Central role of the CNGA4 channel subunit in Ca2+-calmodulin-dependent odor adaptation.

Heteromultimeric cyclic nucleotide-gated (CNG) channels play a central role in the transduction of odorant signals and subsequent adaptation. The contributions of individual subunits to native channel function in olfactory receptor neurons remain unclear. Here, we show that the targeted deletion of the mouse CNGA4 gene, which encodes a modulatory CNG subunit, results in a defect in odorant-dependent adaptation. Channels in excised membrane patches from the CNGA4 null mouse exhibited slower Ca2+-calmodulin-mediated channel desensitization. Thus, the CNGA4 subunit accelerates the Ca2+-mediated negative feedback in olfactory signaling and allows rapid adaptation in this sensory system.

Retinal ganglion cells express a cGMP-gated cation conductance activatable by nitric oxide donors.

We have identified a putative cGMP-gated cation conductance in rat retinal ganglion cells. Both in situ hybridization and polymerase chain reaction amplification detected transcripts in ganglion cells that were highly homologous to the cGMP-gated cation channel expressed in rod photoreceptors. Whole-cell patch-clamp recordings detected a current stimulated by cGMP due to activation of nonselective cation channels. This current had a reversal potential near 0 mV, showed some outward rectification, and could be blocked by Cd2+. The current could also be activated by a phosphodiesterase inhibitor and the nitric oxide donors sodium nitroprusside and S-nitrosocysteine. We propose that nitric oxide released from an identified subpopulation of amacrine cells may activate this channel to modulate ganglion cell activity.

From odor and pheromone transduction to the organization of the sense of smell.

Chemosensory neurons in the mammalian nose detect an array of odors and pheromones that carry essential information about the animal's environment. How the nose organizes this immense amount of information is a major question in sensory biology. New evidence suggests that there are several subpopulations of sensory neurons in the nose that project to different areas in the forebrain. Strikingly, evidence is now emerging that several of these neuronal subpopulations employ distinct second messenger cascades to transduce chemical stimuli. This new understanding of the heterogeneity of chemosensory transduction mechanisms offers the opportunity to use genetically altered animals to specifically target these subpopulations. Such approaches should enable researchers to examine the role that each of these subsystems could play in chemosensory-dependent behaviors.

Cyclic nucleotide gated channels as regulators of CNS development and plasticity.

Cyclic nucleotide gated (CNG) cation channels are critical for signal transduction in vertebrate visual and olfactory systems. Members of the CNG channel gene family have now been cloned from a number of species, from Caenorhabditis elegans to humans. An important advance has been the discovery that CNG channels are present in many neurons of the mammalian brain. CNG channels act as molecular links between G-protein-coupled cascades, Ca2+-signalling systems, and gaseous messenger pathways. Perhaps most striking are recent data implicating CNG channels in both developmental and synaptic plasticity.

Impaired odor adaptation in olfactory receptor neurons after inhibition of Ca2+/calmodulin kinase II.

Odor adaptation in vertebrate olfactory receptor neurons (ORNs) is commonly attributed to feedback modulation caused by Ca(2+) entry through the transduction channels, but it remains unclear and controversial whether this Ca(2+)-mediated adaptation resides in the cAMP-gated channel alone or whether other molecules of the transduction cascade are modulated as well. Attenuation of adenylyl cyclase activity by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) has also been proposed as a mechanism for adaptation. To test this in intact ORNs, we have compared the properties of adaptation induced by a sustained (8 sec) or brief (100 msec) odor stimulus. Although adaptation induced by both types of stimuli occurs downstream from the odor receptors and is Ca(2+)-dependent, only adaptation induced by a sustained pulse involves alterations in the odor response kinetics, consistent with a reduction in the rate of adenylyl cyclase activation. By disrupting CaMKII to block adenylyl cyclase attenuation using a specific peptide inhibitor of CaMKII, autocamtide-2-related inhibitory peptide (AIP), we show that this reaction is necessary for odor adaptation in vivo. With CaMKII disrupted, adaptation induced by a sustained stimulus is significantly impaired: the onset rate of adaptation is decreased by threefold, and the recovery rate from adaptation is increased by up to sixfold. In contrast, adaptation induced by a brief odor pulse is unaffected, demonstrating that the effect of AIP must be highly specific. The results indicate that CaMKII controls the temporal response properties of ORNs during odor adaptation. We propose that CaMKII plays a prominent role in odor perception.

The TRPC2 ion channel and pheromone sensing in the accessory olfactory system.

The mammalian vomeronasal organ (VNO) has emerged as an excellent model to investigate the signaling mechanisms, mode of activation, biological function, and molecular evolution of transient receptor potential (TRP) channels in real neurons and real physiological systems. TRPC2, a member of the canonical TRPC subfamily, is highly localized to the dendritic tip of vomeronasal sensory neurons. Targeted deletion of the TRPC2 gene has established that TRPC2 plays a fundamental role in the detection of pheromonal signals by the VNO. TRPC2-deficient mice exhibit striking behavioral defects in the regulation of sexual and social behaviors. A novel Ca(2+)-permeable, diacylglycerol-activated cation channel found at the dendritic tip of vomeronasal neurons is severely defective in TRPC2 mutants, providing the first clear example of native diacylglycerol-gated cation channels in the mammalian nervous system. The TRPC2 gene has become an important marker for the evolution of VNO-dependent pheromone signaling in primates.

Modulation by cyclic GMP of the odour sensitivity of vertebrate olfactory receptor cells.

Recent evidence has indicated a significant role for the cGMP second messenger system in vertebrate olfactory transduction but no clear functions have been identified for cGMP so far. Here, we have examined the effects of 8-Br-cGMP and carbon monoxide (CO) on odour responses of salamander olfactory receptor neurons using perforated patch recordings. We report that 8-Br-cGMP strongly down-regulates the odour sensitivity of the cells, with a K1/2 of 460 nM. This adaptation-like effect can be mimicked by CO, an activator of soluble guanylyl cyclase, with a K1/2 of 1 microM. Sensitivity modulation is achieved through a regulatory chain of events in which cGMP stimulates a persistent background current due to the activation of cyclic nucleotide-gated channels. This in turn leads to sustained Ca2+ entry providing a negative feedback signal. One consequence of the Ca2+ entry is a shift to the right of the stimulus-response curve and a reduction in saturating odour currents. Together, these two effects can reduce the sensory generator current by up to twenty-fold. Thus, cGMP functions to control the gain of the G-protein coupled cAMP pathway. Another consequence of the action of cGMP is a marked prolongation of the odour response kinetics. The effects of CO/cGMP are long-lasting and can continue for minutes. Hence, we propose that cGMP helps to prevent saturation of the cell's response by adjusting the operational range of the cAMP cascade and contributes to olfactory adaptation by decreasing the sensitivity of olfactory receptor cells to repeated odour stimuli.

Inhibition of the olfactory cyclic nucleotide gated ion channel by intracellular calcium.

When olfactory receptor neurons are exposed to sustained application of odours, the elicited ionic current is transient. This adaptation-like effect appears to require the influx of Ca2+ through the odour-sensitive conductance; in the absence of extracellular Ca2+ the current remains sustained. Odour transduction proceeds through a G-protein-based second messenger system, resulting finally in the direct activation of an ion channel by cyclic AMP. This channel is one possible site for a negative feedback loop using Ca2+ as a messenger. In recordings of single cyclic AMP gated channels from olfactory receptor neurons, the open probability of the channel in saturating cAMP concentrations was dependent on the concentration of intracellular Ca2+. It could be reduced from 0.6 in 100 nm Ca2+ to 0.09 in 3 microM Ca2+. However, as neither the single channel conductance nor the mean open time were affected by Ca+ concentration, this does not appear to be a mechanism of simple channel block. Rather, these results suggest that intracellular Ca2+ acts allosterically to stabilize a closed state of the channel.

Cyclic AMP-gated cation channels of olfactory receptor neurons.

Odor-induced electrical activity in vertebrate olfactory receptor neurons is, at least in part, the result of the direct cyclic AMP-dependent activation of a nonselective cation channel. Single-channel recordings from extraciliary regions of isolated salamander olfactory receptor neurons have greatly improved our knowledge about distinctive properties of the cAMP-gated channel such as channel kinetics, modulation through divalent cations, and pharmacology. Because of the central role of these channels in the transduction cascade, these efforts have led to a better understanding of the physiology of olfactory transduction.

Role of cyclic GMP in olfactory transduction and adaptation.

The detection of odor molecules by vertebrate olfactory receptor neurons (ORNs) involves signal transduction mechanisms that are thought to occur primarily through a cyclic adenosine monophosphate (cAMP)-mediated second messenger pathway. There has been intense debate whether cAMP is the sole second messenger responsible for all excitation and adaptation. The recent identification of a distinct form of odor adaptation that depends on the carbon monoxide/cyclic guanosine monophosphate (cGMP) second messenger system demonstrates that cAMP alone cannot account for all phases of adaptation and that multiple second messenger pathways exist in ORNs to perform distinct but closely related olfactory functions.

Visualizing odor detection in olfactory cilia by calcium imaging.

To visualize odor detection in individual cilia of olfactory sensory neurons we have developed a new approach by using high-resolution calcium imaging techniques. Laser scanning confocal microscopy revealed, for the first time, that odor stimuli induce transient Ca2+ elevations in single olfactory cilia. Pharmacological analysis indicates that these Ca2+ signals depend entirely on Ca2+ entry through activated cyclic nucleotide-gated (CNG) channels. This novel approach enables us to monitor the initial steps leading to olfactory perception in a spatially and temporally resolved manner.

Cyclic GMP evoked calcium transients in olfactory receptor cell growth cones.

Nitric oxide-induced calcium transients in growth cones are believed to be mediated by cyclic nucleotides. Because nitric oxide is thought to influence the development of olfactory receptor cells (ORCs), we have begun to explore the effect of cyclic nucleotides on ORC growth cones. Cultured ORCs were loaded with fluo-3 AM and confocal imaging was employed to monitor calcium transients following cyclic nucleotide-gated channel activation. Application of 8-bromo-cGMP at the growth cone caused transient increases in fluorescence which were restricted to the growth cone and lasted tens of seconds. The signal was abolished by LY83583, an inhibitor of cyclic nucleotide-gated channels. 8-Bromo-cGMP also inhibited further extension of growth cones. The data indicate that ORC growth cones exhibit cGMP-dependent calcium transients that are consistent with those generated by cyclic nucleotide-gated channels.

A calcium-permeable cGMP-activated cation conductance in hippocampal neurons.

Whole-cell patch clamp recordings detected a previously unidentified cGMP-activated membrane conductance in cultured rat hippocampal neurons. This conductance is nonselectively permeable for cations and is completely but reversibly blocked by external Cd2+. The Ca2+ permeability of the hippocampal cGMP-activated conductance was examined in detail, indicating that the underlying ion channels display a high relative permeability for Ca2+. The results indicate that hippocampal neurons contain a cGMP-activated membrane conductance that has some properties similar to the cyclic nucleotide-gated channels previously shown in sensory receptor cells and retinal neurons. In hippocampal neurons this conductance similarly could mediate membrane depolarization and Ca2+ fluxes in response to intracellular cGMP elevation.

Similarities between the effects of lindane (gamma-HCH) and picrotoxin on ligand-gated chloride channels in crayfish muscle membrane.

Effects of lindane (gamma-HCH) and picrotoxin have been tested on excised outside-out patches of crayfish stomach muscle membrane. The pulsed application of 10(-3) M gamma-aminobutyric acid (GABA) elicited an inward CL- current of several 100 pA, which could be reversibly reduced by the simultaneous application of lindane or picrotoxin. The rise time and desensitization time constant of the GABA response did not seem to be affected. The apparent KI was 5 x 10(-6) M for lindane and 15 x 10(-6) M for picrotoxin. Also Cl- currents induced by glutamate or acetylcholine could be reversibly reduced by lindane or picrotoxin. The evaluation of single Cl- channel openings which were elicited by the simultaneous application of glutamate and lindane showed no effect of lindane on the unitary conductance and on the mean open time of these channels. A mechanism for the antagonistic action of lindane and picrotoxin is discussed.

Parallel processing of social signals by the mammalian main and accessory olfactory systems.

The mammalian olfactory system has evolved complex mechanisms to detect a vast range of molecular cues. In rodents, the olfactory system comprises several distinct subsystems. Current interest has focused on the exact role that each of these subsystems plays in detecting molecular information and regulating chemosensory-dependent behaviors. Here, we summarize recent results showing that the mouse main and accessory olfactory systems detect, at least in part, overlapping sets of social chemosignals. These findings give rise to a model that involves parallel processing of the same molecular cues in both systems. Together with previous work, this model will lead to a better understanding of the general organization of chemical communication in mammals and give a new direction for future experiments.

Dual activation of a sex pheromone-dependent ion channel from insect olfactory dendrites by protein kinase C activators and cyclic GMP.

Olfactory transduction is thought to take place in the outer dendritic membrane of insect olfactory receptor neurons. Here we show that the outer dendritic plasma membrane of silkmoth olfactory receptor neurons seems to be exclusively equipped with a specific ion channel activated by low concentrations of the species-specific sex pheromone component. This so-called AC1 channel has a conductance of 56 pS and is nonselectively permeable to cations. The AC1 channel can be activated from the intracellular side by protein kinase C activators such as diacylglycerol and phorbolester and by cGMP but not by Ca2+, inositol 1,4,5-triphosphate, or cAMP. Our results imply that phosphorylation of this ion channel by protein kinase C could be the crucial step in channel opening by sex pheromones.

A calcium-activated nonspecific cation channel from olfactory receptor neurones of the silkmoth Antheraea polyphemus.

Single-channel patch-clamp techniques were used to identify and characterize a Ca2(+)-activated nonspecific cation channel (CAN channel) on insect olfactory receptor neurones (ORNs) from antennae of male Antheraea polyphemus. The CAN channel was found both in acutely isolated ORNs from developing pupae and in membrane vesicles from mature ORNs that presumably originated from inner dendritic segments. Amplitude histograms of the CAN single-channel currents presented well-defined peaks corresponding to at least four channel substates each having a conductance of about 16 pS. Simultaneous gating of the substates was achieved by intracellular Ca2(+) with an EC(50) value of about 80 nmol(-l). Activity of the CAN channel could be blocked by application of amiloride (IC(50)<100 nmoll(-1)). Moreover, in the presence of l µmoll(-1) Ca2+,opening of the CAN channel was totally suppressed by 10 µmoll(-1) cyclic GMP,whereas ATP (1 mmoll(-1)) was without effect. We suggest that the CAN channel plays a specific role in modulation of cell excitability and in shaping the voltage response of ORNs.

The cyclic nucleotide gated channel of olfactory receptor neurons.

Olfactory transduction proceeds through a G-protein coupled cascade that produces the ubiquitous second messenger cyclic AMP. The cyclic AMP causes a change in membrane potential by acting directly on an ion channel that allows cations to flow into the cell. This ion channel is one of a new family of ion channels that are activated by intracellular cyclic nucleotides. However, even though they are activated by binding a ligand their amino acid structure shows that they share a common ancestry with voltage activated channels, especially voltage dependent Ca2+ channels. In olfactory neurons these channels perform a critical role in the transduction of chemical information in the environment into changes in membrane electrical properties that are transmitted to higher order processing centers in the brain.

Divalent cations block the cyclic nucleotide-gated channel of olfactory receptor neurons.

1. The effects of external divalent cations on odor-dependent, cyclic AMP-activated single-channel currents from olfactory receptor neurons of the tiger salamander (Ambystoma tigrinum) were studied in inside-out membrane patches taken from dendritic regions of freshly isolated sensory cells. 2. Channels were reversibly activated by 100 microM cyclic AMP. In the absence of divalent cations, the channel had a linear current-voltage relation giving a conductance of 45 pS. With increasing concentrations of either Ca2+ or Mg2+ in the external solution, the channel displayed a rapid flickering behavior. At higher concentrations of divalent cations, the transitions were too rapid to be fully resolved and appeared as a reduction in mean unitary single-channel current amplitude. 3. This effect was voltage dependent, and on analysis was shown to be due to an open channel block by divalent ions. In the case of Mg2+, the block increased steadily with hyperpolarization. In contrast, for Ca2+ the block first increased with hyperpolarization and then decreased with further hyperpolarization beyond -70 mV, providing evidence for Ca2+ permeation of this channel. 4. This block is similar to that seen in voltage-gated calcium channels. Additionally, the cyclic nucleotide-gated channel shows some pharmacological similarities with L-type calcium channels, including a novel block of the cyclic nucleotide channel by nifedipine (50 microM). 5. Our results indicate that the sensory generator current simultaneously depends on the presence of the second messenger and on the membrane potential of the olfactory neuron.

Membrane currents and mechanisms of olfactory transduction.

The term olfactory transduction refers to the mechanisms that transform chemical information into electrical signals. With the patch-clamp technique it is possible to record those signals and to infer something about the mechanism that produced them. The direct activation of a cation-permeable channel by cAMP is the final step in producing the odour-induced ionic current. Because it occupies a critical position in the transduction process, measurements of the ion channel's activity provide useful insights into the molecular processes underlying olfactory transduction. In addition to its activation by cAMP and cGMP, the channel is modulated by both extracellular and intracellular Ca2+ ions and by extracellular Mg2+ ions, all at physiological concentrations. These effects are probably important in promoting signal reliability. An unusual feature of this channel is its termination kinetics--it can remain active for hundreds of milliseconds after the agonist has been removed. This is likely to add to the integrating properties of the olfactory sensory neuron.