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Signal recognition and chemoelectrical transduction in olfaction
Biosensors & Bioelectronics 9 (1994) 625-632
Signal recognition and chemoelectrical transduction in olfaction
Abstract : The mimicking of olfaction is considered to be a promising approach for the construction of artificial odour-sensing systems . In the nose, the detection of volatile odorants begins when the odorant ligands interact with specific odorant receptors in the ciliar membrane of the olfactor ne rons . A large famil of genes encoding p tative odorant receptors has been identified recentl . Individ al receptor t pes are expressed in s bsets of cells distrib ted in distinct ones of the olfactor epitheli m . Ligand-receptor interaction triggers a rapid m ltistep reaction cascade, res lting in a "p lse" of second messengers that initiates an electrical response from the receptor ne ron . Olfactor signalling is terminated b phosphorlation of receptors via a negative feedback reaction, catal ed b specific kinases . Ke words : receptors, expression, second messenger, desensiti ation
INTRODUCTION Tremendo s efforts have been made recentl , to design artificial olfactor devices, so-called "electronic noses", which in the f t re ma replace h man sensor tests in fields of food, drink, cosmetic and environmental control . Mimicking the biological mechanisms of olfaction is considered to be a promising approach for constr cting artificial odo r-sensing s stems . However, research in this field ver m ch depends on progress in nderstanding the molec lar mechanisms of olfactor signalling in o r nose . The c rrent application of modern biological research methodolog to explore the mechanisms of olfaction has led to some major breakthro ghs in the last few ears . O r sense of smell is able to recogni e and discriminate with sensitivit and acc rac , tho sands of extraneo s volatile molec les of diverse 0956-5663/94/ $07 . 110 1994 Elsevier Science Ltd .
str ct re . Odorant stim li at concentrations as low as a few parts per trillion are detected, and tho sands of distinct odo rs, even stereoisomeric compo nds, are discriminated . This task is accomplished b speciali ed chemosensor ne rons located in the nasal ne roepitheli m ; these cells encode the strength, d ration and q alit of odorant stim li into distinct patterns of afferent ne ronal signals . Th s, the molec lar str ct re of an odorant is converted into a pattern of ne ronal activit which, in t rn, is processed in the olfactor b lb and higher brain centres . Odo r perception is a res lt of complex biochemical and electroph siological reaction cascades (Getchell, 1986 ; Lancet, 1986) . The process of olfaction in air breathing vertebrates begins when volatile odoro s molec les are inhaled ; the inspired air contains low concentrations of h drophobic odorants that m st traverse an aq eo s medi m, the m c s la er 625
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covering the nasal epitheli m, before contacting receptor sites of the olfactor cilia . Processes that infl ence the entr , exit or residence time of odorant molec les in the receptor vicinit have been termed perireceptor events (Getchell et al., 1984) . These a xiliar processes incl de translocation of h drophobic odorants to their receptor sites, which is probabl mediated b sol ble odorant binding proteins (Pevsner & Sn der, 1990) and inactivation of chemostim lants b en me-catal ed degradation or biotransformation (Carr et al., 1990) . The recognition of odorants and the primar events of olfactor signal transd ction occ r in the cilia of olfactor receptor ne rons ; the cilia, extr ding from the dendritic knob, are considered to act as scaffolding for the chemosensor membrane, providing a large s rface area. Upon interaction between odoro s ligands and specific receptor proteins, a m lti-step reaction cascade is initiated, which amplifies the olfactor signal and ltimatel leads to the electrical response of the sensor ne ron . To el cidate the complex molec lar machiner mediating the chemo-electrical transd ction process in olfactor receptor ne rons, the main principles common to the transd ction operations of most sensor modalities are c rrentl nder extensive investigation . These principles incl de detection and discrimination, amplification and encoding of the signal, and termination and adaptation of the transd ction process (Shepherd, 1993) .
RECOGNITION OF ODOROUS LIGANDS After traversing the m c s matrix, odorants reach the chemo-sensor ciliar membrane of olfactor ne rons . An electrical response is elicited in a s bset of these cells, reactive ne rons apparentl being more sensitive towards specific odorants . The olfactor s stem of terrestrial vertebrates responds to a plethora of volatile compo nds (Getchell, 1986) ; altho gh most of them are foreign molec les that are evol tionaril " nknown" to the organism ntil the are enco ntered for the first time, the are readil detected and discriminated . The implicated problems of molec lar recognition appear to be analogo s to those of the imm ne s stem . In fact, two alternative principles have been disc ssed : b analog with colo r vision, the specificit of 626
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odo r detection ma be based on onl a few receptor t pes, each reacting with a wide range of odorants ; alternativel , b analog with the imm ne s stem and the large antibod repertoire, there might be tho sands of distinct receptors, each speciali ed for one or a small n mber of odorants . In the latter case, m ch of the discrimination between odo rs ma occ r in the peripher and th s alleviate inp t processing in the brain . Unravelling the nat re as well as the diversit and specificit of receptors for odorants is, therefore, cr cial to the nderstanding of olfaction.
IDENTIFICATION OF ODORANT RECEPTORS The notion that G-protein co pled transd ction cascades pla a central role in olfactor transd ction has led to the concept that receptors for odorants are members of the G-protein linked receptor s perfamil (Lancet, 1986) . Emplo ing conventional biochemical approaches has res lted in the discover of interesting proteins b t none of them meet the criteria of odorant receptors. A breakthro gh was achieved, however, when B ck & Axel (1991) discovered a novel gene famil encoding pol peptides with seven h drophobic domains ; these are c rrentl considered as potential odorant receptors . These novel receptors displa all the hallmarks of the Gprotein co pled receptor s perfamil b t also possess some niq e motifs : most notabl , the appear to be minimal in str ct re with ver short c toplasmic and extracell ar loops ; in addition, the displa a striking str ct ral diversit in the third, fo rth and fifth transmembrane domains, which are s pposed to form the h drophobic core of these proteins ; this region, in t rn, is s pposed to form the ligand binding site of the receptors (Fig . 1) . In their pioneering st d , B ck & Axel (1991) fo nd that expression of the receptors appears to be restricted to olfactor epitheli m, and genomic anal ses revealed a s rprisingl large n mber of genes encoding these receptors ; this novel m ltigene famil seems to comprise several h ndreds or even tho sands of homologo s genes . Meanwhile, n mero s members of this new receptor famil have been identified in vario s species, incl ding rat (B ck & Axel, 1991 ; Lev et al., 1991 ; Raming et al., 1993), mice (Nef et
Biosensors & Bioelectronics
Signal recognition and chemo-electrical transd ction in olfaction
Fig. 1 . Schematic drawing showing the proposed membrane topolog of an olfactor receptor, based on the str ct ral model of other seven transmembrane domain (7TMD) receptors, s ch as rhodopsin or f3-adrenergic receptors. In analog with other 7TMD receptors, it is ass med that the odo r ligand is bo nd within a pocket formed b the seven transmembrane segments of the pol peptide chain . The pocket of an olfactor receptor ma be able to accept a relativel wide variet of odoro s molec les. High variabilit of the primar str ct re in the transmembrane segments is s pposed to acco nt for the binding selectivit between different receptor s btpes.
al., 1992 ; Ressler et al., 1993), fish (Ngai et al., 1993a) and man (Reed, 1992 ; Lancet et al., 1993; Sch rmans et al., 1993) . The large q antit of
these newl discovered genes, co pled with the fact that the are onl expressed in the olfactor epitheli m, indicated that the ma encode the long so ght odorant receptors . This view was strongl s bstantiated b in sit h bridi ation experiments, which demonstrated that these receptor proteins are indeed expressed in the olfactor receptor ne rons (Ngai et al., 1993a ; Raming et al., 1993 ; Ressler et al., 1993 ; V. Strotmann et al., 1994a,b ; Vassar et al., 1993) . Confirmation that the new proteins described are indeed the receptors for odoro s molec les can onl be provided b f nctional expression of receptor-encoding complementar DNA in s rrogate cells ; this sho ld demonstrate that the pol peptide prod cts can mediate odorant activation of G-protein pathwa s . The bac lovirs-Sf9 cell s stem was chosen for the heterologo s
expression of odorant receptor genes . Upon extensive screening to find s itable odoro s ligands, it was demonstrated that receptor proteins expressed in Sf9 cells interact and, in t rn, are activated b certain odorants, and f nctionall co ple to the second messenger cascade of host cells (Raming et al., 1993) . The graded response to several o t of a set of odo rs indicates that this receptor has a relativel broad b t selective ligand specificit . This observation is in line with previo s s ggestions that olfactor ne rons ma express onl one receptor t pe b t still respond to different odorants (Lancet, 1986) . F nctional expression of odorant receptors ma allow the s ccessive reconstit tion of the complete olfactor signalling cascade in s rrogate cells and th s allow novel approaches to explore the f nctional role of each molec lar element in signal recognition and transd ction. A f nctional characteri ation of an arra of receptors will reveal if individ al receptors are broadl or narrowl 627
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t ned for certain odo rs, and which t pe of G-protein or second messenger pathwa the activate . Q estions concerning the genomic organi ation of the large famil of genes encoding the odorant receptors, along with the factors that ma reg late the expression of defined s bsets of these genes in individ al olfactor ne rons, are of great interest and nder intensive investigation (Lancet et al., 1993 ; Margolis, 1993 ; Wang & Reed, 1993) . TOPOGRAPHIC LOCALIZATION OF RECEPTORS Perception of the odorants enco ntered b an organism req ires the identification of the s bset of receptor ne rons which responds to a given odorant . The position of individ al cells in the nasal ne roepitheli m and/or their projection patterns to the olfactor b lb ma be sed to identif the responsive ne rons (Macka -Sim et al., 1982; Ka er, 1991 ; Shepherd, 1991) . The identification of the odorant receptor genes has provided molec lar probes that allow investigations into whether olfactor ne rons expressing a specific receptor t pe are spatiall segregated in the olfactor epitheli m . Recent st dies on different species emplo ing a limited n mber of receptor probes led to q ite variable res lts . In catfish olfactor epitheli m, no discernible pattern of receptor expression was observed (Ngai et al ., 1993b) . In mice, the distrib tion of odorant receptor mRNA s ggested that the nasal cavit was divided into several expression ones, b t with a random distrib tion of positive cells within a given one (Ressler et al., 1993) . In rat, ver similar expression ones were observed (Strotmann et al., 1994a,b ; Vassar et al., 1993) ; in addition, for one partic lar receptor t pe a spatial segregation of reactive cells in a ver restricted region was fo nd (Strotmann et al., 1992) . St dies are now being carried o t to investigate whether these differences are d e to species variations, or represent some of the man principles nderl ing the topographic organi ation of the chemosensor epitheli m . SIGNAL TRANSDUCTION The primar events of odo r detection occ r in the olfactor cilia on the dendritic knobs of 628
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the primar receptor ne rons . Proced res for isolating olfactor cilia have greatl facilitated molec lar st dies of olfaction, as m ch as those for retinal rods have contrib ted to phototransd ction st dies . Olfactor cilia preparations possess high levels of aden late c clase, which is stim lated b certain odorants in a GTP-dependent manner (Pace et al ., 1986 ; Sklar et al ., 1986) . These observations have led to the concept that cAMP ma pla a ke role in olfactor signal transd ction . This view was consolidated b the discover of a c clic n cleotide gated ion channel in the membrane of olfactor cilia (Nakam ra & Gold, 1987) . B emplo ing molec lar cloning approaches, olfactor specific isoforms of elements forming the cAMP-cascade have been identified: a specific G-protein, "G 0,f" (Jones & Reed, 1989), an olfactor aden late c clase, "aden late c clase III" (Bakal ar & Reed, 1990), and an olfactor c clic n cleotide gated channel (Dhallan et al., 1990 ; L dwig et al ., 1990 ; Go lding et al., 1992) . The reason wh olfactor ne rons express specific rather than the conventional isoforms of these proteins is not clear . It has been s ggested that these s bt pes ma contrib te to a high signal to noise ratio for signal amplification (Bakal ar & Reed, 1990) . Altho gh most of the molec lar elements essential for the cAMP cascade were identified, and altho gh en me activit meas rements implicated that cAMP ma be involved in the transd ction process, it was a matter of debate as to whether stim lation of c clase activit reall res lted in a significant b ild- p of second messenger levels and whether the kinetics wo ld be fast eno gh to elicit the electrical response of olfactor receptor ne rons, which occ rs on a s bsecond time scale . These problems were approached b sing a rapid q ench techniq e to monitor the odorant-ind ced formation of cAMP in isolated cilia preparations on a s bsecond time scale . The fast kinetic methodolog emplo ed also allowed biochemical assa s to be performed with low odo r doses . Application of odorants, s ch as isomenthon or citralva, elicited a rapid elevation of cAMP-levels ; a peak concentration, several fold higher than the basal level, was reached after 50 ms ; thereafter, the concentration ret rned to pre-stim lated levels within a few h ndred ms (Breer et al ., 1990) . This "p lse" of cAMP precedes the electrical response and th s ma mediate the chemo-electrical transd ction in receptor cells .
Biosensors & Bioelectronics
Signal recognition and chemo-electrical transd ction in olfaction
A s rve of n mero s odoro s compo nds indicates that all odorants previo sl shown to
of different odo r stim li ma alread begin at the level of primar reactions in the olfactor
activate aden late c clase (Sklar et al., 1986) ind ce a rapid cAMP-response; odorants that fail to elicit c clic n cleotide acc m lation prod ce another second messenger response : the instead ind ce an increase in inositol 1,4,5-trisphosphate (IP3) (Boekhoff et al., 1990) . The odorantind ced IP3-signals displa similar rapid kinetics ;
receptor cells .
the transient "p lse" of IP 3 also precedes a t pical electrical response . IP3-gated ion channels have been discovered in the plasma membrane of olfactor ne rons from vario s species (Restrepo et al ., 1990 ; Fadool & Ache, 1992) . Understanding exactl how the intracell lar IP3signal is propagated and converted into an electrical response of the cell req ires f rther investigation . Assa ing a whole range of odorants on isolated cilia indicated that odorants ma be categori ed into one of two classes, each class activating a different transd ction pathwa (Breer and Boekhoff, 1991) ; however, a recent st d indicates that in c lt red receptor cells a given odorant ma activate both pathwa s (Ronnet et al., 1993) . The notion that two alternative pathwa s exist for the chemo-electrical transd ction of olfactor stim li opens the wa for new concepts towards an nderstanding of olfactor sensor processing (Fig . 2) . Coexistence of both pathwa s within the same receptor cell raises the possibilit of an efficient interaction between the two s stems, th s providing the potential for considerable positive and negative "cross talk", which wo ld affect the generation of electric responses . The existence of two alternative transd ction cascades wo ld be of partic lar relevance if the co ld be related to the observed alternative excitator or inhibitor responses of olfactor ne rons to odo r stim lation (Dionne, 1992) . S ch a correlation
TERMINATION OF OLFACTORY SIGNALLING (DESENSITIZATION)
An essential prereq isite for the precise reaction of chemosensor ne rons to iterative stim lation is the characteristic phasic response of the cells . The basis for this characteristic feat re is a rapid termination of the odo r ind ced primar reaction, i . e. the rapid "switch-off" of the second messenger cascade initiated b an odorant-activated receptor . This is reflected in the kinetics of odorant-ind ced rapid and transient second messenger response . The "switching off" reaction cannot be attrib ted to odorant-inactivation or to activation of catabolic en mes (Boris et al ., 1992), b t rather resembles the waning of second messenger responses to cell-s rface receptor activation in other cells (desensiti ation) . This feat re, common to man forms of transmembrane signalling, has been attrib ted to phosphor lation of liganded receptor proteins (Lefkowit et al ., 1990) . Recent st dies emplo ing specific kinase inhibitors have indicated that, in the olfactor s stem, termination of the odorant-ind ced primar reaction involves a kinase that is stim lated b the second messengers generated in the active cascade : switching off the cAMP-generating cas-
has been clearl demonstrated in recent elegant st dies on olfactor cells from lobster, in which the cAMP-s stem mediates h perpolari ation and inhibitor responses, whereas the IP 3-pathwa s
cade involves protein kinase A (PKA) ; the pathwa prod cing IP3 and DAG (diac lgl cerol) is controlled b protein kinase C (PKC) (Boekhoff & Breer, 1992) . In an extensive st d , it was previo sl shown that specific ciliar pol peptides are rapidl and transientl phosphorlated pon odorantstim lation . A toradiographic anal sis s ggested that the pol peptides labelled d ring odorantstim lation ma in fact be the receptors for
leads to depolari ation and excitator responses (Fadool & Ache, 1992) . There is some preliminar evidence s ggesting that in vertebrates, the f nctional role of the two second messengers ma be reverse : cAMP evokes an inward c rrent and, as a res lt, excites the cell, whereas IP 3 evokes an o tward c rrent and thereb inhibits the spontaneo s activit of the cells (Bacigalopo et al., in press) . In an case, a convergent integration
odorants (Boekhoff et al., 1992) . This notion was recentl confirmed sing receptor-specific antipeptide antibodies, which allowed the imm noprecipitation of the phosphor lated proteins (Krieger et al., 1993) . Th s, the emerging pict re s ggests that the rapid "switch-off' reaction for the olfactor transd ction cascade is in line with desensiti ation of other signalling s stems . 629
Na+
Ion channel
Receptors
Aden l l c clase
G-protein
4q
Ion channel
Fig. 2. Diagram depicting the two second messenger pathwa s implicated in the chemo-electrical transd ction events of olfactor receptor ne rons . Both cascades are initiated b odorant molec les interacting with G -protein co pled receptors. In one case, receptor/ligand interaction enhances the generation of cAMP, which in t rn activates c clic n cleotide gated cation channels in the plasma membrane . In the other case, receptor binding enhances h drol sis of phosphatid linositol prod cing IP3i which is s pposed to target IP3-gated channels in the membrane .
Na+
04
Odorants
Biosensors & Bioelectronics
Signal recognition and chemo-electrical transd ction in olfaction
CONCLUSION Recent st dies have led to a more refined nderstanding of olfactor ne rons and the mechanisms involved in odo r detection . The collaborative efforts of electroph siologists and biochemists have shown that olfactor receptor ne rons accomplish their task of odorant recognition and chemo-electrical signal transd ction b recr iting molec lar elements common to man other transmembrane signalling s stems. This new information ma contrib te greatl to the development of artificial olfactor devices; s ch odo rsensing s stems, or "electronic noses", are eagerl awaited for the efficient monitoring of volatile compo nds .
ACKNOWLEDGEMENTS The work from this laborator was s pported b the De tsche Forsch ngsgemeinschaft .
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B ck, L. & Axel, R . (1991) . A novel m ltigene famil ma encode odorant receptors : a molec lar basis for odo r recognition. Cell, 65, 175-187 . Carr, W.E.S., Gleeson, R.A. & Trapido-Rosenthal, H.G . (1990) . The role of perireceptor events in chemosensor processes. Trends Ne rosci., 13, 212-215 . Dhallan, R .S., Ya , K.W., Schrader, K .A. & Reed, R.R. (1990) . Primar str ct re and f nctional expression of a c clic n cleotide-activated channel from olfactor ne rons. Nat re, 347, 184-187 . Dionne, V.E. (1992) . Chemosensor responses in isolated olfactor receptor ne rons from Nect r s mac los s . J. Gen. Ph siol., 99, 415-433 . Fadool, D .A . & Ache, B.W. (1992) . Plasma membrane inositol 1,4,5-trisphosphate-activated channels mediate signal transd ction in lobster olfactor ne rons. Ne ron, 9, 907-918 . Getchell, T .V . (1986) . F nctional properties of vertebrate olfactor receptor ne rons . Ph siol. Rev., 66, 772-817 . Getchell, T.V., Margolis, F.L. & Getchell, M .L . (1984) . Perireceptor and receptor events in vertebrate olfaction . Prog. Ne robiol., 23, 317-345 . Go lding, E.H ., Ngai, J., Kramer, R .H ., Colicos, S ., Axel, R ., Siegelba m, S .A . & Chess, A . (1992) . Molec lar cloning and single-channel properties of the c clic n cleotide-gated channel from catfish olfactor ne rons . Ne ron, 8, 45-52. Jones, D.T . & Reed, R .R. (1989) . Golf: an olfactor ne ron specific G-protein involved in odorant signal transd ction . Science, 244, 790-795 . Ka er, J .S. (1991) . Contrib tions of topograph and parallel processing to odo r coding in the vertebrate olfactor pathwa . Trends Ne rosci., 14, 79-85 . Krieger, J., Raming, K ., Strotmann, J ., Wanner, I ., Boekhoff, I ., Schleicher, S ., de Ge s, P. & Breer, H . (1994) . Probing odorant receptors with seq ence-specific antibodies . E r. J. Biochem . 219, 829-835 . Lancet, D . (1986) . Vertebrate olfactor reception . Ann. Rev ., 9, 329-355 . Lancet, D ., Gross-Isseroff, R ., Margalit, T ., Seidemann, E . & Ben-Arie, N . (1993) . Olfaction : from signal transd ction and termination to h man genome mapping . Chem. Senses, 18, 217-225 . Lefkowit , R.J ., Ha sdorff, W.P . & Caron, M .G . (1990). Role of phosphor lation in desensiti ation of the /3-adrenoreceptor . Trends Pharmacol . Sci., 11, 190-194 . Lev , N.S ., Bakal ar, H .A . & Reed, R .R . (1991) . Signal transd ction in olfactor ne rons. J. Steroid Biochem . Mol. Biol., 39, 633-637 . L dwig, J ., Margalit, T ., Eismann, E ., Lancet, D . & Ka pp, U .B . (1990) . Primar str ct re of cAMPgated channel from bovine olfactor epitheli m . FEBS Lett ., 270, 24-29. 631
H. Breer Macka -Sim, A., Shaman, P . & Mo lton, D .G . (1982) . Topographic coding of olfactor q alit : odorant specific patterns of epithelial responsivit in the salamander . J. Ne roph siol., 48, 584-596 . Margolis, F.L. (1993) . Reg lation of olfactor ne ron gene expression . C totechnolog , 11, 17-22. Nakam ra, T . & Gold, G .H. (1987) . A c clicn cleotide gated cond ctance in olfactor receptor cilia . Nat re, 325, 442-444 . Nef, P., Hermans-Borgme er, I ., Artieres-Pin, H ., Beasle , L ., Dionne, V.E. & Heinemann, S .F. (1992) . Spatial pattern of receptor expression in the olfactor epitheli m . Proc. Nat! . Acad. Sci. USA, 89, 8948-8952 . Ngai, J ., Dowling, M.M ., B ck, L., Axel, R. & Chess, A. (1993a) . The famil of genes encoding odorant receptors in the channel catfish . Cell, 72, 657-666. Ngai, J., Chess, A., Dowling, M.M., Necles, N., Macgno, E .R. & Axel, R. (1993b) . Coding of olfactor information : topograph of odorant expression in the catfish olfactor ephitheli m . Cell, 72, 667-680 . Pace, U., Hanski, E., Salomon, Y . & Lancet, D . (1986). Odorant-sensitive aden late c clase ma mediate olfactor reception . Nat re, 325, 442-444 . Pevsner, J . & Sn der, S.H. (1990) . Odorant binding protein: odorant transport f nction in the nasal epitheli m . Chem. Senses, 15, 217-222 . Raming, K., Krieger, J ., Strotmann, J., Boekhoff, I ., K bick, S ., Ba mstark, C. & Breer, H . (1993) . Cloning and expression of odorant receptors . Nat re, 361, 353-356 . Reed, R.R . (1992) . Mechanisms of sensitivit and specificit in olfaction . Cold Spring Harbor S mposia on Q antitative Biolog , 57, 501-504 . Ressler, K.J ., S llivan, S .L. & B ck, L .B . (1993) . A onal organi ation of odorant receptor gene expression in the olfactor epitheli m. Cell, 73, 597-609 . Restrepo, D ., Mi amoto, T., Br ant, B .P . & Teeter, J .H . (1990) . Odo r stim li trigger infl x of calci m into olfactor ne rons of the channel catfish . Science, 249, 1166-1168 .
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Biosensors & Bioelectronics Ronnet, G .V ., Cho, H., Hester, L.D ., Wood, S.F. & Sn der, S .H . (1993) . Odorants differentiall enhance phosphoinoside t rnover and aden l l c clase in olfactor receptor ne ronal c lt res . J. Ne rosci., 13, 1751-1758 . Sch rmans, S ., M scatelli, F ., Miot, F., Mattei, M .G., Vassart, G . & Parmentier, M . (1993) . The OLFR1 gene encoding the HGMPO7E p tative olfactor receptor maps to the 17p13-p12 region of the h man genome and reveals an Mspl restriction fragment length pol morphism . C togenet Cell Genet, 63, 200-204 . Shepherd, G .M . (1991) . Comp tational str ct re of the olfactor s stem . In: Olfaction : A Model S stem for Comp tational Ne roscience . (J.L . Davis & H . Eichba m, eds .) MIT Press, Cambridge . Shepherd, G.M . (1993) . C rrent iss es in the molec lar biolog of olfaction . Chem. Senses, 18, 191-198. Sklar, P .D ., Anholt, R .H . & Sn der, S .H. (1986) . The odorant-sensitive aden late c clase of olfactor receptor cells : different stim lation b distinct classes of odorants. J. Biol. Chem., 261, 15538-15543 . Strotmann, J ., Wanner, I ., Krieger, J ., Raming, K . & Breer, H . (1992) . Expression of odorant receptors in spatiall restricted s bsets of chemosensor ne rons. Ne roReport, 3, 1053-1056 . Strotmann, J ., Wanner, I ., Helfrich, T., Beck, A ., Meinken, C., K bick, S . & Breer, H . (1994a) . Olfactor ne ron expressing distinct odorant receptors s bt pes are spatiall segregated in the nasal ne roepitheli m . Cell Tiss e Res ., 276, 429-438 . Strotmann, J ., Warner, J., Helfrich, T . & Breer, H . (1994b) . Rostro-ca dal patterning of receptorexpressing olfactor ne rones in the rat nasal cavit . Cell Tiss e Res ., 278, 11-20 . Vassar, R ., Ngai, J . & Axel, R . (1993) . Spatial segregation of odorant receptor expression in the olfactor epitheli m . Cell, 73, 597-609 . Wang, M.M. & Reed, R .R. (1993) . Coordinate reg lation of olfactor ne ronal gene expression . Chem. Senses, 18, 199-202 .
Signal recognition and chemoelectrical transduction in olfaction
Abstract : The mimicking of olfaction is considered to be a promising approach for the construction of artificial odour-sensing systems . In the nose, the detection of volatile odorants begins when the odorant ligands interact with specific odorant receptors in the ciliar membrane of the olfactor ne rons . A large famil of genes encoding p tative odorant receptors has been identified recentl . Individ al receptor t pes are expressed in s bsets of cells distrib ted in distinct ones of the olfactor epitheli m . Ligand-receptor interaction triggers a rapid m ltistep reaction cascade, res lting in a "p lse" of second messengers that initiates an electrical response from the receptor ne ron . Olfactor signalling is terminated b phosphorlation of receptors via a negative feedback reaction, catal ed b specific kinases . Ke words : receptors, expression, second messenger, desensiti ation
INTRODUCTION Tremendo s efforts have been made recentl , to design artificial olfactor devices, so-called "electronic noses", which in the f t re ma replace h man sensor tests in fields of food, drink, cosmetic and environmental control . Mimicking the biological mechanisms of olfaction is considered to be a promising approach for constr cting artificial odo r-sensing s stems . However, research in this field ver m ch depends on progress in nderstanding the molec lar mechanisms of olfactor signalling in o r nose . The c rrent application of modern biological research methodolog to explore the mechanisms of olfaction has led to some major breakthro ghs in the last few ears . O r sense of smell is able to recogni e and discriminate with sensitivit and acc rac , tho sands of extraneo s volatile molec les of diverse 0956-5663/94/ $07 . 110 1994 Elsevier Science Ltd .
str ct re . Odorant stim li at concentrations as low as a few parts per trillion are detected, and tho sands of distinct odo rs, even stereoisomeric compo nds, are discriminated . This task is accomplished b speciali ed chemosensor ne rons located in the nasal ne roepitheli m ; these cells encode the strength, d ration and q alit of odorant stim li into distinct patterns of afferent ne ronal signals . Th s, the molec lar str ct re of an odorant is converted into a pattern of ne ronal activit which, in t rn, is processed in the olfactor b lb and higher brain centres . Odo r perception is a res lt of complex biochemical and electroph siological reaction cascades (Getchell, 1986 ; Lancet, 1986) . The process of olfaction in air breathing vertebrates begins when volatile odoro s molec les are inhaled ; the inspired air contains low concentrations of h drophobic odorants that m st traverse an aq eo s medi m, the m c s la er 625
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covering the nasal epitheli m, before contacting receptor sites of the olfactor cilia . Processes that infl ence the entr , exit or residence time of odorant molec les in the receptor vicinit have been termed perireceptor events (Getchell et al., 1984) . These a xiliar processes incl de translocation of h drophobic odorants to their receptor sites, which is probabl mediated b sol ble odorant binding proteins (Pevsner & Sn der, 1990) and inactivation of chemostim lants b en me-catal ed degradation or biotransformation (Carr et al., 1990) . The recognition of odorants and the primar events of olfactor signal transd ction occ r in the cilia of olfactor receptor ne rons ; the cilia, extr ding from the dendritic knob, are considered to act as scaffolding for the chemosensor membrane, providing a large s rface area. Upon interaction between odoro s ligands and specific receptor proteins, a m lti-step reaction cascade is initiated, which amplifies the olfactor signal and ltimatel leads to the electrical response of the sensor ne ron . To el cidate the complex molec lar machiner mediating the chemo-electrical transd ction process in olfactor receptor ne rons, the main principles common to the transd ction operations of most sensor modalities are c rrentl nder extensive investigation . These principles incl de detection and discrimination, amplification and encoding of the signal, and termination and adaptation of the transd ction process (Shepherd, 1993) .
RECOGNITION OF ODOROUS LIGANDS After traversing the m c s matrix, odorants reach the chemo-sensor ciliar membrane of olfactor ne rons . An electrical response is elicited in a s bset of these cells, reactive ne rons apparentl being more sensitive towards specific odorants . The olfactor s stem of terrestrial vertebrates responds to a plethora of volatile compo nds (Getchell, 1986) ; altho gh most of them are foreign molec les that are evol tionaril " nknown" to the organism ntil the are enco ntered for the first time, the are readil detected and discriminated . The implicated problems of molec lar recognition appear to be analogo s to those of the imm ne s stem . In fact, two alternative principles have been disc ssed : b analog with colo r vision, the specificit of 626
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odo r detection ma be based on onl a few receptor t pes, each reacting with a wide range of odorants ; alternativel , b analog with the imm ne s stem and the large antibod repertoire, there might be tho sands of distinct receptors, each speciali ed for one or a small n mber of odorants . In the latter case, m ch of the discrimination between odo rs ma occ r in the peripher and th s alleviate inp t processing in the brain . Unravelling the nat re as well as the diversit and specificit of receptors for odorants is, therefore, cr cial to the nderstanding of olfaction.
IDENTIFICATION OF ODORANT RECEPTORS The notion that G-protein co pled transd ction cascades pla a central role in olfactor transd ction has led to the concept that receptors for odorants are members of the G-protein linked receptor s perfamil (Lancet, 1986) . Emplo ing conventional biochemical approaches has res lted in the discover of interesting proteins b t none of them meet the criteria of odorant receptors. A breakthro gh was achieved, however, when B ck & Axel (1991) discovered a novel gene famil encoding pol peptides with seven h drophobic domains ; these are c rrentl considered as potential odorant receptors . These novel receptors displa all the hallmarks of the Gprotein co pled receptor s perfamil b t also possess some niq e motifs : most notabl , the appear to be minimal in str ct re with ver short c toplasmic and extracell ar loops ; in addition, the displa a striking str ct ral diversit in the third, fo rth and fifth transmembrane domains, which are s pposed to form the h drophobic core of these proteins ; this region, in t rn, is s pposed to form the ligand binding site of the receptors (Fig . 1) . In their pioneering st d , B ck & Axel (1991) fo nd that expression of the receptors appears to be restricted to olfactor epitheli m, and genomic anal ses revealed a s rprisingl large n mber of genes encoding these receptors ; this novel m ltigene famil seems to comprise several h ndreds or even tho sands of homologo s genes . Meanwhile, n mero s members of this new receptor famil have been identified in vario s species, incl ding rat (B ck & Axel, 1991 ; Lev et al., 1991 ; Raming et al., 1993), mice (Nef et
Biosensors & Bioelectronics
Signal recognition and chemo-electrical transd ction in olfaction
Fig. 1 . Schematic drawing showing the proposed membrane topolog of an olfactor receptor, based on the str ct ral model of other seven transmembrane domain (7TMD) receptors, s ch as rhodopsin or f3-adrenergic receptors. In analog with other 7TMD receptors, it is ass med that the odo r ligand is bo nd within a pocket formed b the seven transmembrane segments of the pol peptide chain . The pocket of an olfactor receptor ma be able to accept a relativel wide variet of odoro s molec les. High variabilit of the primar str ct re in the transmembrane segments is s pposed to acco nt for the binding selectivit between different receptor s btpes.
al., 1992 ; Ressler et al., 1993), fish (Ngai et al., 1993a) and man (Reed, 1992 ; Lancet et al., 1993; Sch rmans et al., 1993) . The large q antit of
these newl discovered genes, co pled with the fact that the are onl expressed in the olfactor epitheli m, indicated that the ma encode the long so ght odorant receptors . This view was strongl s bstantiated b in sit h bridi ation experiments, which demonstrated that these receptor proteins are indeed expressed in the olfactor receptor ne rons (Ngai et al., 1993a ; Raming et al., 1993 ; Ressler et al., 1993 ; V. Strotmann et al., 1994a,b ; Vassar et al., 1993) . Confirmation that the new proteins described are indeed the receptors for odoro s molec les can onl be provided b f nctional expression of receptor-encoding complementar DNA in s rrogate cells ; this sho ld demonstrate that the pol peptide prod cts can mediate odorant activation of G-protein pathwa s . The bac lovirs-Sf9 cell s stem was chosen for the heterologo s
expression of odorant receptor genes . Upon extensive screening to find s itable odoro s ligands, it was demonstrated that receptor proteins expressed in Sf9 cells interact and, in t rn, are activated b certain odorants, and f nctionall co ple to the second messenger cascade of host cells (Raming et al., 1993) . The graded response to several o t of a set of odo rs indicates that this receptor has a relativel broad b t selective ligand specificit . This observation is in line with previo s s ggestions that olfactor ne rons ma express onl one receptor t pe b t still respond to different odorants (Lancet, 1986) . F nctional expression of odorant receptors ma allow the s ccessive reconstit tion of the complete olfactor signalling cascade in s rrogate cells and th s allow novel approaches to explore the f nctional role of each molec lar element in signal recognition and transd ction. A f nctional characteri ation of an arra of receptors will reveal if individ al receptors are broadl or narrowl 627
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t ned for certain odo rs, and which t pe of G-protein or second messenger pathwa the activate . Q estions concerning the genomic organi ation of the large famil of genes encoding the odorant receptors, along with the factors that ma reg late the expression of defined s bsets of these genes in individ al olfactor ne rons, are of great interest and nder intensive investigation (Lancet et al., 1993 ; Margolis, 1993 ; Wang & Reed, 1993) . TOPOGRAPHIC LOCALIZATION OF RECEPTORS Perception of the odorants enco ntered b an organism req ires the identification of the s bset of receptor ne rons which responds to a given odorant . The position of individ al cells in the nasal ne roepitheli m and/or their projection patterns to the olfactor b lb ma be sed to identif the responsive ne rons (Macka -Sim et al., 1982; Ka er, 1991 ; Shepherd, 1991) . The identification of the odorant receptor genes has provided molec lar probes that allow investigations into whether olfactor ne rons expressing a specific receptor t pe are spatiall segregated in the olfactor epitheli m . Recent st dies on different species emplo ing a limited n mber of receptor probes led to q ite variable res lts . In catfish olfactor epitheli m, no discernible pattern of receptor expression was observed (Ngai et al ., 1993b) . In mice, the distrib tion of odorant receptor mRNA s ggested that the nasal cavit was divided into several expression ones, b t with a random distrib tion of positive cells within a given one (Ressler et al., 1993) . In rat, ver similar expression ones were observed (Strotmann et al., 1994a,b ; Vassar et al., 1993) ; in addition, for one partic lar receptor t pe a spatial segregation of reactive cells in a ver restricted region was fo nd (Strotmann et al., 1992) . St dies are now being carried o t to investigate whether these differences are d e to species variations, or represent some of the man principles nderl ing the topographic organi ation of the chemosensor epitheli m . SIGNAL TRANSDUCTION The primar events of odo r detection occ r in the olfactor cilia on the dendritic knobs of 628
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the primar receptor ne rons . Proced res for isolating olfactor cilia have greatl facilitated molec lar st dies of olfaction, as m ch as those for retinal rods have contrib ted to phototransd ction st dies . Olfactor cilia preparations possess high levels of aden late c clase, which is stim lated b certain odorants in a GTP-dependent manner (Pace et al ., 1986 ; Sklar et al ., 1986) . These observations have led to the concept that cAMP ma pla a ke role in olfactor signal transd ction . This view was consolidated b the discover of a c clic n cleotide gated ion channel in the membrane of olfactor cilia (Nakam ra & Gold, 1987) . B emplo ing molec lar cloning approaches, olfactor specific isoforms of elements forming the cAMP-cascade have been identified: a specific G-protein, "G 0,f" (Jones & Reed, 1989), an olfactor aden late c clase, "aden late c clase III" (Bakal ar & Reed, 1990), and an olfactor c clic n cleotide gated channel (Dhallan et al., 1990 ; L dwig et al ., 1990 ; Go lding et al., 1992) . The reason wh olfactor ne rons express specific rather than the conventional isoforms of these proteins is not clear . It has been s ggested that these s bt pes ma contrib te to a high signal to noise ratio for signal amplification (Bakal ar & Reed, 1990) . Altho gh most of the molec lar elements essential for the cAMP cascade were identified, and altho gh en me activit meas rements implicated that cAMP ma be involved in the transd ction process, it was a matter of debate as to whether stim lation of c clase activit reall res lted in a significant b ild- p of second messenger levels and whether the kinetics wo ld be fast eno gh to elicit the electrical response of olfactor receptor ne rons, which occ rs on a s bsecond time scale . These problems were approached b sing a rapid q ench techniq e to monitor the odorant-ind ced formation of cAMP in isolated cilia preparations on a s bsecond time scale . The fast kinetic methodolog emplo ed also allowed biochemical assa s to be performed with low odo r doses . Application of odorants, s ch as isomenthon or citralva, elicited a rapid elevation of cAMP-levels ; a peak concentration, several fold higher than the basal level, was reached after 50 ms ; thereafter, the concentration ret rned to pre-stim lated levels within a few h ndred ms (Breer et al ., 1990) . This "p lse" of cAMP precedes the electrical response and th s ma mediate the chemo-electrical transd ction in receptor cells .
Biosensors & Bioelectronics
Signal recognition and chemo-electrical transd ction in olfaction
A s rve of n mero s odoro s compo nds indicates that all odorants previo sl shown to
of different odo r stim li ma alread begin at the level of primar reactions in the olfactor
activate aden late c clase (Sklar et al., 1986) ind ce a rapid cAMP-response; odorants that fail to elicit c clic n cleotide acc m lation prod ce another second messenger response : the instead ind ce an increase in inositol 1,4,5-trisphosphate (IP3) (Boekhoff et al., 1990) . The odorantind ced IP3-signals displa similar rapid kinetics ;
receptor cells .
the transient "p lse" of IP 3 also precedes a t pical electrical response . IP3-gated ion channels have been discovered in the plasma membrane of olfactor ne rons from vario s species (Restrepo et al ., 1990 ; Fadool & Ache, 1992) . Understanding exactl how the intracell lar IP3signal is propagated and converted into an electrical response of the cell req ires f rther investigation . Assa ing a whole range of odorants on isolated cilia indicated that odorants ma be categori ed into one of two classes, each class activating a different transd ction pathwa (Breer and Boekhoff, 1991) ; however, a recent st d indicates that in c lt red receptor cells a given odorant ma activate both pathwa s (Ronnet et al., 1993) . The notion that two alternative pathwa s exist for the chemo-electrical transd ction of olfactor stim li opens the wa for new concepts towards an nderstanding of olfactor sensor processing (Fig . 2) . Coexistence of both pathwa s within the same receptor cell raises the possibilit of an efficient interaction between the two s stems, th s providing the potential for considerable positive and negative "cross talk", which wo ld affect the generation of electric responses . The existence of two alternative transd ction cascades wo ld be of partic lar relevance if the co ld be related to the observed alternative excitator or inhibitor responses of olfactor ne rons to odo r stim lation (Dionne, 1992) . S ch a correlation
TERMINATION OF OLFACTORY SIGNALLING (DESENSITIZATION)
An essential prereq isite for the precise reaction of chemosensor ne rons to iterative stim lation is the characteristic phasic response of the cells . The basis for this characteristic feat re is a rapid termination of the odo r ind ced primar reaction, i . e. the rapid "switch-off" of the second messenger cascade initiated b an odorant-activated receptor . This is reflected in the kinetics of odorant-ind ced rapid and transient second messenger response . The "switching off" reaction cannot be attrib ted to odorant-inactivation or to activation of catabolic en mes (Boris et al ., 1992), b t rather resembles the waning of second messenger responses to cell-s rface receptor activation in other cells (desensiti ation) . This feat re, common to man forms of transmembrane signalling, has been attrib ted to phosphor lation of liganded receptor proteins (Lefkowit et al ., 1990) . Recent st dies emplo ing specific kinase inhibitors have indicated that, in the olfactor s stem, termination of the odorant-ind ced primar reaction involves a kinase that is stim lated b the second messengers generated in the active cascade : switching off the cAMP-generating cas-
has been clearl demonstrated in recent elegant st dies on olfactor cells from lobster, in which the cAMP-s stem mediates h perpolari ation and inhibitor responses, whereas the IP 3-pathwa s
cade involves protein kinase A (PKA) ; the pathwa prod cing IP3 and DAG (diac lgl cerol) is controlled b protein kinase C (PKC) (Boekhoff & Breer, 1992) . In an extensive st d , it was previo sl shown that specific ciliar pol peptides are rapidl and transientl phosphorlated pon odorantstim lation . A toradiographic anal sis s ggested that the pol peptides labelled d ring odorantstim lation ma in fact be the receptors for
leads to depolari ation and excitator responses (Fadool & Ache, 1992) . There is some preliminar evidence s ggesting that in vertebrates, the f nctional role of the two second messengers ma be reverse : cAMP evokes an inward c rrent and, as a res lt, excites the cell, whereas IP 3 evokes an o tward c rrent and thereb inhibits the spontaneo s activit of the cells (Bacigalopo et al., in press) . In an case, a convergent integration
odorants (Boekhoff et al., 1992) . This notion was recentl confirmed sing receptor-specific antipeptide antibodies, which allowed the imm noprecipitation of the phosphor lated proteins (Krieger et al., 1993) . Th s, the emerging pict re s ggests that the rapid "switch-off' reaction for the olfactor transd ction cascade is in line with desensiti ation of other signalling s stems . 629
Na+
Ion channel
Receptors
Aden l l c clase
G-protein
4q
Ion channel
Fig. 2. Diagram depicting the two second messenger pathwa s implicated in the chemo-electrical transd ction events of olfactor receptor ne rons . Both cascades are initiated b odorant molec les interacting with G -protein co pled receptors. In one case, receptor/ligand interaction enhances the generation of cAMP, which in t rn activates c clic n cleotide gated cation channels in the plasma membrane . In the other case, receptor binding enhances h drol sis of phosphatid linositol prod cing IP3i which is s pposed to target IP3-gated channels in the membrane .
Na+
04
Odorants
Biosensors & Bioelectronics
Signal recognition and chemo-electrical transd ction in olfaction
CONCLUSION Recent st dies have led to a more refined nderstanding of olfactor ne rons and the mechanisms involved in odo r detection . The collaborative efforts of electroph siologists and biochemists have shown that olfactor receptor ne rons accomplish their task of odorant recognition and chemo-electrical signal transd ction b recr iting molec lar elements common to man other transmembrane signalling s stems. This new information ma contrib te greatl to the development of artificial olfactor devices; s ch odo rsensing s stems, or "electronic noses", are eagerl awaited for the efficient monitoring of volatile compo nds .
ACKNOWLEDGEMENTS The work from this laborator was s pported b the De tsche Forsch ngsgemeinschaft .
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Biosensors & Bioelectronics Ronnet, G .V ., Cho, H., Hester, L.D ., Wood, S.F. & Sn der, S .H . (1993) . Odorants differentiall enhance phosphoinoside t rnover and aden l l c clase in olfactor receptor ne ronal c lt res . J. Ne rosci., 13, 1751-1758 . Sch rmans, S ., M scatelli, F ., Miot, F., Mattei, M .G., Vassart, G . & Parmentier, M . (1993) . The OLFR1 gene encoding the HGMPO7E p tative olfactor receptor maps to the 17p13-p12 region of the h man genome and reveals an Mspl restriction fragment length pol morphism . C togenet Cell Genet, 63, 200-204 . Shepherd, G .M . (1991) . Comp tational str ct re of the olfactor s stem . In: Olfaction : A Model S stem for Comp tational Ne roscience . (J.L . Davis & H . Eichba m, eds .) MIT Press, Cambridge . Shepherd, G.M . (1993) . C rrent iss es in the molec lar biolog of olfaction . Chem. Senses, 18, 191-198. Sklar, P .D ., Anholt, R .H . & Sn der, S .H. (1986) . The odorant-sensitive aden late c clase of olfactor receptor cells : different stim lation b distinct classes of odorants. J. Biol. Chem., 261, 15538-15543 . Strotmann, J ., Wanner, I ., Krieger, J ., Raming, K . & Breer, H . (1992) . Expression of odorant receptors in spatiall restricted s bsets of chemosensor ne rons. Ne roReport, 3, 1053-1056 . Strotmann, J ., Wanner, I ., Helfrich, T., Beck, A ., Meinken, C., K bick, S . & Breer, H . (1994a) . Olfactor ne ron expressing distinct odorant receptors s bt pes are spatiall segregated in the nasal ne roepitheli m . Cell Tiss e Res ., 276, 429-438 . Strotmann, J ., Warner, J., Helfrich, T . & Breer, H . (1994b) . Rostro-ca dal patterning of receptorexpressing olfactor ne rones in the rat nasal cavit . Cell Tiss e Res ., 278, 11-20 . Vassar, R ., Ngai, J . & Axel, R . (1993) . Spatial segregation of odorant receptor expression in the olfactor epitheli m . Cell, 73, 597-609 . Wang, M.M. & Reed, R .R. (1993) . Coordinate reg lation of olfactor ne ronal gene expression . Chem. Senses, 18, 199-202 .