Biochemistry of Olfaction Lesson 1-2 (PDF)

Biochemistry of Olfaction Olfaction Human beings can detect and distinguish thousands of different compounds by smell, often with considerable sensitivity and specificity. Most odorants are small organic compounds with sufficient volatility that they can be carried as vapors into the nose. For exam...

Biochemistry of Olfaction Olfaction Human beings can detect and distinguish thousands of different compounds by smell, often with considerable sensitivity and specificity. Most odorants are small organic compounds with sufficient volatility that they can be carried as vapors into the nose. For example, a major component responsible for the odor of almonds is the simple aromatic compound benzaldehyde, whereas the sulfhydryl compound 3-methylbutane-1-thiol is a major component of the odor of skunks. What properties of these molecules are responsible for their odors? First, the shape of the molecule rather than its other physical properties is crucial. We can most clearly see the importance of shape by comparing molecules such as those responsible for the odors of spearmint and caraway. These compounds are identical in essentially all physical properties such as hydrophobicity because they are exact mirror images of one another. Thus, the odor produced by an odorant depends not on a physical property but on the compound’s interaction with a specific binding surface, most likely a protein receptor. Second, some human beings (and other animals) suffer from specific anosmias; that is, they are incapable of smelling specific compounds even though their olfactory systems are otherwise normal. Such anosmias are often inherited. These observations suggest that mutations in individual receptor genes lead to the loss of the ability to detect a small subset of compounds. Odorants are detected in a specific region of the nose, called the main olfactory epithelium, that lies at the top of the nasal cavity. Approximately 1 million sensory neurons line the surface of this region. Cilia containing the odorant- binding protein receptors project from these neurons into the mucous lining of the nasal cavity. Biochemical studies in the late 1980s examined isolated cilia from rat olfactory epithelia that had been treated with odorants. Exposure to the odorants increased the cellular level of cyclic AMP, and this increase was observed only in the presence of GTP. On the basis of what was known about signal transduction systems, the participation of cAMP and GTP strongly suggested the involvement of a G protein and, hence, 7TM receptors. Indeed, Randall Reed purified and cloned a G-protein a subunit, termed G (olf ), which is uniquely expressed in olfactory cilia. The involvement of 7TM receptors suggested a strategy for identifying the olfactory receptors themselves. Olfactory signal transduction. Olfactory signal transduction. Olfactory signal transduction. Typically, an odorant binds to the olfactory receptor (OR) in the ciliary membrane of an olfactory neuron, activating an olfaction-specific G protein (G olf ) which converts GTP to GDP and activates the adenylyl cyclase type III (ACIII) to convert ATP to cAMP. The second messenger cAMP gates the olfactory cyclic nucleotide-gated ion channel (CNG) resulting in the intake of Ca 2+ which lead to the activation of a Ca 2+ -activated Cl − channel (CaCC) and subsequent Cl − efflux which further depolarizes the cell. Ca 2+ also stimulates the activity of a phosphodiesterase (PDE). Ca 2+ is extruded by a Na + /Ca 2+ exchanger. cAMP also activates cAMP-dependent Protein Kinase (PKA) resulting on gene transcription. OR stimulation also leads to the cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) into 1,4,5-inositol trisphosphate (IP3) and diacylglycerol (DAG) via phospholipase C (PLC). DAG gates the non-specific cation current through the type 2 canonical transient receptor potential channel (TRPC2) and IP3 binds the IP3 receptor (IP3R), the increase in intracellular Ca 2+ amplifies the chemosignalinduced current by activating the non-selective ion channel (CaNS) and the calcium-activated big conductance potassium ion channels (BK) resulting in cell depolarization. In non-olfactory tissues, ORs can activate various signalling pathways. ORs are members of the G protein–coupled receptor (GPCR) superfamily, and share with other members a number of stereotypical motifs, including an apparent seven-transmembrane domain topology. However, ORs exhibit unique sequence motifs not seen in other GPCRs, and which, along with their expression in OSNs (olfactory sensory neurons), help to define them as a distinct group of receptor proteins. Analyses of the human and mouse genomes has revealed ~350 functional OR genes in humans (with an equivalent number of OR pseudogenes) and ~1200 in mice, making this the largest mammalian gene family. OR genes are found in clusters within the genome on nearly every chromosome, suggesting a large number of local tandem duplications during evolution. The large number of mammalian ORs has enormous implications for the way odorants are detected and encoded by the olfactory system. The odor-dependent second-messenger cascade is a classic cyclic nucleotide-based system with some interesting modifications. A G protein that is likely to couple odorant receptors to other intracellular elements of the cascade, Gαolf, has been identified. An isoform of Gαs, it is highly enriched in OSN cilia. OSNs also contain an adenylyl cyclase isoform, type III (ACIII), that is highly enriched in olfactory cilia. An important characteristic of this isozyme is that, when expressed in a mammalian cell line, its basal activity is extremely low, while in its stimulated state it has a catalytic rate higher than other known cyclases. These properties could confer a high signal-to-noise ratio on the system, being quiescent in the absence of stimulus but able to rapidly generate large amounts of the second messenger adenosine 3’5’-cyclic monophosphate (cAMP) upon odor exposure. Deletion of the gene encoding ACIII elicits a general anosmia in mice. ACIII is also inhibited by Ca 2+, providing an opportunity for negative feedback upon intracellular elevations of this signaling molecule. cAMP activates CNG. It was subsequently determined that the channel responsible is comprised of three distinct olfactory specific subunits, CNGA2, CNGA4 and CNGB1b; all are members of the cyclic nucleotide-gated (CNG) channel family. These channels are homologous with voltage-sensitive channels, such as K+ and Ca 2+ channels. CNGA2 is obligatory for channel function both in vitro and in vivo, while CNGA4 and CNGB1b serve important modulatory. The olfactory CNG channel is selective for cations; is sensitive to both cAMP and cGMP, with a Kd of 20 μM or Kd of 5 μM, respectively; requires at least three molecules of cyclic nucleotide to bind for activation; and is required for odor signaling in OSNs. The activation of tens to hundreds of these channels and the subsequent influx of cations, including both Na + and Ca 2+, leads to depolarization of the cell membrane. OSNs have one additional level of amplification that is rather unusual. A Ca 2+activated Cl - conductance is also present on the cilia and is opened during the odor response by the Ca 2+ ions flowing into the cilia through the cAMP-gated channel; this channel was recently identified as anoctamin 2 (ANO2). Curiously, olfactory neurons maintain a very high intracellular Cl- concentration, perhaps as high as 125 mM, so that the driving force for Cl- ions is outward. Thus, activation of this Cl- conductance further depolarizes the cell. Changing ion concentrations in the mucus could affect the driving forces on ions, so the olfactory neuron supplies its own driving force by maintaining a high intracellular Cl- concentration. The olfactory transduction pathway provides several amplification steps between odorant binding and signal generation The termination of a response in the continued presence of an agonist is characteristic of many signaling systems and is variously known as adaptation or desensitization. The CNG channel is permeable to cations, especially Ca 2+. Thus, increased channel activation results in influx of Ca 2+ and a transient rise in the intracellular Ca 2+ concentration. Intracellular Ca 2+ concentrations of 1 to 3 μM have been found to lead to a decrease in the open probability of the ion channel, even in the presence of high concentrations of cAMP. Mediated by the intermediate Ca 2+-binding protein calmodulin, this Ca 2+-dependent desensitization of the channel is a major contributor to short-term adaptation and is dependent on the presence of the CNGA4 subunit, which increases the kinetics of Ca 2+/calmodulin-mediated desensitization of the native channel nearly 70-fold. This is an attractive mechanism for mediating a rapid but short-lasting form of adaptation since it is dependent on the influx of Ca 2+ during the response to an odor. Other mechanisms have also been implicated in odor adaptation, including phosphodiesterase-mediated hydrolysis of cAMP; cAMP-dependent phosphorylation of ciliary proteins via protein kinase A; G protein receptor kinase activity (GRK3), possibly via phosphorylation of the OR; Ca 2+/ calmodulin kinase II (CaMKII) phosphorylation of ACIII; cGMP; and carbon monoxide. These latter three mechanisms have been particularly linked to longer-lasting forms of adaptation, on the order of tens of seconds (for CaMKII) or minutes (CO, cGMP). Together with the short-term adaptation described previously, these various molecular mechanisms provide the OSN with a number of ways to fine- tune odor responses over time.  The vomeronasal organ is an accessory chemosensing system that plays a major role in the detection of semiochemicals Vomeronasal organ is vestigial in humans. A model for chemosensory transduction in vomeronasal sensory neurons. In contrast to the transduction cascade in OSNs, the mechanism of vomeronasal transduction is less well characterized. Vomeronasal sensory neurons express V1R, V2R or FPR (third class of GPCRs, members of the formyl peptide receptor (FPR) family receptors) and either Gαi2 or Gαo. The TRPC2 (TRPC - transient receptor potential (TRP) family channel) channel subunit is expressed in all VSNs, and may be part of a multimeric channel complex. Ca 2+ ions are represented as purple balls; Na + ions as blue balls. The native VSN channel containing VR, vomeronasal receptor (V1R, V2R or FPR); TRPC2 is directly gated by the lipid messenger diacylglyerol PIP2, phosphatidylinositol 4,5-bisphosphate; IP3, in a pheromone-dependent manner. inositol 1,4,5-trisphosphate. DAG, diacylglycerol. However, the activation of vomeronasal receptors has still not been directly linked to diacylglycerol production via any G protein or phospholipase isoforms. Biochemistry of Taste - receptors, G proteins and second messenger- effector enzymes. Taste Is a Combination of Senses That Function by Different Mechanisms The inability to taste food is a common complaint when nasal congestion reduces the sense of smell. Thus, smell greatly augments our sense of taste (also known as gustation ), and taste is, in many ways, the sister sense to olfaction. Nevertheless, the two senses differ from each other in several important ways. First, we are able to sense several classes of compounds by taste that we are unable to detect by smell; salt and sugar have very little odor, yet they are primary stimuli of the gustatory system. Second, whereas we are able to discriminate thousands of odorants, discrimination by taste is much more modest. Five primary tastes are perceived: bitter, sweet, sour, salty, and umami (the taste of glutamate and aspartate from the Japanese word for “deliciousness”). These five tastes serve to classify compounds into potentially nutritive and beneficial (sweet, salty, umami) or potentially harmful or toxic (bitter, sour). Tastants (the molecules sensed by taste) are quite distinct for the different groups. The simplest tastant, the hydrogen ion, is perceived as sour. Other simple ions, particularly sodium ion, are perceived as salty. The taste called umami is evoked by the amino acids glutamate and aspartate, the former often encountered as the flavor enhancer monosodium glutamate (MSG). In contrast, tastants perceived as sweet and, particularly, bitter are extremely diverse. Many bitter compounds are alkaloids or other plant products, many of which are toxic. However, they do not have any common structural elements or other common properties. Carbohydrates such as glucose and sucrose are perceived as sweet, as are other compounds including some simple peptide derivatives, such as aspartame, and even some proteins. These differences in specificity among the five tastes are due to differences in their underlying biochemical mechanisms. The sense of taste is, in fact, a number of independent senses all utilizing the same organ, the tongue, for their expression. Tastants are detected by specialized structures called taste buds, which contain approximately 150 cells, including sensory neurons.  Our sense of taste can detect and discriminate among various ionic stimuli—for example, Na + as salty, H +as sour, sugars as sweet and alkaloids as bitter. Five primary tastes are perceived: bitter, sweet, sour, salty, and umami (the taste of glutamate and aspartate from the Japanese word for “deliciousness”). These five tastes serve to classify compounds into potentially nutritive and beneficial (sweet, salty, umami) or potentially harmful or toxic (bitter, sour). Tastants (the molecules sensed by taste) are quite distinct for the different groups. Fig. (A) The tongue, the primary organ of taste, consists of small structures known as papillae (raised bumps) where taste buds reside. Depending on their shape papillae are classified into four groups: circumvallate, fungiform, foliate and filiform (B) Each taste bud harbors a set of elongated taste receptor cells that contain taste receptors that sense substances with different taste qualities. Upon detecting a substance, taste receptor cells transmit the information to gustatory nerves in contact with the tissue, which further transmit the information to the central nervous system, ultimately reaching the brain. Fingerlike projections called microvilli, which are rich in taste receptors, project from one end of each sensory neuron to the surface of the tongue. Nerve fibers at the opposite end of each neuron carry electrical impulses to the brain in response to stimulation by tastants. Structures called taste papillae contain numerous taste buds.  Sweet, bitter and umami taste involve G protein coupled receptors Type 1 Taste Receptors (T1Rs) Recognize Sweet and Umami Stimuli The first taste-specific GPCRs were identified in 1999 but remained orphan receptors (i.e., their physiological ligands were unknown) for several years. Now called T1R1 and T1R2, these receptors are Class C GPCRs and contain a large extracellular N-terminus that contains the orthosteric ligand-binding site. (Several allosteric sites are found on other parts of the proteins). The T1Rs are differentially distributed across the gustatory epithelium: T1R1 and T1R2 are expressed in distinct populations of TRCs (T1R1 is found predominantly on the anterior tongue and T1R2 mostly on the posterior tongue), but are always coexpressed with T1R3. The T1Rs function as heteromeric receptors (likely dimers), with T1R2 and T1R3 combining to form a receptor for sweet tasting compounds including sugars, sweeteners, and some D-amino. In contrast, T1R1/T1R3 heteromers are insensitive to sweet-tasting stimuli but do respond to umami stimuli, including some L-amino acids. Furthermore, T1R1/ T1R3 responses are potentiated by 5’-ribonucleotides, a characteristic of umami taste. Schematic drawing of the multiple binding sites between sweet and umami taste receptors and ligands. The N- terminal extracellular domain of T1R2 is required for the binding of aspartame, neotame, saccharin and sucrose. The transmembrane domains of T1R3 are required for the binding of cyclamate, lactisole, saccharin and artificial sweeteners. The N-termini of T1R3 near the first transmembrane region is required for certain sweet-tasting proteins such as brazzein. L-Glutamate and IMP bind to the T1R1 subunit, respectively. In addition, the transmembrane domain of T1R1 or TIR2 is responsible for coupling to G proteins  Type 2 Taste Receptors (T2Rs) Mediate Responses to Bitter-Tasting Stimuli A distinct receptor family is involved in the detection of bitter tastants. Sweet, bitter and umami tasting stimuli are transduced by a G-protein–coupled signaling cascade α-Gustducin functions in sweet, bitter and umami taste In TRCs, α-gustducin associates with the G protein β and γ subunits Gβ3 and Gγ13. It is these subunits that activate the effector enzyme phospholipase C-β2 (PLCβ2), which can produce the second IP3 messengers and diacylglycerol through the cleavage of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate  A model for the major signaling mechanisms for the transduction of sweet, bitter and umami stimuli. Note that stimuli of each of these taste qualities interact with GPCRs: bitter stimuli with T2Rs, and sweet and umami stimuli with T1Rs. α-Gustducin has been implicated in the transduction of all three types of stimuli. Ca 2+-activated cation channel is present in taste cells: the TRP channel type M5 (TRPM5), is essential for normal sweet, bitter and umami taste function. Finally, cAMP has also been suggested to play a role in taste transduction, perhaps as a mediator of taste adaptation. Gβγ Signaling Taste receptor binding results in Gβγ activation of phospholipase C β2 (PLCβ2), which cleaves the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) into the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Although the function of DAG in taste cells is unclear, IP3 is cytosolic and binds to the type 3 IP3 receptor (IP3R3), located on the endoplasmic reticulum, causing a release of Ca2+ from intracellular stores and subsequent Ca2+-dependent activation of the monovalent-selective cation channel, transient receptor potential channel M5 (TRPM5). Influx of Na+ through TRPM5 depolarizes the membrane, leading to activation of voltage-gated Na+ channels, generation of action potentials There are currently two models proposed for sweet taste transduction. The first pathway is a GPCRGs- cAMP pathway. This pathway starts with sucrose and other sugars activating Gs inside the cell through a membrane-bound GPCR. The activated Gas activates adenylyl cyclase to generate cAMP. From this point, one of two pathways can be taken. cAMP may act directly to cause an influx of cations through cAMP- gated channels or cAMP can activate protein kinase A, which causes the phosphorylation of K+ channels, thus closing the channels, allowing for depolarization of the taste cell, subsequent opening of voltage- gated Ca2+ channels and causing neurotransmitter release. The second pathway is a GPCR-Gq/Gβγ-IP3 pathway which is used with artificial sweeteners. Artificial sweeteners bind and activate GPCRs coupled to PLCβ2 by either α-Gq or Gβγ. The activated subunits activate PLCβ2 to generate IP3 and DAG. IP3 and DAG elicit Ca2+ release from the endoplasmic reticulum and cause cellular depolarization. An influx of Ca2+ triggers neurotransmitter release. While these two pathways coexist in the same TRCs, it is unclear how the receptors selectively mediate cAMP responses to sugars and IP3 responses to artificial sweeteners So, the sense of taste in vertebrates reflects the activity of gustatory neurons clustered in taste buds on the surface of the tongue. For example, sweet tasting molecules are those that bind receptors in “sweet” taste buds. In taste sensory neurons, GPCRs are coupled to the heterotrimeric G protein gustducin. When the tastant molecule binds its receptor, gustducin is activated and stimulates cAMP production by adenylyl cyclase. The resulting elevation of [cAMP] activates PKA, which phosphorylates K+ channels in the plasma membrane, causing them to close and sending an electrical signal to the brain. Other taste buds specialize in detecting bitter, sour, salty, or umami (savory) tastants, using various combinations of second messengers and ion channels in the transduction mechanisms. Salts and acids are transduced by direct interaction with ion channels Several salts, including both NaCl and KCl, can elicit a perception of salty taste. At least two distinct molecularmechanisms mediate salt taste in mammals: one is Na+specific, while the other is responsive to all salty-tasting stimuli. The presumed mechanism for Na+ taste had long been the direct influx of Na+ through amiloride-sensitive epithelial sodium channels (ENaCs): the α, β and γ subunits of the ENaC channel have been localized to the apical membranes of TRCs (ENaCs function as heterooligomeric complexes), and the Na+- specific component of salt taste is blockable by amiloride at micromolar concentrations. Sour taste is a function of the acidity of a solution, depending primarily on the proton concentration and to a lesser extent on the particular anion involved. Several mechanisms have been proposed to mediate sour taste, including proton or pH-dependent gating of ion channels, direct flux of protons through ion channels or intracellular acidification of ion channels or other proteins. To date only a single, very specialized sour taste mechanism has been identified: the membrane bound carbonic anhydrase isoform Car4 is expressed on the surface of a subset of TRCs and mediates the sour taste of carbonation, likely through the generation of protons through the catalysis of carbon dioxide and water. Carbonic anhydrases (CAs) reversibly catalyze the conversion of CO2 into bicarbonate ions and free protons. Car4 is a mammalian carbonic anhydrase that functions as an extracellular, lycosylphosphatidylinositol (GPI)–anchored enzyme