Electrical
signals
in olfactory transduction
Stuart Firestein Yale University Olfactory system, direct
Medical
transduction which
results
activation
of
to threshold
the transduction has been olfaction,
in the
occupied
second messenger
Current
Opinion
their
pathways
Underlying these two forms of membrane potential change, the slow generator potential and the fast action potential, are two kinds of channels which provide the pathways for ionic currents. The odor-elicited current results from the activation of a cyclic-nucleotidegated channel in the specialized cilia that emanate from the single dendrite of the receptor neuron. Underlying action potential generation is a family of somatic voltagegated Na+, K+ and Ca2+ channels that are similar to those found in other spiking neurons [l-3]. Thus, the production of a signal can be conveniently divided into two phases, a slow odor-induced depolarization, followed by rapid spike generation and propagation. This review is concerned with the first of these, the sensory current that brings the cell to firing threshold. current
Ionic currents resulting from the action of odors on cell surface receptors were first recorded using the whole-cell patch-clamp technique [4,5]. The favorable morphology of the isolated olfactory receptor neuron assures that the slow currents elicited by odors are usually well clamped. Several important features of the odor response have
messenger
of CAMP. by CAMP
@ Current
of the central considerable
behavior
role in effort
at a molecular
have also been proposed
in
1992, 2:444-448
been observed in experiments in which odor containing solutions were pressure ejected into the vicinity of isolated receptor neurons and the resulting ion currents recorded. Site of transduction
The site of olfactory transduction, long suspected to be in the cilia, was unequivocally confirmed by carefully directing pulses of odor at the cilia, the dendrite and the cell body [4,6]. Only those pulses of odor reaching the cilia elicited an ionic current. The cilia were shown to be highly specialized for transduction as high concentrations of KC1 elicited currents only when directed at the dendritic and somatic regions of the cell and not at the cilia. Recently, these observations have been confirmed in experiments that also determined that the distribution of odor-sensitive channels along the cilia was likely to be uniform [ 71. Electrical
characteristics
In the range of - 30 mV to + 20 mV the current-voltage relation of the odor-elicited current is nearly linear, and the current reverses polarity just positive to OmV. This is suggestive of a non-selective cation conductance and, indeed, Kurahashi [4] has shown that most monovalent cations are nearly equally permeant. Significant rectification of the current can be seen in the voltage range negative to - 40 mV, and also at positive potentials (F Zufall and S Firestein: Biop@sical Journal [AM-] 1992, 61:162X) [4]. It now seems probable that this apparent voltage-dependence is actually due to divalent cation blocking effects. The role of Ca2+ and Mg2+ ions will be discussed below in the context of single-channel recordings. Kinetic
features
and second
messenger
system
The current elicited by a brief ( < looms) pulse of odor has a characteristic time course which includes a relatively
protein; W-inositol Biology
The
is the
brings the membrane
Abbreviations C protein--CTP-binding
USA
for these is less well developed.
in Neurobiology
Olfactory transduction is the process by which chemical information, in the guise of odor molecules, is transformed into cellular electrical activity. The nature of this electrical activity is a change in membrane potential as a result of the flow of ionic current. In olfactory neurons the membrane potential change comes in two varieties, a generator potential and an action potential. The first is a gradual depolarization which shifts the membrane from its resting potential, around - 55 mV, to the threshold for action potential generation, near -25mV. It is the action potential which is propagated centrally to the first synapse in the system at the olfactory bulb, and which constitutes the final step in peripheral signal transduction.
that
Because
by these channels
evidence
Introduction
The odor-elicited
cilia membrane
understanding
but the physiological
second
production
the slow depolarization
toward
Connecticut,
a G-protein-coupled
odor-dependent
for spike generation.
cascade
directed
level. Alternative
involves in the
ion channels
final step in producing potential
School, New Haven,
1,4,5-trisphosphate.
Ltd ISSN 0959-4388
Electrical
long latency of I5&500 ms, a slow rise time to a peak amplitude, and an exponential decay with a time constant that depends on the response magnitude. The amplitude of the odor-elicited current is a function of stimulus intensity and duration, but the dynamic range of an individual cell appears to be rather narrow, spanning only an order of magnitude of stimulus concentration. Of considerable interest is the observation that the rising phase and peak of the odor response actually occur as the stimulus intensity is decreasing, due to diffusion of the odor molecules away from the cell surface. Finally, the response to sustained stimulus application is transient, returning to near baseline levels within l&12 s, indicating an adaptation mechanism [6,8]. This assortment of response features can most easily be understood as the result of an integrating step, such as the modulation of an intracellular second messenger. In the last few years all of the molecular components of a CAMP-generating second messenger cascade have been biochemically identified (see review by H Breer, this issue, pp 43!@443) [9]. Physiological confirmation of the operation of such a system has come from patchclamp studies using CAMP and its analogues, as well as phosphodiesterase inhibitors and GTP-binding protein (G protein) modulators. Direct injection of either CAMP or cGMP through the patch pipette, or the application of membrane permeable analogues induces a large inward current that is electrically similar to the odor induced current [10,11**,12]. Thus, there is now strong evidence generated from complementary methodologies that odors cause a G-protein-mediated increase in CAMP, which leads to the activation of a non-specific cationic conductance pathway in the cilia, The cyclic-nucleotide-gated
channel
It has been known since 1987 that a sizeable cyclicnucleotide-activated conductance pathway is present in the ciliary membrane [Is], but it has not been possible to resolve single-channel activity in membrane patches. On the other hand, single channels that are activated by either CAMPor cGMP have been observed in membrane patches taken from the dendrite and soma [ 14,151. Recently, in cell-attached patches from the dendrite, it has been shown conclusively that these channels are activated during the odor response and that they are also gated by intracellular CAMP [ 16*,17**], thus providing a direct link between the odor-induced production of CAMPand the odor-sensitive current. A thorough characterization of the single channel in excised patches of dendritic membrane has been undertaken by Zufall et al. [ 17**]. In confirmation of previous observations from macroscopic currents the single than nel could be activated by either CAMP or cGMP with a Kp of 20 PM and 4 l.tM, respectively. For both nuc!eotide ligands the Hill coefficient was 2.7 and the single-channel conductance was about 45 pS. Neither the conductance nor the open probability showed any significant voltage dependence over the range - 100 mV to + 100 mV. The open time distribution could be described by a single exponential, while the closed time distribution was best described by at least three exponentials, suggesting that
signals in olfactory
transduction
Firestein
there is probably a single open state and at least three closed states. The three closed states were reflected in the single-channel records as long closings that separated bursts of openings, short interburst closings, and a rapid flickering due to incompletely resolved closings. At least three cloned versions of the olfactory channel have been identified. Dhallan et al. [ 181 first cloned the rat channel, followed by Ludwig et al. [ 191 (bovine channel), and most recently Goulding et al [ 20**] have identified a clone in the catfish. Although originating in diverse species all of these channels possess a high sequence homology, appear functionally similar, and are apt to be alike structurally. The olfactory cyclic-nucleotide-gated channel also bears significant homology (about 70 % at the amino acid level) with the cGMP-gated channel of vertebrate photoreceptors [ 211. This makes the difference in cyclicnucleotide sensitivity between the two channels a particularly compelling molecular question. The photoreceptor channel is highly selective for cGMP, while the olfactory channel is activated about equally well by either nucleotide. A1though a putative binding site for the cyclic nucleotide has been identified as a string of some 80 amino acids near the carboxyl terminus, the specific residues responsible for the binding differences are unknown [ 221. A related question of physiological interest is whether cGMP plays any role in olfactory transduction. As of this time no odor-sensitive guanylate cyclase activity has been meas ured in olfactory neurons, and so it appears that CAMP is the physiological ligand (but see below). Role of divalent
cations
This is one of the more controversial areas of olfactory transduction research, with many, and sometimes incompatible, actions being claimed for Ca*+. It is now clear that the cAMP-gated channel is permeable to Cal’ ions, producing a Ca*+ influx during the odor response (F Zufall et al., unpublished data) [4]. On the other hand, Ca2+ ions can also act as open channel blockers, in much the same way as seen at voltage-gated Ca*+ than nels . This is not the only similarity shared with voltagegated Ca2+ channels, Goulding et al. [ 20**] have shown that basic pH relieves some of the open channel block by H+ ions, and replacement of Na+ with Li+ also significantly relieves the block (S Firestein, unpublished data). Further, some Ca*+ channel blockers, such as nifedipine, also block the CAMP-gated channel. All of these results suggest a surprising similarity between this ligand-gated channel and the voltage-sensitive L-type Ca*+ channel. Mg*+, the other divalent cation present in significant concentrations in physiological solutions, also acts as an open channel blocker, but unlike Ca*+ does not appear to have any significant permeability. The Mg*+ block may explain the strong outward rectification seen in the current-voltage relation of the odor-elicited current. In the absence of divalent cations the channel possesses an ohmic current-voltage relation. The physiological significance of the divalent block is curious At the normal resting potential, about - 55 mV, the channels would be mostly blocked by physiological lev-
445
Sensory
systems
els of Ca2+ and Mg2+, and a great many would have to be activated to produce a significant depolarization, Yau and Baylor [23], considering a similar problem in photoreceptors, have pointed out that this might actu ally serve to increase signaling reliability. This is because the variance associated with the random behavior of a large population of small channels is much less than that generated by the activity of a few large channels. Because olfactory neurons also fire action potentials, a second increase in signaling reliability is gained by the progressive relief of the block by depolarization. As the membrane depolarizes, activated channels are capable of conducting more current, further depolarizing the cell. The effect is to create a kind of response threshold, in which a signal capable of opening enough channels to create a small initial depolarization, begins a regenerative process that rapidly brings the membrane to the potential required for spike generation, As it is now clear that Ca2+ permeates the open channel, it is logical to ask what effects may be mediated by an increase in intracellular Ca2+, Recently Kleene et al. [ 241 have been able to draw an entire cilium into a patch pipette, gain a giga-ohm seal at the base of the cilium and then separate it entirely from the cell. This results in what is essentially an inside-out patch, but the patch consists of an entire intact cilium. In addition to the cAMP-gated conductance they present evidence for a Ca2 + -activated Clconductance. The K,/, for Ca2+ activation is 5 PM and the current-voltage relation shows strong outward rectification. The physiological significance of this current is, however, unclear pending further investigation. Schild et al. [ 251 have identified what they believe to be a Ca2+ -activated nonspecific cation conductance, but the evidence for this is less direct. What does seem clear from their report is that there is likely to be both a Ca2+ -ATPase and a Na+-Ca2+ exchanger in olfactory neurons. It must be emphasized, however, that there is as yet no definitive
Adenylate cyclase
evidence to link either of these Ca2+ effects to the odor response. When a maintained step of odor stimulus is presented to an isolated olfactory neuron the elicited current is seen to be transient, reflecting a strong and rapid adapta tion process. Kurahashi and Shibuya [8] showed that this could be abolished by removing extracellular Ca2+. One mechanism for this has been demonstrated by Zufall et al. [26-l in recordings of single cAMP-gated channels in excised membrane patches. The open probability of the channel, but not its conductance, was reduced more than tenfold by intracellular concentrations of Ca2+ between 1 uM and 3 PM. This is distinct from the open channel block caused by extracellular Ca2+, and appeared to be mediated by an allosteric mechanism, for example Ca2+ binding to some intracellular site on the channel protein and stabilizing a closed state. This would be an attractive feedback mechanism for mediating rapid desensitization of the odor response.
Other
second
messengers
There is mounting molecular and biochemical evidence for an inositol 1,45trisphosphate (IP3) pathway, either independent of, or in addition to, the CAMP signaling pathway (Fig.1). The physiological data associating IP3 with odor transduction is less conclusive, if no less tan talizing. Breer (this issue, pp 439-443) has outlined the biochemical evidence for these alternative second messenger pathways. Here, I will concentrate on the electrophysiological evidence. An increase in IP3 should result in an increase in intracellular Ca2+, and indeed Restrepo, Teeter and colleagues [27,28] have measured such an increase in response to certain amino acids in the catfish. In addition, they have recorded the activation of a channel by IP3 in bilayers to which olfactory cilia vesicles were fused. No such channel
Phospholipase
C ?
Fig.1. Probable (CAMP) and possible (IP,) pathways Action
potential
current eration.
for
the
odor-induced
leading to action potential
ionic gen-
Electrical
signals
in olfactory
transduction
Firestein
has yet been observed in plasma membrane patches from intact cells, however. This is important as the cilia contin no Ca2+ storing organelles, requiring that the IP3 induce a Ca2+ influx through the plasma membrane. mile a plasma membrane IP3 receptor would be novel, there is no reason to believe it unlikely. Cunningham and Reed [ 291 note promising preliminary data from their lab showing possible IP3 receptor immunoreactivity in the cilia membrane.
presence correspondingly difficult to establish. One interesting possibility may be that IP3 produced in the cilia increases intracellular Ca2 + , which activates the guanylate cyclase known to be present in olfactory neurons, resulting in the generation of cGMP. This cGMP would be able to activate the same channel as the CAMP.Thus, two pathways would produce two different cyclic nucleotides, but both could work through the same channel.
As further evidence for an odor-induced Ca2+ influx Restrep0 et al. cite the inhibition of the electro-olfactogram response to odors by the removal of extracellular Ca2 + This appears, however, to be in contradiction to the effects of Ca2+ shown by Kurahashi and Shibuya [8] and Zufall et al. [26-l, in which removal of extracellular Ca2+ enhanced the response to odors. The discrepancy could be due to species differences, but a more likely explanation is that in non-voltage clamped preparations the removal of Ca2+ serves to strongly depolarize cells, inactivating the Na+ current, and thereby making the neurons refractory to action potential generation. This has also been pointed out by Frings, Benz and Lindemann [30*] who, while recording unclamped action potentials in olfactory cilia, observed first an increase and then a decrease in neuronal excitability when Ca2+ was removed from the bathing medium.
Future directions
Including IP3 in the patch pipette does not induce a current, at least in salamander neurons [ll**] , nor does it interfere with the odor-elicited current. On the other hand inclusion of CAMPin the pipette always induces a current. In catfish, and more recently in rat olfactory neurons, Restrep0 and Teeter have reported (4th Annual Meeting of the Association for Chemoreception Sciences, Sarasota, Florida: April 1992, Abstract 144) a depolarization upon patch rupture and intracellular dialysis of 10 PM IP3, but the effect was rather slow (on a time scale of minutes) and not robust. An alternative explanation might be that IP3 was releasing Ca2+ from somatic internal stores and effecting membrane potential through mechanisms unrelated to odor transduction. Although this review is concerned with vertebrate olfactory transduction, it might be noted in passing that the most conclusive evidence for an odor-sensitive plasma membrane IP3 receptor was recently presented in studies with lobster cultured olfactory neurons (DA Fadool et al.: 4th Annual Meeting of the Association for Chemoreception Sciences, Sarasota, Florida, April 1992, Abstract 147) in which IP3-activated channels, first observed during application of odors, were then recorded in patches taken directly from the plasma membrane. Some consideration might be given to the likely size of the IP3-regulated Ca2 + influx. Olfactory cilia are less than 0.5 mm in diameter and have a high surface to volume ratio. In a typical cell with ten cilia, each 3Opm long, a Ca2+ current of only about 0.6~~ could transiently raise the ciliary Ca2+ concentration to between ~/.LMand 10pM. The Ca2+ buffering capacity of the cilia is unknown, but presumably depends entirely on the action of membrane pumps and exchangers. Thus, depending on the exact role of intracellular Ca2+, relatively few IP,-gated channels may be required and their
The field of olfactory transduction has experienced remarkable progress over the past 5 years, and in that short time a consensus view of the broad outlines of olfactory transduction has been established [31*-l. Nonetheless, it would be premature to congratulate ourselves on having unlocked the mysteries of olfaction. As more results are reported it is becoming clear that there are several critical discrepancies in the data that cannot be ascribed solely to differences in techniques and preparations. A full appreciation of olfactory transduction will require their resolution. Among the priorities are an understanding of the role of IPj and the possible interaction between this molecule and the cyclic-nucleotide pathway. The unequivocal identification of an IP3 receptor in the plasma membrane of a vertebrate olfactory neuron would also be of general interest to the neurobiology community. Likewise, it might be expected that the cyclic-nucleotide-gated channel will soon be found in other neuronal populations besides sensory neurons. It has already been established that the cGMP channel can be found in retinal bipolar cells [32], and the new appreciation of the nitric-oxide-mediated stimulation of guanylate cyclase in neuronal cell populations means that cGMP channels are possible targets for this pathway as well. References Papers of view, have . of .. of
and recommended
particular interest, published been highlighted as: - special interest outstanding interest
reading
within the annual period of re-
1.
TROTIER D: A Patch
2.
FIRESTEIN S, WERBUNF: Gated Currents in Isolated Olfactory Receptor Neurons of the Larval Tiger Salamander. Proc Nut1 Acud Sci USA 1987, 84629245296.
3.
PAIN RYK, GESTELANDRC: Somatic Sodium Channels of Frog Olfactory Receptor Neurones are Inactivated at Rest. Pjugers Arch 1991, 418:504-511.
4.
KURAHA~HI T: Activation by Odorants of Cation-Selective Conductance in the Olfactory Receptor Cell Isolated from the Newt. J f%ysiol 1989,419:177-192.
5.
FIRESTEIN S, WEREX~NF: Odor-Induced Membrane Currents in Vertebrate Olfactory Receptor Neurons. Science 1989, 244:7%-?2.
6.
FIRESTEINS, SHEPHERDGM, WEREZINFS: Time Course of the Membrane Current Underlying Sensory Transduction in Salamander Olfactory Receptor Neurones. J Pbysiol (Londl 199Q,430:13>158.
Clamp Analysis of Membrane Currents in Salamander OIfactory Cells. Pflugers Arch 1986, 407:58%595.
447
448
Sensory
systems
7
LOWE G, GOLD GH: The Spatial Distributions of Odorant Sensitivity and Odorant-Induced Currents in Salamander Olfactory Receptor Cells. J P&-i01 1991, 442:147-l&.
8.
KURAIIASHIT, SHIBLP(AT: Calcium Dependent Adaptive Properties in the Solitary Olfactory Receptor Cells of the Newt. Brain Res 1990, 515:261-268.
9. 10.
REED R:
SignaIling 1992, 8:205-209.
Pathways
KWAHASHI T: The Response AMP in Isolated Olfactory P&-i01 1990, 430:355- 371.
in Odorant
Detection.
native channels (from dendrite patches) were compared and some differences were seen, Similarities between these channels and voltages dependent L-type CaZ+ channels were noted both in their amino acid sequence and their functional characteristics. 21.
Neuron 22.
Induced Receptor
FIRESTEINS, DARROUIJ B, SHEP~IERUGM: Activation of the Sensory Current in Salamander Olfactory Receptor Neurons Depends on a G-Protein Mediated CAMP Second Messenger System. Neuron 1991, 68255835. The effect of the various elements of the CAMP second messenger system on the odor~induced current were tested m experiments designed to show the contribution of their biochemistry to the shape of the phy siological response. 12.
FR~NGSS, LINDEMANN B: Current Recording from Sensory Cilia of Olfactory Receptor Cells In Situ. 1. The Neuronal Response to Cyclic Nucleotides. / Ge?z P!ysioll991, 97:1-16.
13.
NAKAMIIRA T, GOLD Gli: A Cyclic-Nucleotide Gated Conductance in Olfactory Receptor Cilia. Nature 1987, 325:442-444.
14.
KIIRAHASIIIT, KANEKOA: High Density CAMP-Gated Channels at the Ciliary Membrane in the Olfactory Receptor Cell. Neuroreport 1991, 2:5-8
15.
SLIZIIKIN: Single Cyclic Nucleotide-Activated Ion Channel Activity in Olfactory Receptor CeU Soma Membrane. Neurosci Res [Supfd] 1990, 12:11+126.
16 .
FIRESTEINS, ZUFALLF, SHEPHERD GM: Single Odor Sensitive Channels in Olfactory Receptor Neurons are also Gated by Cyclic Nucleotides. ./ Neurosci 1991, 11:3565+3572. On-cell patches from the dendritic membrane and apical knob exhih ited channel activity in response to pulses of odorant delivered to the cilia The same channel could then he seen to he activated by CAMP, demonstrating that the same conductance was sensitive to externally applied odors and gated by intracellular cyclic nucleotide. ZI~FAI~F, FIKESTMN S, SHEPHERD GM: Analysis of Single Cyclic Nucleotide Gated Channels in OIfactory Receptor Cells. ,/ Neurosci 1991, 11.3573-3580. A complete analysis of the cyclic-nucleotide-sensitive channel in sala mander olfactov neurons, including kinetic a.. well as steady state 17. ..
ddU.
18.
I&ILL&N RS, YA~I KW, SCHRA~I:RKA, WED RR: Primary Structure and Functional Expression of a Cyclic NucleotideActivated Channel from Olfactory Neurons. Nuture 1990, 347:186187.
19.
LllDWlG J, M4RC;ALlT ‘I’, EISMAN
20.
GXIILXNG
E, IANCETD, Structure of CAMP-Gated Channel from Epithelium. FE‘BS Lcff 1990, 270:24-29.
KMJI’P
LIB: Primary Bovine Olfactory
NGAI J, KKAMER KH, Coucos S, AXEI. R, . . SIEGEIAAUMSA, 011% A: Molecular Cloning and SingleChannel Properties of the Cyclic Nucleotide-Gated Channel from Catfish Olfactory Neurons. Neuron 1992, 8:45-58. The cat&h olfactory cyclic-nucleotide~gated channel was cloned, sea quenced and expressed in oocytes. Recordings from the cloned and
Channels EpitheUum.
of VerTrends
ALTENHOFEN
W, L~JDWIGJ, EISMANN E, KRAUS W, BONIGK UB: Control of Ligand Specificity in Cyclic Nucleotide-Gated Channels from Rod Photoreceptors and Olfactory Epithelium. Proc Nat1 Acad Sci USA 1991, 88:986%9872. KAUPP
23.
YA~I K W, BAYLOR DA: Cyclic GMP-Activated Conductance of Retinal Photoreceptor Cells. Anrzu Ret’ Neurosci 1989, 12328932%
24.
KUXNE
25.
SCHIIII D, BISCHOFBERGEK J: Ca*+ Cation Conductance in OIfactory E~cl, Brain Res 1991, 84:1X7-194.
SJ, GESEL+NL) RC: Calcium-Activated Chloride Conductance in Frog Olfactory Cilia. ,/ Nruraui 1991, 11~36243629. Modulates an Unspecific Cilia of Xenopus laevis.
26. ??.
ZUFAL, F, S~IEPHEKD GM,
FIRESTEIN S: Inhibition of the OIfactory Cyclic Nucleotide Gated Ion Channel by Intracellular Calcium. Proc K Sot Land [Biol] 1991, 246.225-230. Intracellular CaL + concentrations of 1 PM act to stabilize a closed state of the cyclic-nucleotide-gated channel by reducing the open probability. This would be an attractive negative feedback mechanism which could be important in the rapid phase of adaptation.
27.
RESTRCPOD, MNAMOTO T, BRYANT BP, TEETER JH: Odor Stimuli Trigger Influx of Calcium into Olfactory Neurons of the Channel Catfish. Science 1990, 249:116&l 168.
28.
RESTREPO D, BOYLE AG: Stimulation of Olfactory Receptors Alters Regulation of [Cad in OIfactory Neurons of the Catfish (Ictalums punctatus). J Membr Biol 1991, 120:223-232.
29.
CIINNINGHAIVI AM, RI
signals
in olfactory transduction
Stuart Firestein Yale University Olfactory system, direct
Medical
transduction which
results
activation
of
to threshold
the transduction has been olfaction,
in the
occupied
second messenger
Current
Opinion
their
pathways
Underlying these two forms of membrane potential change, the slow generator potential and the fast action potential, are two kinds of channels which provide the pathways for ionic currents. The odor-elicited current results from the activation of a cyclic-nucleotidegated channel in the specialized cilia that emanate from the single dendrite of the receptor neuron. Underlying action potential generation is a family of somatic voltagegated Na+, K+ and Ca2+ channels that are similar to those found in other spiking neurons [l-3]. Thus, the production of a signal can be conveniently divided into two phases, a slow odor-induced depolarization, followed by rapid spike generation and propagation. This review is concerned with the first of these, the sensory current that brings the cell to firing threshold. current
Ionic currents resulting from the action of odors on cell surface receptors were first recorded using the whole-cell patch-clamp technique [4,5]. The favorable morphology of the isolated olfactory receptor neuron assures that the slow currents elicited by odors are usually well clamped. Several important features of the odor response have
messenger
of CAMP. by CAMP
@ Current
of the central considerable
behavior
role in effort
at a molecular
have also been proposed
in
1992, 2:444-448
been observed in experiments in which odor containing solutions were pressure ejected into the vicinity of isolated receptor neurons and the resulting ion currents recorded. Site of transduction
The site of olfactory transduction, long suspected to be in the cilia, was unequivocally confirmed by carefully directing pulses of odor at the cilia, the dendrite and the cell body [4,6]. Only those pulses of odor reaching the cilia elicited an ionic current. The cilia were shown to be highly specialized for transduction as high concentrations of KC1 elicited currents only when directed at the dendritic and somatic regions of the cell and not at the cilia. Recently, these observations have been confirmed in experiments that also determined that the distribution of odor-sensitive channels along the cilia was likely to be uniform [ 71. Electrical
characteristics
In the range of - 30 mV to + 20 mV the current-voltage relation of the odor-elicited current is nearly linear, and the current reverses polarity just positive to OmV. This is suggestive of a non-selective cation conductance and, indeed, Kurahashi [4] has shown that most monovalent cations are nearly equally permeant. Significant rectification of the current can be seen in the voltage range negative to - 40 mV, and also at positive potentials (F Zufall and S Firestein: Biop@sical Journal [AM-] 1992, 61:162X) [4]. It now seems probable that this apparent voltage-dependence is actually due to divalent cation blocking effects. The role of Ca2+ and Mg2+ ions will be discussed below in the context of single-channel recordings. Kinetic
features
and second
messenger
system
The current elicited by a brief ( < looms) pulse of odor has a characteristic time course which includes a relatively
protein; W-inositol Biology
The
is the
brings the membrane
Abbreviations C protein--CTP-binding
USA
for these is less well developed.
in Neurobiology
Olfactory transduction is the process by which chemical information, in the guise of odor molecules, is transformed into cellular electrical activity. The nature of this electrical activity is a change in membrane potential as a result of the flow of ionic current. In olfactory neurons the membrane potential change comes in two varieties, a generator potential and an action potential. The first is a gradual depolarization which shifts the membrane from its resting potential, around - 55 mV, to the threshold for action potential generation, near -25mV. It is the action potential which is propagated centrally to the first synapse in the system at the olfactory bulb, and which constitutes the final step in peripheral signal transduction.
that
Because
by these channels
evidence
Introduction
The odor-elicited
cilia membrane
understanding
but the physiological
second
production
the slow depolarization
toward
Connecticut,
a G-protein-coupled
odor-dependent
for spike generation.
cascade
directed
level. Alternative
involves in the
ion channels
final step in producing potential
School, New Haven,
1,4,5-trisphosphate.
Ltd ISSN 0959-4388
Electrical
long latency of I5&500 ms, a slow rise time to a peak amplitude, and an exponential decay with a time constant that depends on the response magnitude. The amplitude of the odor-elicited current is a function of stimulus intensity and duration, but the dynamic range of an individual cell appears to be rather narrow, spanning only an order of magnitude of stimulus concentration. Of considerable interest is the observation that the rising phase and peak of the odor response actually occur as the stimulus intensity is decreasing, due to diffusion of the odor molecules away from the cell surface. Finally, the response to sustained stimulus application is transient, returning to near baseline levels within l&12 s, indicating an adaptation mechanism [6,8]. This assortment of response features can most easily be understood as the result of an integrating step, such as the modulation of an intracellular second messenger. In the last few years all of the molecular components of a CAMP-generating second messenger cascade have been biochemically identified (see review by H Breer, this issue, pp 43!@443) [9]. Physiological confirmation of the operation of such a system has come from patchclamp studies using CAMP and its analogues, as well as phosphodiesterase inhibitors and GTP-binding protein (G protein) modulators. Direct injection of either CAMP or cGMP through the patch pipette, or the application of membrane permeable analogues induces a large inward current that is electrically similar to the odor induced current [10,11**,12]. Thus, there is now strong evidence generated from complementary methodologies that odors cause a G-protein-mediated increase in CAMP, which leads to the activation of a non-specific cationic conductance pathway in the cilia, The cyclic-nucleotide-gated
channel
It has been known since 1987 that a sizeable cyclicnucleotide-activated conductance pathway is present in the ciliary membrane [Is], but it has not been possible to resolve single-channel activity in membrane patches. On the other hand, single channels that are activated by either CAMPor cGMP have been observed in membrane patches taken from the dendrite and soma [ 14,151. Recently, in cell-attached patches from the dendrite, it has been shown conclusively that these channels are activated during the odor response and that they are also gated by intracellular CAMP [ 16*,17**], thus providing a direct link between the odor-induced production of CAMPand the odor-sensitive current. A thorough characterization of the single channel in excised patches of dendritic membrane has been undertaken by Zufall et al. [ 17**]. In confirmation of previous observations from macroscopic currents the single than nel could be activated by either CAMP or cGMP with a Kp of 20 PM and 4 l.tM, respectively. For both nuc!eotide ligands the Hill coefficient was 2.7 and the single-channel conductance was about 45 pS. Neither the conductance nor the open probability showed any significant voltage dependence over the range - 100 mV to + 100 mV. The open time distribution could be described by a single exponential, while the closed time distribution was best described by at least three exponentials, suggesting that
signals in olfactory
transduction
Firestein
there is probably a single open state and at least three closed states. The three closed states were reflected in the single-channel records as long closings that separated bursts of openings, short interburst closings, and a rapid flickering due to incompletely resolved closings. At least three cloned versions of the olfactory channel have been identified. Dhallan et al. [ 181 first cloned the rat channel, followed by Ludwig et al. [ 191 (bovine channel), and most recently Goulding et al [ 20**] have identified a clone in the catfish. Although originating in diverse species all of these channels possess a high sequence homology, appear functionally similar, and are apt to be alike structurally. The olfactory cyclic-nucleotide-gated channel also bears significant homology (about 70 % at the amino acid level) with the cGMP-gated channel of vertebrate photoreceptors [ 211. This makes the difference in cyclicnucleotide sensitivity between the two channels a particularly compelling molecular question. The photoreceptor channel is highly selective for cGMP, while the olfactory channel is activated about equally well by either nucleotide. A1though a putative binding site for the cyclic nucleotide has been identified as a string of some 80 amino acids near the carboxyl terminus, the specific residues responsible for the binding differences are unknown [ 221. A related question of physiological interest is whether cGMP plays any role in olfactory transduction. As of this time no odor-sensitive guanylate cyclase activity has been meas ured in olfactory neurons, and so it appears that CAMP is the physiological ligand (but see below). Role of divalent
cations
This is one of the more controversial areas of olfactory transduction research, with many, and sometimes incompatible, actions being claimed for Ca*+. It is now clear that the cAMP-gated channel is permeable to Cal’ ions, producing a Ca*+ influx during the odor response (F Zufall et al., unpublished data) [4]. On the other hand, Ca2+ ions can also act as open channel blockers, in much the same way as seen at voltage-gated Ca*+ than nels . This is not the only similarity shared with voltagegated Ca2+ channels, Goulding et al. [ 20**] have shown that basic pH relieves some of the open channel block by H+ ions, and replacement of Na+ with Li+ also significantly relieves the block (S Firestein, unpublished data). Further, some Ca*+ channel blockers, such as nifedipine, also block the CAMP-gated channel. All of these results suggest a surprising similarity between this ligand-gated channel and the voltage-sensitive L-type Ca*+ channel. Mg*+, the other divalent cation present in significant concentrations in physiological solutions, also acts as an open channel blocker, but unlike Ca*+ does not appear to have any significant permeability. The Mg*+ block may explain the strong outward rectification seen in the current-voltage relation of the odor-elicited current. In the absence of divalent cations the channel possesses an ohmic current-voltage relation. The physiological significance of the divalent block is curious At the normal resting potential, about - 55 mV, the channels would be mostly blocked by physiological lev-
445
Sensory
systems
els of Ca2+ and Mg2+, and a great many would have to be activated to produce a significant depolarization, Yau and Baylor [23], considering a similar problem in photoreceptors, have pointed out that this might actu ally serve to increase signaling reliability. This is because the variance associated with the random behavior of a large population of small channels is much less than that generated by the activity of a few large channels. Because olfactory neurons also fire action potentials, a second increase in signaling reliability is gained by the progressive relief of the block by depolarization. As the membrane depolarizes, activated channels are capable of conducting more current, further depolarizing the cell. The effect is to create a kind of response threshold, in which a signal capable of opening enough channels to create a small initial depolarization, begins a regenerative process that rapidly brings the membrane to the potential required for spike generation, As it is now clear that Ca2+ permeates the open channel, it is logical to ask what effects may be mediated by an increase in intracellular Ca2+, Recently Kleene et al. [ 241 have been able to draw an entire cilium into a patch pipette, gain a giga-ohm seal at the base of the cilium and then separate it entirely from the cell. This results in what is essentially an inside-out patch, but the patch consists of an entire intact cilium. In addition to the cAMP-gated conductance they present evidence for a Ca2 + -activated Clconductance. The K,/, for Ca2+ activation is 5 PM and the current-voltage relation shows strong outward rectification. The physiological significance of this current is, however, unclear pending further investigation. Schild et al. [ 251 have identified what they believe to be a Ca2+ -activated nonspecific cation conductance, but the evidence for this is less direct. What does seem clear from their report is that there is likely to be both a Ca2+ -ATPase and a Na+-Ca2+ exchanger in olfactory neurons. It must be emphasized, however, that there is as yet no definitive
Adenylate cyclase
evidence to link either of these Ca2+ effects to the odor response. When a maintained step of odor stimulus is presented to an isolated olfactory neuron the elicited current is seen to be transient, reflecting a strong and rapid adapta tion process. Kurahashi and Shibuya [8] showed that this could be abolished by removing extracellular Ca2+. One mechanism for this has been demonstrated by Zufall et al. [26-l in recordings of single cAMP-gated channels in excised membrane patches. The open probability of the channel, but not its conductance, was reduced more than tenfold by intracellular concentrations of Ca2+ between 1 uM and 3 PM. This is distinct from the open channel block caused by extracellular Ca2+, and appeared to be mediated by an allosteric mechanism, for example Ca2+ binding to some intracellular site on the channel protein and stabilizing a closed state. This would be an attractive feedback mechanism for mediating rapid desensitization of the odor response.
Other
second
messengers
There is mounting molecular and biochemical evidence for an inositol 1,45trisphosphate (IP3) pathway, either independent of, or in addition to, the CAMP signaling pathway (Fig.1). The physiological data associating IP3 with odor transduction is less conclusive, if no less tan talizing. Breer (this issue, pp 439-443) has outlined the biochemical evidence for these alternative second messenger pathways. Here, I will concentrate on the electrophysiological evidence. An increase in IP3 should result in an increase in intracellular Ca2+, and indeed Restrepo, Teeter and colleagues [27,28] have measured such an increase in response to certain amino acids in the catfish. In addition, they have recorded the activation of a channel by IP3 in bilayers to which olfactory cilia vesicles were fused. No such channel
Phospholipase
C ?
Fig.1. Probable (CAMP) and possible (IP,) pathways Action
potential
current eration.
for
the
odor-induced
leading to action potential
ionic gen-
Electrical
signals
in olfactory
transduction
Firestein
has yet been observed in plasma membrane patches from intact cells, however. This is important as the cilia contin no Ca2+ storing organelles, requiring that the IP3 induce a Ca2+ influx through the plasma membrane. mile a plasma membrane IP3 receptor would be novel, there is no reason to believe it unlikely. Cunningham and Reed [ 291 note promising preliminary data from their lab showing possible IP3 receptor immunoreactivity in the cilia membrane.
presence correspondingly difficult to establish. One interesting possibility may be that IP3 produced in the cilia increases intracellular Ca2 + , which activates the guanylate cyclase known to be present in olfactory neurons, resulting in the generation of cGMP. This cGMP would be able to activate the same channel as the CAMP.Thus, two pathways would produce two different cyclic nucleotides, but both could work through the same channel.
As further evidence for an odor-induced Ca2+ influx Restrep0 et al. cite the inhibition of the electro-olfactogram response to odors by the removal of extracellular Ca2 + This appears, however, to be in contradiction to the effects of Ca2+ shown by Kurahashi and Shibuya [8] and Zufall et al. [26-l, in which removal of extracellular Ca2+ enhanced the response to odors. The discrepancy could be due to species differences, but a more likely explanation is that in non-voltage clamped preparations the removal of Ca2+ serves to strongly depolarize cells, inactivating the Na+ current, and thereby making the neurons refractory to action potential generation. This has also been pointed out by Frings, Benz and Lindemann [30*] who, while recording unclamped action potentials in olfactory cilia, observed first an increase and then a decrease in neuronal excitability when Ca2+ was removed from the bathing medium.
Future directions
Including IP3 in the patch pipette does not induce a current, at least in salamander neurons [ll**] , nor does it interfere with the odor-elicited current. On the other hand inclusion of CAMPin the pipette always induces a current. In catfish, and more recently in rat olfactory neurons, Restrep0 and Teeter have reported (4th Annual Meeting of the Association for Chemoreception Sciences, Sarasota, Florida: April 1992, Abstract 144) a depolarization upon patch rupture and intracellular dialysis of 10 PM IP3, but the effect was rather slow (on a time scale of minutes) and not robust. An alternative explanation might be that IP3 was releasing Ca2+ from somatic internal stores and effecting membrane potential through mechanisms unrelated to odor transduction. Although this review is concerned with vertebrate olfactory transduction, it might be noted in passing that the most conclusive evidence for an odor-sensitive plasma membrane IP3 receptor was recently presented in studies with lobster cultured olfactory neurons (DA Fadool et al.: 4th Annual Meeting of the Association for Chemoreception Sciences, Sarasota, Florida, April 1992, Abstract 147) in which IP3-activated channels, first observed during application of odors, were then recorded in patches taken directly from the plasma membrane. Some consideration might be given to the likely size of the IP3-regulated Ca2 + influx. Olfactory cilia are less than 0.5 mm in diameter and have a high surface to volume ratio. In a typical cell with ten cilia, each 3Opm long, a Ca2+ current of only about 0.6~~ could transiently raise the ciliary Ca2+ concentration to between ~/.LMand 10pM. The Ca2+ buffering capacity of the cilia is unknown, but presumably depends entirely on the action of membrane pumps and exchangers. Thus, depending on the exact role of intracellular Ca2+, relatively few IP,-gated channels may be required and their
The field of olfactory transduction has experienced remarkable progress over the past 5 years, and in that short time a consensus view of the broad outlines of olfactory transduction has been established [31*-l. Nonetheless, it would be premature to congratulate ourselves on having unlocked the mysteries of olfaction. As more results are reported it is becoming clear that there are several critical discrepancies in the data that cannot be ascribed solely to differences in techniques and preparations. A full appreciation of olfactory transduction will require their resolution. Among the priorities are an understanding of the role of IPj and the possible interaction between this molecule and the cyclic-nucleotide pathway. The unequivocal identification of an IP3 receptor in the plasma membrane of a vertebrate olfactory neuron would also be of general interest to the neurobiology community. Likewise, it might be expected that the cyclic-nucleotide-gated channel will soon be found in other neuronal populations besides sensory neurons. It has already been established that the cGMP channel can be found in retinal bipolar cells [32], and the new appreciation of the nitric-oxide-mediated stimulation of guanylate cyclase in neuronal cell populations means that cGMP channels are possible targets for this pathway as well. References Papers of view, have . of .. of
and recommended
particular interest, published been highlighted as: - special interest outstanding interest
reading
within the annual period of re-
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Induced Receptor
FIRESTEINS, DARROUIJ B, SHEP~IERUGM: Activation of the Sensory Current in Salamander Olfactory Receptor Neurons Depends on a G-Protein Mediated CAMP Second Messenger System. Neuron 1991, 68255835. The effect of the various elements of the CAMP second messenger system on the odor~induced current were tested m experiments designed to show the contribution of their biochemistry to the shape of the phy siological response. 12.
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