coupled receptors, activate transducin, which is also present in taste cells (351), which in turn activates a cAMP-specific PDE. The ensuing drop in cAMP concentration activates the cNMP-suppressible inward current, leading to membrane depolarization and a rise of [Ca²⁺]_i. The molecular identity of the cNMP-suppressible conductance is unknown. The A3 subunit, which is expressed in cone photoreceptors (39, 416) and a small subpopulation of OSNs (273), has been cloned from taste buds of rat (277). This CNG channel isoform is activated rather than inactivated by cyclic nucleotides and, therefore, unlikely to form the cNMP-suppressible conductance alone.

A precedent for chemosensory signaling using cGMP-selective CNG channels exists in the nematode C. elegans (74, 210). The tax-2 and tax-4 genes of C. elegans encode two distinct CNG channel subunits required for chemosensation and thermosensation. Tax-2 and tax-4 mutants display defects in the chemotaxis to volatile compounds and salts, as well as in thermotaxis. Mutations in either gene affect similar behavioral responses, and tax-2 and tax-4 are expressed in the same olfactory, gustatory, and thermosensory neurons, suggesting that the native channel is composed of these two subunits.

The ceB (Tax-2) and ceA (Tax-4) proteins have been localized to sensory neurons that also express some of the 29 different receptor GC genes of C. elegans. The homomeric ceA channel and the heteromeric ceA/ceB channel highly prefer cGMP over cAMP. Both observations suggest that the CNG channels mediate an electrical response that relies on a cGMP- rather than a cAMP-signaling pathway.

In addition to their function in sensory transduction, both the tax-2 and tax-4 genes play a role in the guidance of sensory axon outgrowth during development and the maintenance of normal axon morphology throughout the adult stage (74, 75). In mammals, CNG channels may serve a similar function because CNG channel activity is present in growth cones of cultured OSNs (181), and the A4 subunit was immunohistochemically localized to growing fibers of cultured hippocampal neurons (46).

The congruence of developmental and behavioral defects in C. elegans mutants might be explained by either a primary defect in neuronal connectivity that perturbs normal behavior or by the fact that the correlated neuronal activity is required to refine synaptic connections during development like in sensory systems of vertebrates (75). This latter hypothesis was specifically tested in mice lacking a functional cAMP-sensitive CNG channel due to targeted disruption of the A2 subunit gene. Two groups reported that the peripheral olfactory projections are in part influenced by neuronal activity (438, 439), whereas another group concluded that the olfactory CNG channel, and by inference chemosensory activity, is not required for generating synaptic specificity in the olfactory bulb (244).

C.  CNG Channels in the Brain

1.  cGMP-sensitive currents in retinal neurons and glia cells

Bipolar cells are retinal interneurons that receive synaptic input from photoreceptors. Glutamate, released from the synaptic terminal in the dark, hyperpolarizes ON-bipolar cells (301, 375) through activation of a metabotropic glutamate receptor (mGluR6; Ref. 292) and subsequent suppression of a nonselective cation current. When glutamate release ceases in the light, the bipolar cells depolarize (ON-response). The nonselective cation current is enhanced when cGMP was introduced into the cell through a patch pipette, indicating that the current might flow through CNG channels. The mGluR6 receptor is thought to signal through a G protein to a PDE. Activation of the PDE would stimulate cGMP hydrolysis, thereby closing the CNG channel. This led to the concept that a cGMP-signaling pathway similar to that in the photoreceptor outer segment operates in ON-bipolar cells. However, subsequent experiments did not support this concept (300). When intracellularly perfused with the hydrolysis-resistant analogs 8-pCPT-cGMP and 8-BrcGMP or the PDE inhibitor IBMX, bipolar cells continue to respond to glutamate, and no difference was observed in the kinetics and the amplitude of the response (300), whereas similar treatments of rods result in profoundly altered kinetics and size of light responses (355, 442). These results argue against regulation of the glutamate-sensitive current by PDE activity. Moreover, the properties of the presumptive cGMP-sensitive channel do not match those of known CNG channels. For example, the cGMP-sensitive current in bipolar cells is not blocked by extracellular Ca²⁺ (376). Finally, several different antibodies against known subunits of retinal CNG channels (A1, A2, A3, and B1a, see sect. V) do not label ON-bipolar cells (408; F. Müller, unpublished observations). These results argue against the presence of a photoreceptor-type CNG channel in ON-bipolar cells.

There is also some suggestion of a rodlike CNG channel in retinal ganglion cells (2, 190). About one-half of the ganglion cells in the rat retina respond to stimulation by NO with an inward current (190). This current is mimicked by either intracellular perfusion with cGMP or extracellular application of the membrane-permeable analogs 8-BrcGMP and 8-pCPT-cGMP, suggesting that the cGMP-sensitive current flows through CNG channels. This conclusion appears to be supported by in situ hybridization and PCR that detect transcripts coding for an A1 subunit in ganglion cells (2).

However, an electrophysiological study in salamander (164) found no evidence that treatment with membrane-permeant cGMP analogs, IBMX, or NO donors led to the activation of CNG channels. Moreover, the cGMP-sensitive current displays properties that do not match the properties of known CNG channels. First, the blockage of the current by divalent cations is weak and does not result in a strong outward rectification, i.e., the voltage dependence of blockage is weak. Second, the current in ganglion cells reverses at roughly 0 mV under biionic conditions (Cs⁺ inside, Na⁺ outside), whereas CNG channels would display a distinctively more positive V_rev under similar ionic conditions (see sect. VIII on ion selectivity). Third, under these ionic conditions, the current (I)-voltage (V_m) relations of CNG channels display a distinctive inward rectification, due to the smaller conductance of CNG channels for Cs⁺ compared with Na⁺ (see, for example, Refs. 112, 255, 271, 309, 416), whereas the cGMP-sensitive current in ganglion cells is rectifying in the outward direction (2). Moreover, antibodies against known CNG channel subunits do not label ganglion cells in the mammalian retina (408; Müller, unpublished observations). With the proviso that CNG channels in photoreceptors and ganglion cells are antigenically similar, a low channel density cannot explain this failure. As an example, in the rod outer segment, 1-2% of open CNG channels sustain a current of 30 pA (297, 434), whereas the cGMP-sensitive conductance in ganglion cells carries currents up to 500 pA.

8-BrcGMP and NO donors also enhance whole cell currents in acutely dissociated or cultured retinal Müller cells and stimulate Ca²⁺ influx (228), whereas 8-BrcAMP has no effect on whole cell currents. A fraction of the cGMP-sensitive current seemed to be carried by a Ca²⁺-activated K⁺ channels and another fraction by a nonselective cation channel. Transcripts encoding a CNG channel subunit have been amplified from cultured human Müller cells using primers specific for the A1 subunit of the cGMP-gated channel of rod photoreceptors. These pieces of evidence have been interpreted to indicate that Müller cells express a rodlike CNG channel. Again, immunohistochemical studies do not support the presence of a rodlike CNG channel in retinal glia cells (408; Müller, unpublished observations).

A variety of different transcripts encoding CNG channel subunits have been detected in several brain areas by in situ hybridization, cloning of cDNA, and PCR (see sect. VI); however, studies on the functional characterization of neuronal CNG channels in situ are sparse. Two laudable examples are the work by Leinders-Zufall et al. (233) and Bradley et al. (46). At rest (80 mV), a whole cell inward current is evoked in hippocampal neurons upon superfusion with 8-BrcGMP. Although this basic observation is shared by both reports, the underlying currents appear to be different.

In one study, the I-V_m relation of the 8-BrcGMP-activated current was almost linear in the presence of 2.5 mM Ca²⁺ and 1 mM Mg²⁺ in the bath (233). The current was 1.5- to 2-fold larger in the absence of divalent cations, but the shape of the I-V_m relation was largely unchanged. This result contrasts with the strong blockage of known CNG channels by extracellular divalent cations, whether Ca²⁺ or Mg²⁺, which produces a pronounced outward rectification of currents in photoreceptors and OSNs. Furthermore, the I-V_m relation has been recorded with 140 mM Cs⁺ in the pipette and 140 mM Na⁺ in the bath. Under these biionic conditions, known CNG channels show a distinctively positive V_rev (~20 mV), and the I-V_m relation is slightly inwardly rectifying (112, 255, 271, 309, 416); in contrast, the currents in hippocampal neurons reverse either at ~0 mV or at 40 mV (Figs. 2C and 3C, respectively, of Ref. 233) and rectify slightly in the outward direction.

Leinders-Zufall et al. (233) also attempted to study Ca²⁺ permeability by estimating the Ca²⁺ current flowing through this channel relative to the Na⁺ current. To this end, the authors compared the current amplitudes in Na⁺ and in various choline/Ca²⁺ mixtures. At 30, 100, and 1,000 µM Ca²⁺, the current ratio I_Ca/I_Na was roughly 0.25, 0.5, and 0.7, respectively, implying that Ca²⁺ carries almost as much current as Na⁺ at more than 100-fold lower concentrations. Similar experiments with CNG channels in rods and heterologously expressed subunits yielded entirely different results. At 70-100 mM external Ca²⁺, I_Ca amounts to only 1-2% of I_Na in the native rod CNG channel, to roughly 10% in the A2 homomeric channel, and to 14% in the Drosophila channel (22, 65, 97). In conclusion, the currents recorded from hippocampal neurons (233) do not match the properties of currents flowing through known CNG channels.

The I-V_m relation reported by Bradley et al. (46) is slightly outwardly rectifying in the absence of external divalent cations, and 1 mM Mg²⁺ blocked the inward current at 80 mV almost completely. Although the currents have not been further characterized, this piece of evidence is consistent with the idea that the current is carried by CNG channels.

A cAMP-activated cation current is widely distributed among central molluscan neurons (for references, see Refs. 191, 192, 382). In neurons of Helix, Aplysia, and Pleurobranchaea, the current persists in the presence of inhibitors of PKA (165, 192, 382), arguing that the cAMP action on channels is direct and does not involve phosphorylation. In excised membrane patches, perfusion with 1 mM cAMP produces single-channel events of ~40 pS (382). These two observations have been taken as evidence that the underlying channels belong to the class of CNG channels. The unit conductance of 40 pS was determined in the presence of 60 mM divalent cations, and the maximum P_o at saturating cAMP concentrations was only 6.3% (at 0 mV; Ref. 382). Known CNG channels, whether vertebrate or invertebrate, are strongly blocked by 60 mM divalents, and no single-channel recordings would be feasible.

Cyclic nucleotides are key elements of cellular signaling in sperm of both vertebrates and invertebrates (for reviews, see Refs. 118, 406). cAMP and cGMP mediate several cellular responses, including acrosomal exocytosis, swimming behavior, and chemoattraction. The swimming behavior of sperm is controlled by factors, usually short peptides, that are secreted by the egg or cellular structures of the oviduct. In a few species, primarily marine invertebrates with external fertilization, the amino acid sequence of the peptides and their cognate membrane receptors on the surface of the sperm have been identified (119, 275, 406). For example, speract, a short peptide from the sea urchin Strongylocentrotus purpuratus, activates a receptor-type GC and thereby stimulates a rise of the intracellular cGMP concentration (118, 406). Speract also gives rise to an increase of [Ca²⁺]_i. These observations suggest that cGMP activates a Ca²⁺ conductance, although a direct causal relationship has not been established rigorously. Owing to their high Ca²⁺ permeability, CNG channels are among the prime candidates for the Ca²⁺-entry pathway. Unfortunately, in sea urchin, where their potential function would have been so obvious, no CNG channels have been detected by homology screening (R. Gauss and U. B. Kaupp, unpublished observations). Instead, Gauss et al. (120) identified the first member of the family of pacemaker channels (or HCN channels), which are controlled by both cyclic nucleotides and voltage. The HCN channel in S. purpuratus is highly selective for cAMP and is not Ca²⁺ permeable. Its function in spermatozoa is not known.

The search for CNG channels was more successful in mammalian sperm. The testicular expression of several CNG channel subunits (A3, B1, and B3) has been suggested by cloning of cDNA from testis libraries or by Northern analysis (34, 35, 122, 416, 417). Antibodies specific for the A3 and B1 subunits labeled the flagellum of mature sperm and precursor cells in cross-sections of seminiferous tubules (417). Heterologous expression of the A3 subunit cloned from testis produces channels that are cGMP sensitive and cGMP selective: the K_1/2 for cAMP is ~200-fold higher than the K_1/2 for cGMP (8.3 and 1,720 µM, respectively; Ref. 416). Therefore, these channels might be involved in a cGMP-stimulated Ca²⁺ influx into intact sperm (417). cGMP-stimulated channel activity was detected in small vesicles that might have been derived from cytoplasmic droplets and in patches excised from osmotically swollen sperm. The low success rate of establishing a giga-seal resistance and of detecting channel activity prevented a more thorough characterization of the native channel in situ. Cyclic nucleotide-mediated Ca²⁺ influx into sperm was studied by confocal laser scanning microscopy (417). The 8-bromo- and 8-pCPT-analogs of cGMP were delivered from caged compounds by brief flashes of ultraviolet light. Photolysis of both caged compounds evokes a Ca²⁺ influx into sperm; the respective derivatives of cAMP are much less effective. The Ca²⁺ influx depends on the presence of extracellular Ca²⁺ and was greatly reduced at high extracellular Mg²⁺ concentrations.

Because knock-out mice lacking the A3 subunit are fertile (33), at this point we can only speculate about a functional role of cGMP-selective CNG channels in sperm. CNG channels might be involved in some aspect of motility control or, more specifically, in chemotactic swimming behavior or in the process of capacitation or acrosomal exocytosis. Another concern is the failure so far to identify a receptor-type GC in mammalian sperm, leaving alone its cognate ligand. There is also no evidence in sperm for the presence of the Ca²⁺-regulated GCs (GC-E and GC-F) known from retinal photoreceptors.

Mouse sperm express two other channels (CatSper1 and CatSper2) that bear similarity with a single repeat of the four-repeat structure of voltage-activated Ca²⁺ channels (332, 336). Targeted disruption of the CatSper1 gene results in male sterility; moreover, the cAMP-induced Ca²⁺ influx is abolished in mutant mice. The CatSper channels are unrelated to CNG or HCN channels; in particular, they are lacking in a cAMP/cGMP-binding domain. The cloned CatSper channel genes so far have resisted functional expression, suggesting that they might require additional subunits to become functional. An intriguing possibility is that CNG and CatSper subunits coassemble to form Ca²⁺-permeable and cyclic nucleotide-sensitive ion channels.

Xu et al. (427) report the expression of transcripts coding for the CNG channel from rod photoreceptors (A1 and B1 subunits) in a human alveolar cell line (A549). Furthermore, the authors compared whole cell currents recorded from the alveolar cell line to whole cell currents from A1-transfected HEK293 cells and from untransfected controls in the presence and absence of 8-BrcGMP. The averaged current amplitude recorded from one set of alveolar cells in the presence of 8-BrcGMP was about twofold larger than the averaged amplitudes recorded from another set of alveolar cells without 8-BrcGMP. A problem with this approach is that it does not allow correcting for leak currents in one and the same cell. Moreover, when working with transfected cell lines, the fluorescence of the cotransfected green fluorescent protein (GFP) does not always correlate with channel activity. Therefore, the contribution of the cGMP-activated current to the total whole cell current is not known. Moreover, the blockage of CNG channels by L-cis-diltiazem in rods and cones and OSNs is strongly voltage dependent (112, 154, 266), whereas the blockage in A549 cells is not. This study would have been more convincing had the cGMP-sensitive currents been unequivocally identified in excised membrane patches or in the whole cell configuration by infusion of cGMP from the recording pipette. CNG channels have also been involved in liquid homeostasis of lung in 6-mo-old sheep but not in 6-wk-old sheep on the basis of a pharmacological study using dichlorobenzil, amiloride, and pimozide, which, among other channels, also block CNG channels (179, 180)

Vitalis et al. (405) report the identification by PCR of transcripts for the CNG channel subunits A2, A4, and B1 in a neuronal cell line (GT1) secreting the gonadotropin-releasing hormone (GnRH). These th