Electrical signals in olfactory transduction.

Olfactory transduction involves a G-protein-coupled second messenger system, which results in the odor-dependent production of cAMP. The direct activation of ion channels in the cilia membrane by cAMP is the final step in producing the slow depolarization that brings the membrane potential to threshold for spike generation. Because of the central role in the transduction cascade occupied by these channels considerable effort has been directed toward understanding their behavior at a molecular level. Alternative second messenger pathways have also been proposed in olfaction, but the physiological evidence for these is less well developed.     

Calcium and olfactory transduction

1. Inorganic cations, organic calcium antagonists, and calmodulin antagonists were applied to olfactory epithelia of frogs (Rana pipiens) while recording electroolfactogram (EOG) responses. 2. Inorganic cations inhibited EOGs in a rank order, reflecting their calcium channel blocking potency: La3+ greater than Zn2+ greater than Cd2+ greater than Al3+ greater than Ca2+ greater than Sr2+ greater than Co2+ greater than Ba2+ greater than Mg2+. Barium ion significantly enhanced EOGs immediately following application. 3. Diltiazem and verapamil produced dose-dependent EOG inhibition. 4. Calmodulin antagonists inhibited EOGs without correlation to their anti-calmodulin potency.     

Odour transduction in olfactory receptor neurons

The molecular mechanisms that control the binding of odorant to olfactory receptors and transduce this signal into membrane depolarization are reviewed. They are compared in vertebrates and insects for interspecific (allelochemicals) and intraspecific (pheromones) olfactory signals. Attempts to develop quantitative models of these multistage signalling networks are presented. Computational analysis of olfactory transduction is still in its infancy and appears as a promising area for future developments.     

Properties of cyclic nucleotide-gated channels mediating olfactory transduction. Activation, selectivity, and blockage.

Cyclic nucleotide-gated channels (cng channels) in the sensory membrane of olfactory receptor cells, activated after the odorant-induced increase of cytosolic cAMP concentration, conduct the receptor current that elicits electrical excitation of the receptor neurons. We investigated properties of cng channels from frog and rat using inside-out and outside-out membrane patches excised from isolated olfactory receptor cells. Channels were activated by cAMP and cGMP with activation constants of 2.5-4.0 microM for cAMP and 1.0-1.8 for cGMP. Hill coefficients of dose-response curves were 1.4-1.8, indicating cooperativity of ligand binding. Selectivity for monovalent alkali cations and the Na/Li mole-fraction behavior identified the channel as a nonselective cation channel, having a cation-binding site of high field strength in the pore. Cytosolic pH effects suggest the presence of an additional titratable group which, when protonated, inhibits the cAMP-induced current with an apparent pK of 5.0-5.2. The pH effects were not voltage dependent. Several blockers of Ca2+ channels also blocked olfactory cng channels. Amiloride, D 600, and diltiazem inhibited the cAMP-induced current from the cytosolic side. Inhibition constants were voltage dependent with values of, respectively, 0.1, 0.3, and 1 mM at -60 mV, and 0.03, 0.02, and 0.2 mM at +60 mV. Our results suggest functional similarity between frog and rat cng channels, as well as marked differences to cng channels from photoreceptors and other tissues.     

Sensitivity and specificity amplification in signal transduction

Intracellular signal transduction pathways transmit signals from the cell surface to various intracellular destinations, such as cytoskeleton and nucleus through a cascade of protein-protein interactions and activation events, leading to phenotypic changes such as cell proliferation, differentiation, and death. Over the past two decades, numerous signaling proteins and signal transduction pathways have been discovered and characterized. There are two major classes of signaling proteins: phosphoproteins (e.g., mitogen-activated protein kinases) and guanosine triphosphatases (GTPases; e.g., Ras and G proteins). They both function as molecular switches by addition and removal of one or more high-energy phosphate groups. This review discusses developments that seek to quantify the signal transduction processes with kinetic analysis and mathematical modeling of the signaling phosphoproteins and GTPases. These studies have provided insights into the sensitivity and specificity amplification of biological signals in integrated systems.     

Calcium,the two-faced messenger of olfactory transduction and adaptation

Exposure of olfactory receptor cells to odour stimulates the influx of Ca(2+) through cyclic nucleotide-gated channels into the small volume within the cilia, the site of olfactory transduction. The consequent rise in intraciliary Ca(2+) concentration has two opposing effects: activation of an unusual excitatory Cl(-) conductance, and negative feedback actions on various stages of the odour transduction mechanism. Recent studies are beginning to unravel how Ca(2+) performs this dual function, and how the spatial and temporal dynamics of Ca(2+) modulate the odour response. The feedback actions of Ca(2+) on different elements of the transduction cascade seem to occur on different timescales, and are therefore responsible for shaping different parts of the receptor current response to odour stimulation.     

Temporal cooperativity and sensitivity amplification in biological signal transduction

Sensitivity amplification in signal transduction modules regulated by phosphorylation-dephosphorylation cycles and GTPases results from a new type of cooperativity which is fundamentally different from that of allosterism. This type of cooperativity, termed temporal cooperativity [Qian, H. (2003) Biophys. Chem. 105, 585-593], is analyzed in this paper through stochastic models for molecular interactions. Mathematical analysis is developed through a series of models with different levels of complexity, from which a simple conceptual model based on linear cooperativity is derived. The following are shown: (i) When both kinase and phosphatase are nonsaturating, the distribution of the number of activated substrate molecules is binomial. With increasing kinase activity, the peak of the distribution continuously moves toward 100% activation. (ii) When the kinase is saturated, i.e., zeroth order, the distribution is Poisson. (iii) When both enzymes are saturated, the distribution is geometric. Ultrasensitivity corresponds to an abrupt switching of the peak position of the distribution from 0 to 100%. The theory is applicable to a wide range of processes in cell signaling including the specificity and sensitivity of T-cell activation.     

Extracellular transduction events under pulsed stimulation in moth olfactory sensilla

In natural conditions, pheromones released continuously by female moths are broken in discontinuous clumps and filaments. These discontinuities are perceived by flying male moths as periodic variations in the concentration of the stimulus, which have been shown to be essential for location of females. We study analytically and numerically the evolution in time of the activated pheromone-receptor (signaling) complex in response to periodic pulses of pheromone. The 13-reaction model considered takes into account the transport of pheromone molecules by pheromone binding proteins (PBP), their enzymatic deactivation in the perireceptor space and their interaction with receptors at the dendritic membrane of neurons in Antheraea polyphemus sensitive to the main pheromone component. The time-averaged and periodic properties of the temporal evolution of the signaling complex are presented, in both transient and steady states. The same time-averaged response is shown to result from many different pulse trains and to depend hyperbolically on the time-averaged pheromone concentration in air. The dependency of the amplitude of the oscillations of the signaling complex on pulse characteristics, especially frequency, suggests that the model can account for the ability of the studied type of neuron to resolve repetitive pulses up to 2 Hz, as experimentally observed. Modifications of the model for resolving pulses up to 10 Hz, as found in other neuron types sensitive to the minor pheromone components, are discussed.     

The role of the coreceptor Orco in insect olfactory transduction

Insects sense odorants with specialized odorant receptors (ORs). Each antennal olfactory receptor neuron expresses one OR with an odorant binding site together with a conserved coreceptor called Orco which does not bind odorants. Orco is necessary for localization of ORs to dendritic membranes and, thus, is essential for odorant detection. It forms a spontaneously opening cation channel, activated via phosphorylation by protein kinase C. Thereafter, Orco is also activated via cyclic adenosine monophosphate (cAMP). Orco forms homo—as well as heteromers with ORs with unknown stoichiometry. Contradictory publications suggest different mechanisms of olfactory transduction. On the one hand, evidence accumulates for the employment of more than one G protein-coupled olfactory transduction cascade in different insects. On the other hand, results from other studies suggest that the OR–Orco complex functions as an odorant-gated cation channel mediating ionotropic signal transduction. This review analyzes conflicting hypotheses concerning the role of Orco in insect olfactory transduction. In conclusion, in situ studies in hawkmoths falsify the hypothesis that Orco underlies odorant-induced ionotropic signal transduction in all insect species. Instead, Orco forms a metabotropically gated, slow cation channel which controls odorant response threshold and kinetics of the sensory neuron.     

The electrochemical basis of odor transduction in vertebrate olfactory cilia

Most vertebrate olfactory receptor neurons share a common G-protein-coupled pathway for transducing the binding of odorant into depolarization. The depolarization involves 2 currents: an influx of cations (including Ca2+) through cyclic nucleotide-gated channels and a secondary efflux of Cl- through Ca2+-gated Cl- channels. The relation between stimulus strength and receptor current shows positive cooperativity that is attributed to the channel properties. This cooperativity amplifies the responses to sufficiently strong stimuli but reduces sensitivity and dynamic range. The odor response is transient, and prolonged or repeated stimulation causes adaptation and desensitization. At least 10 mechanisms may contribute to termination of the response; several of these result from an increase in intraciliary Ca2+. It is not known to what extent regulation of ionic concentrations in the cilium depends on the dendrite and soma. Although many of the major mechanisms have been identified, odor transduction is not well understood at a quantitative level.     

Modification of transduction mechanisms in the frog's olfactory mucosa using a thiol reagent as olfactory stimulus

The effects of the thiol-specific reagent N-ethylmaleimide (NEM)used in the vapour phase have been tested on the olfactory epitheliumof the frog when recording the electro-olfactogram (EOG) andspike activity from single receptor cells. The reagent was deliveredalone or mixed with the odorant isoamyl acetate. At low concentrationthe reagent induced slow potentials resembling simple EOGs.At higher concentrations (20% of the saturated vapour) threenegative and one positive slow components were observed in theresponse. A complex relationship was found between the amplitudeof the slow potential and the concentration of the reagent.Repeated stimulations at high concentration caused the suppressionof the negative voltage transients and the development of thepositive component. NEM vapour elicited spike discharges insome of the recorded units, with the responses resembling thoseevoked by usual odorants. After long-lasting stimulations (30 sec) with NEM, the receptorsfailed to respond to both reagent and odorant. This suppressionof response could be partly prevented by exposing the olfactoryepithelium to the odorant vapour before and during the exposureto the reagent (protection). The results indicate that NEM acts on the olfactory epitheliumin several ways, including an odorant-like action on olfactoryreceptor sites. An effect on the supporting cells is also suggested.Hypotheses for explaining the protection mechanism are considered.     

Failure to implicate cAMP in transduction in lobster olfactory receptor cells

The possible role of adenosine 3',5'-cyclic monophosphate (cAMP)in olfactory transduction in the spiny lobster was investigatedusing radioimmunoassay of cAMP and intracellular recording.Application of forskolin or 1-isobutyl-3-methylxanthine increasedcAMP levels in intact sensilla containing the chemoreceptiveouter dendritic segments of the lobster olfactory receptor cell,thereby demonstrating adenylate cyclase and phosphodiesteraseactivity in the sensilla. A complex odor mixture and identifiedexcitatory odor molecules failed to stimulate the productionof cAMP, however In intracellular recordings, superfusion ofthe outer dendritic segments with forskolin, 1-isobutyl-3-methylxanthineand cyclic nucleotide analogs had no direct effect on odor-responsivecells. These compounds did cause infrequent enhancements (sixof 42 cells) of odor-evoked receptor potentials, but processesother than transduction are the most likely causes of this effect.We conclude that cAMP-dependent transduction mechanisms areunlikely to mediate most odor responses in lobsters, in contrastto transduction mechanisms in amphibians and rats.     

Nanovesicle-based bioelectronic nose platform mimicking human olfactory signal transduction

We developed a nanovesicle-based bioelectronic nose (NBN) that could recognize a specific odorant and mimic the receptor-mediated signal transmission of human olfactory systems. To build an NBN, we combined a single-walled carbon nanotube-based field effect transistor with cell-derived nanovesicles containing human olfactory receptors and calcium ion signal pathways. Importantly, the NBN took advantages of cell signal pathways for sensing signal amplification, enabling ≈ 100 times better sensitivity than that of previous bioelectronic noses based on only olfactory receptor protein and carbon nanotube transistors. The NBN sensors exhibited a human-like selectivity with single-carbon-atomic resolution and a high sensitivity of 1 fM detection limit. Moreover, this sensor platform could mimic a receptor-meditated cellular signal transmission in live cells. This sensor platform can be utilized for the study of molecular recognition and biological processes occurring at cell membranes and also for various practical applications such as food screening and medical diagnostics.     

Plasma membrane inositol 1,4,5-trisphosphate-activated channels mediate signal transduction in lobster olfactory receptor neurons.

Inositol 1,4,5-trisphosphate (IP3) selectively evokes an inward (excitatory) current in cultured lobster olfactory receptor neurons (ORNs) and directly activates two types of channels in cell-free patches of plasma membrane from the ORNs. The IP3-activated channels have kinetic properties of odor-activated channels in the ORNs and pharmacological properties of intracellular IP3-activated channels in other systems. An antibody directed against an intracellular, cerebellar IP3 receptor recognizes a protein with a molecular weight similar to the mammalian receptor in the ORNs. The antibody selectively increases odor-evoked inward currents and IP3-activated unitary currents in the ORNs. The data provide further evidence for IP3 as an olfactory second messenger and implicate at least one and possibly two novel plasma membrane IP3 receptors in olfactory transduction.