Proc. Natd. Acad. Sci. USAVol. 88, pp. 8520-8524, October 1991Neurobiology

Dual activation of a sex pheromone-dependent ion channel frominsect olfactory dendrites by protein kinase C activators andcyclic GMP

(olfactory transduction/second messengers/invertebrates)

FRANK ZUFALL* AND HANNS HATTPhysiologisches Institut der Technischen UniversitMt Munchen, Biedersteinerstr. 29, D-8000 Munich 40, Federal Republic of Germany

Communicated by Vincent G. Dethier, May 13, 1991 (receivedfor review February 1, 1991)

ABSTRACT Olfactory transduction is thought to takeplace in the outer dendritic membrane of insect olfactoryreceptor neurons. Here we show that the outer dendriticplasma membrane of silkmoth olfactory receptor neuronsseems to be exclusively equipped with a specific ion channelactivated by low concentrations of the species-specific sexpheromone component. This so-called AC1 channel has aconductance of 56 pS and is nonselectively permeable tocations. The AC1 channel can be activated from the intracel-lular side by protein kinase C activators such as diacylglyceroland phorbolester and by cGMP but not by Ca2+, inositol1,4,5-trisphosphate, or cAMP. Our results imply that phos-phorylation of this ion channel by protein kinase C could be thecrucial step in channel opening by sex pheromones.

A central question concerning olfaction is the identity of themolecular mechanism that underlies chemo-electrical trans-duction. In vertebrates, there is now considerable evidencethat the binding of some odor molecules to specific receptorproteins in olfactory cilia stimulates an adenylate cyclase,which leads to a membrane depolarization by the opening ofa cylic nucleotide-gated cation channel (1-8). An alternativesignaling pathway, which includes the opening of Ca2l chan-nels by inositol 1,4,5-trisphosphate, may be used for thedetection of some other odor molecules (9).Among invertebrates, male noctuid moths, which have

long been known for their remarkable ability to detectextremely low concentrations of female sex pheromones (10,11), represent the best studied olfactory model system,although the chemo-electrical transduction mechanism re-mains elusive. The male silkmoth (Antheraea polyphemus)detects female sex pheromone components via specializedolfactory receptor neurons (ORNs) innervating long, sexu-ally dimorphic trichoid sensilla on its antennae (11). It isgenerally assumed that the lipophilic pheromone moleculesenter the olfactory hair through pore tubules (12) and are thentransported via pheromone-binding proteins (13) through theaqueous sensillum lymph to the outer dendritic membrane ofORNs, where they may bind to specific receptor proteins(14). Recent biochemical studies described a pheromone-stimulated phospholipase C (15) and a transient, odor-induced accumulation of inositol trisphosphate in antennalpreparations (16). Moreover, a pheromone-induced increasein cGMP through the activation of a guanylate cyclase hasbeen reported (17). These biochemical events may trigger theopening of specific ion channels, thereby depolarizing themembrane.To investigate these ion channels, we recently examined

excitable properties of ORNs by the patch-clamp technique.ORNs were either freshly isolated or maintained in primary

cell culture (18-20). The soma membrane ofORNs respondedto pheromone stimulation with the opening of a 66-pS Ca2+-activated K+ channel and a 60-pS Ca2+-activated nonspecificcation channel. A channel directly gated by cAMP or cGMPwas not found in these membranes (20). One important crite-rion that must be met for an ion channel to be involved in theprimary transduction process is that it is present in thedendritic membrane of ORNs.We now report single-channel recordings from the outer

dendritic plasma membrane of ORNs in situ. Single-channelcurrents were activated by low concentrations of a specificsex pheromone. The sex pheromone-dependent ion channelcould also be stimulated from the intracellular side by acti-vators of protein kinase C and by cGMP. Our data stronglyindicate that phosphorylation of the ion channel by proteinkinase C could be the crucial step in sex pheromone trans-duction.

MATERIALS AND METHODSAntennae were obtained from adult male silkmoths (A.polyphemus) kindly provided by T. Keil, Max-Planck-Institut, Seewiesen, F.R.G. Dendrites protruded from the cutend of single trichoid hairs (21).

Patch-clamp recordings closely followed the methods de-scribed in a previous paper (20). Seals of >10 GU wereobtained with fire-polished borosilicate glass microelec-trodes, without enzymatic treatment of the preparation.Electrode resistance was usually about 15 MM. The exposeddendrites were bathed at room temperature in a solution thatmimicked sensillum lymph (11), 150 mM KCI/20mM NaCl/2mM CaC12/5 mM glucose/10 mM Hepes, pH 7.1. The samesolution was used as pipette filling solution except in oneseries of experiments, where all K+ was replaced by Na+.Pheromone application onto dendrites during cell-attachedrecordings was achieved by pressure ejection from "puffer"pipettes. Excised patches were transferred to a microcham-ber equipped with a liquid filament switch for fast solutionchange (within 200 us) (22). The inner surface of the mem-brane was bathed in EGTA-buffered "control" solution, 150mM KCI/1 mM NaCl/1 mM CaCl2/10 mM EGTA/10 mMHepes/5 mM MgATP, pH 7.1.

Single-channel currents were recorded with a List EPC7patch-clamp amplifier and stored on video tape. For single-channel analysis, records were digitized at the indicatedsampling rate and filter corner (-3 decibels) frequency andstored on a Hewlett-Packard HP 9800 microcomputer sys-tem. The event-detection program described by Dudel andFranke (23) was used. The mean open probability, p, wascalculated according to the equation p = I/(N x i), where I

Abbreviations: ORN, olfactory receptor neuron; AC1, (6E,11Z)-6,11-hexadecadienyl acetate.*To whom reprint requests should be addressed.

8520

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 88 (1991) 8521

is the mean current, N is the number of channels in the patch,and i is the single-channel amplitude. Since we never saw

superpositions of single-channel currents in all our record-ings, N was assumed to be 1. Mean current was calculated byintegrating current flow during channel openings and dividingthe integral by the total time of the sample.A sample of (6E,11Z)-6,11-hexadecadienyl acetate (AC1),

the main component of silkmoth pheromone, was kindlysupplied by H.-J. Bestmann (University of Erlangen,F.R.G.). All other chemicals were obtained from Sigma. Asin a previous study with lipophilic compounds (24), stocksolutions were prepared in dimethyl sulfoxide (DMSO) andstored frozen. Dilutions to the indicated values were madeimmediately before use, using ultrasonication, to a finalmaximal DMSO concentration of 0.1% (vol/vol). ThisDMSO concentration had no effect on ionic channels incontrol recordings and did not activate pheromone-stimulated channels when applied alone. Likewise, no chan-nel activity was observed using bombykal (1 ng/ml) (n = 5),a structurally similar sex pheromone component of thesphinx moth (Manduca sexta).

RESULTS

First, a gigaohm seal in the cell-attached configuration was

established at the outer dendritic membrane ofORNs (Fig. 1).Under control conditions without pheromone application, wenever saw any single-channel openings (n = 61). Likewise, no

voltage-gated channels and Ca2+- or ATP-gated channels(inside-out patches) were found (n = 14). Thus, when at rest

FIG. 1. Light micrograph (phase-contrast optics) showing thelocation of the patch pipette (p) during seal formation on the plasmamembrane of an olfactory dendrite (d) (outer dendritic segment)protruding from the cut end of an antennal hair (h) (sensillumtrichodeum) of the silkmoth A. polyphemus. (Bar = 10 Aim.)

the outer dendritic segment has an extremely tight membranewith a high input resistance.We next incubated the dendrites with a low concentration

(0.1 ng/ml) of the main pheromone component, AC1 (25) for1-60 min. Under these conditions, single-channel eventswere detected in about one-third ofthe cell-attached dendriticmembrane patches (second trace of Fig. 2A). The currentswere inwardly directed and occurred as brief, isolated open-ings with a mean open time of 0.12 + 0.04 ms (mean ± SD,n = 7) (single exponential fit of open-time distribution) and avery low mean open probability, p - 0.001.

Further evidence for pheromone dependence of thesesingle-channel events was obtained by investigating the effectof pheromone concentration. A 100 times higher concentra-tion of the pheromone (10 ng/ml) was focally applied to thedendrite during the cell-attached recording (Fig. 2A, seearrow). After a short delay (about 100 ms) the frequency ofevents increased dramatically (p = 0.24). The amplitude ofevents was unchanged, but activity fused into long periods ofrapid opening and closing (bursts). Since the pheromone hadno direct access to the channels within the patch pipette, thisexperiment implicated a second messenger-mediated channelactivation. The increased activity could not be reversedduring the lifetime of the patch (10-20 min). This irrevers-ibility may be due to the lipophilic properties of the phero-mone and the absence of pheromone-binding proteins andpheromone-degrading enzyme, which may be used in theintact hair to terminate the pheromone response (13). Sincethe pheromone effect with higher concentrations could not bereversed, no attempts were made to obtain a quantitativedose-response relationship.The channel density in the outer dendritic membrane

seemed to be low. Even with the highest pheromone con-centration, we saw no superpositions of openings, indicatingthat there was only one active channel in the patch. Fig. 2Bshows single-channel openings of the pheromone-dependention channel (AC1 channel) at higher time resolution and atthree different membrane potentials. A typical "flicker"behavior is obvious, and this also occurred with Mg2+-freeextracellular solution. To determine the main conductancestate of the AC1 channel, amplitude probability densityfunctions (26) were calculated (Fig. 2E). The distribution hasa shoulder that resulted from subconductance states thathave not been analyzed further. The current-voltage relation(Fig. 2C) is linear between -80 mV and 80 mV, with areversal potential of 0 mV giving an approximate single-channel conductance (y) of 56 pS, which was independent ofwhether Na' or K+ was the main cation in the extracellularsolution. This result suggests that the AC1 channel does notselect between Na' and K+.

Kinetic analysis of unitary currents elicited by AC1 (Fig.2D) showed that with higher pheromone concentration, opentimes had two time constants (,1 = 0.14 + 0.02 ms and r2 =1.48 ± 0.21 ms) and a mean burst length of 5.15 ± 1.42 ms (n= 3). A burst was defined as a group of openings interruptedby closings of <4 ms as indicated by the closed-time distri-bution (data not shown).Having established the existence ofa specific, pheromone-

dependent ion channel in the outer dendritic membrane, itwas of interest to find out whether this channel could beactivated by intracellular constituents (as was implied by theexperiment of Fig. 2A). For this purpose dendrites werepretreated with a low concentration (0.1 ng/ml) of the pher-omone AC1 for 7 min. Only those patches that contained theactive AC1 channel (in the cell-attached configuration) wereexcised to the inside-out configuration. This protocol assuredthat exactly the same ion channel first activated by thepheromone was subsequently tested for second-messengereffects. In the inside-out configuration the AC1 channel wasstill active with nearly the same open probability (p = 0.0008)

Neurobiology: Zufall and Hatt

8522 Neurobiology: Zufall and Hatt

A control

I- wr iwrp

B60 mV

-30 mV

-80 mV

2pA~~~~~~~~~pD ~A-1 Ors

1000

10-

.G ~ ~ ~ Il

2.i ° Open time, ms 9W 60r

I -

16' 48Burst length, ms

C

E0.1

If

80 -1'f4

Currf

FIG. 2. Recordings and evaluations of single-_ y r channel currents in cell-attached patches, activated by

the silkmoth sex pheromone component AC1. Inwardcurrents are shown as downward deflections. (A) Toptrace, control recording without pheromone pretreat-ment; middle and bottom trace, continuous recordingafter pretreatment of the preparation with AC1 (0.1ng/ml). Isolated openings of single-channel currentsoccur at a low rate. Current amplitudes are attenuatedin this low time-resolution plot due to the low sampling

J20A rate. After focal application (arrow) of a 100 times200ms higher concentration of AC1 (10 ng/ml), a substantial

increase in both open probability and burst length of5 single-channel currents is obvious. Membrane poten-

tial was -60 mV. Records were low-pass-filtered at 2**t kfkz (8-pole Bessel) and sampled at 5 kHz. (B) Exam-

ples ofAC1 (10 ng/ml)-inducod single-channel cirrentsat higher time resolution (bandwidth of recording, 0-4kHz; sampling rate, 20 kHz) and at three different

oJ/membrane potentials as indicated. Note the typicalflickering between open and closed states. (C) Cur-rent-voltage relation of AC,-induced single-channel

50 currents. Each point of the curve was obtained byVoltage. mV taking the current value at the maximum probability

density from distributions as shown in E. Data are fromthree different experiments. *, High K+ inside thepatch pipette; A, high Na'. (D) Kinetic analysis ofAC1(10 ng/ml)-in4uced channel openings from the record-ing shown in A. Sampling rate and filter frequency were

s5 as in B. Open-time distribution was fitted by the sum oftwo exponentials with time constapts of 0.15 and 1.37ms, respectively. The ordipate scale is logarithmic;binwidth is 100 us. Distribution pf burst lengths wasfitted by an exponential with a time constant of5.23 Ms.Some bursts of several hundred milliseconds may havecontributed to an additional, longer time constant thatcould not be determined because of too few events.Binwidth is 500 ;Ls. A burst was defined as a group ofopenings interrupted by closings of <4 ms as indicatedby the closed-time distribution (data not shown). (E)Probability density function of AC,-induced openingsat -60 mV. The peak of the distribution at -4.5 p-A

.4: td a 0 corresponds to the main conductance state. Binwidth isnta0.2 pA. Total number of detected events in the distri-amplitude,pAbution is 3465.

and mean open time (0.11 ms), provided that the intracellularsolution contained MgATP (5 mM) (Fig. 3A). After switchingto MgATP-free solution, the activity declined to zero withina few hundred milliseconds (Fig. 3 A and B). The MgATPeffect was fully reversible. The AC1 channel was not Ca2+-gated (raising the intracellular Ca2' concentration to >1 ,uMhad no effect). These results, together with the fact that noATP-activated channels were found in control patches, in-dicated that the AC1 channel was probably in a phosphoryl-ated state before inside-out formation. We therefore at-tempted to activate protein kinases. Since phospholipase Chas been shown to be a key enzyme in antennal fractions afterpheromone stimulation (15), we tested the effect of the Ca2+-,phospholipid-, and diacylglycerol-dependent protein kinaseC (27). The AC1 channel increased its open probability to p= 0.26 + 0.08 (n = 4) after application of a syntheticdiacylglycerol, 1,2-dioctanoylglycerol (0.1 Ag/ml; Fig. 3 Aand B). Moreover, long bursts of openings (mean burstlength, 4.38 ms) with kinetic behavior similar to that shownwith high pheromone concentration (see Fig. 2 A and D)appeared. The rate of opening was greatly reduced afterswitching to a MgATP-free solution, although dioctanoyl-glycerol was still present (Fig. 3B), suggesting phosphataseactivity in the excised membrane. The effect of dioctanoyl-glycerol could be repeated, though the response was smaller

and was washed out after 5 min with MgATP-containingcontrol solution (Fig. 3B). Similar results (n = 3) wereachieved by the application of phorbol 12-myristate 13-acetate (100 nM; see Fig. 5), which activates protein kinasedirectly (27, 28). In control experiments without pheromonepreincubation, dioctanoylglycerol had no effect (n = 6) oninside-out patches.We also tested the effect of other second-messenger can-

didates. Surprisingly, application of cGMP altered the kinet-ics and open probability of the AC1 channel (previouslyactivated by pheromone), similar to activators of proteinkinase C (n = 6). Fig. 4A shows an example ofan AC1 channelwhose activity was reversibly increased by application of 50A&M 3'5'-cGMP to the intracellular side. The cGMP-mediatedactivation was reduced in MgATP-free solution (data notshowp). The concentration dependence of the cGMP effectwas determined (Fig. 4B). The normalized mean current wasplotted as a function of3'5'-cGMP concentration and fitted tothe Hill equation with K1/2 = 55 AM and a cooperativityfactor n = 1.4.

Fig. 5 summarizes the effects of all the tested second-messenger candidates. Surprisingly, the AC1 channel did notdiscriminate between 3'5'-cGMP and 2'3'-cGMP, whereas8-bromo-3'5'-cGMP was less effective. cAMP and inositoltrisphosphate were without measurable effects (n = 9). No

0.1 ng/ml AC1

II '' 'I 'I 10 ng/ml AC

IT__171 T -Tw I- w I I I * I | I | it I | I L[I L1

Proc. Natl. Acad. Sci. USA 88 (1991)

II 4

I

Proc. Natl. Acad. Sci. USA 88 (1991) 8523

cell-attached0.1 ng/ml AC,

inside-outcontrol (MgATP)

AT-fTVeeDOG + MgATP

B 0.8C I

._

0

I MgATPcontrol

Q ATP-Q free

0

0 _

0 60

DOG. I DOG+

MgATP MgATP

11l I

I- .

120 180Time, s

Car1JpA200ms

controlafter 5 man wash

3330240

FIG. 3. Properties of AC1 (0.1 ng/ml)-activatedchannels in an inside-out patch. (A) Original recordingsshowing the AC1 channel before (cell-attached) andafter (inside-out) patch excision and the effect ofMgATP and 1,2-dioctanoylglycerol (DOG) on the AC1channel. The low activity (second trace, 5 mM MgATP)decreased to zero in MgATP-free solution (third trace)and increased drastically in the presence of DOG (0.1,ug/ml; lower trace). Membrane potential, -60 mV;bandwidth, 0-2 kHz; sampling rate, 5 kHz. (B) Thesame experiment as partially shown in A. The openprobability of single-channel currents was plotted as afunction of time for this continuous recording. Bin-width, 100 ms.

attempts were made in this study to test whether channelactivation by protein kinase C activators and cGMP ana-logues results in additive effects. A direct cGMP-gated con-ductance in the absence of an odorant stimulus (withoutpheromone preincubation), as shown for vertebrate olfactorycilia (4), was not observed (n = 7).

DISCUSSIONIntegration and processing of electrical signals in individualneurons depend critically on the spatial distribution of ionchannels on the cell surface. By using an in situ preparation,we have shown that the outer dendritic membrane of silk-moth ORNs, which was previously not accessible to patch-clamp recordings, seems to be exclusively equipped with aspecific ion channel (AC1 channel) activated by a species-specific sex pheromone component. Activation of the AC1channel occurred in the absence of pheromone-binding pro-teins, indicating that the pheromone need not be complexedwith these proteins to elicit a response. Since none of ourpreviously described channels from ORN somata in vitro(18-20) has been found in the dendrites in situ, channeldistribution in ORNs must be highly asymmetric, as has alsobeen demonstrated for vertebrate olfactory (29) and tastecells (30). Thus, occupation of outer dendritic membrane byjust one type of ion channel provides an extreme functionaladaptation that optimizes the role of ORNs in the detectionof only a few specific molecules that have an extraordinarysignificance for reproductive behavior.

Several lines of evidence strongly indicate that the de-scribed AC1 channel mediates olfactory transduction in thesilkmoth: (i) application of very low concentrations of pher-omone leads to the opening of a previously covert class of ionchannels, (it) only the species-specific pheromone leads tochannel opening, (iii) both open probability and burst lengthof the AC1 channel critically depend on the pheromone

concentration, and (iv) openings ofthe AC1 channel resembleextracellularly recorded, so-called elementary receptorpotentials (11).One striking feature ofour study is the low channel density

in the outer dendritic segment. The total number of channelsper dendrite can be estimated to be between 140 and 700, ifone assumes a pipette diameter of 0.2-1 Am2, a dendriticsurface area of 420 Am2 (11), and a homogeneous channeldistribution. Also we take into account that no superpositionsof single-channel currents occurred and that about everythird patch contained a channel. From this the high and lowvalues for the channel density would be 1.6 and 0.33 .m2,

respectively.The sensitivity of the AC1 channel to activators of protein

kinase C and to cGMP strongly supports the involvement ofa second-messenger cascade in the transduction process,which surely provides the basis for the extremely low detec-tion threshold of sex pheromones (11). We also provideevidence for extensive cross-talk between two different sec-ond-messenger pathways, as suggested in other systems (31,32). Our results are provocative in light of the recent obser-vation of an odor-activated phospholipase C in antennalfractions (15), an enzyme that catalyzes the hydrolysis ofphosphatidylinositol bisphosphate to produce diacylglyceroland inositol trisphosphate (33). Since we provide directevidence for an ATP-dependent stimulatory effect ofthe AC1channel by diacylglycerol and phorbol ester, but not by Ca2+or inositol trisphosphate, we conclude that phosphorylationof the ion channel by a protein kinase C could be the crucialstep in this transduction cascade. In contrast to other proteinkinases known so far, most protein kinases C have to undergoa conversion from a cytosolic or membrane-bound form to amembrane-inserted form, a so-called translocation, beforebeing fully activatable by diacylglycerol (34). This strikingproperty of protein kinases C may account for our inability toactivate the AC1 channel with dioctanoylglycerol or even

A

I

Neurobiology: Zufall and Hatt

8524 Neurobiology: Zufall and Hatt

A

B

control

cGMP ::~~~~~~~-7T--coto o

200ms

1

1 10

cGMP concentration, pM

FIG. 4. Effect of cGMP on the activity of the AC1 channel. (A)Original recording showing the activity of the AC1 channel in aninside-out patch with control solution (5 mM MgATP) (top trace),during the presence of 50 A.&M 3'5'-cGMP (middle trace), and again incontrol solution (bottom trace). (B) Concentration dependence ofthe3'5'-cGMP-induced increase in open probability of the AC1 channel.Normalized mean current was plotted as a function of cGMPconcentration in a double logarithmic plot. The Hill equation, g =

CM/(C" + K172), where C is the cGMP concentration and n is the Hillcoefficient, was fitted by eye to the data points. The values of K112and n used for this curve were 55 ,uM and 1.4, respectively.

cGMP without a previous preincubation with the pheromone.As in other systems (34), the translocation of protein kinasemay be promoted by the reported transient, odor-activatedincrease (time to peak, <50 ms) in inositol trisphosphate (16).Whether the cGMP-mediated activation of the AC1 chan-

nel is used as a transduction mechanism remains unknown.Recent biochemical evidence (17) seems to exclude this ideaand, rather, favors a modulatory role for cGMP in silkmothORNs. Clearly, more experiments are needed to resolve thisquestion. Since it has been shown that the electroantenno-

1.

CL

ci

C,cCI

1 2 3 4 5 6 7 8

FIG. 5. Effects of presumed second messenger candidates on

AC,-induced single-channel activity in inside-out patches. Bar 1,control; bar 2, 1,2-dioctanoylglycerol (0.1 ,ug/ml); bar 3, phorbol12-myristate 13-acetate (100 ,uM); bar 4, cAMP (100 uM); bar 5,inositol 1,4,5-trisphosphate (1 ;LM); bar 6, 2'3'-cGMP (100 .uM); bar7, 3'5'-cGMP (100 uM); bar 8, 8-bromo-3',5'-cGMP (100 ;iM). Dataare from 23 experiments.

gram comprises two different decay time constants afterpheromone stimulation (11), it is conceivable that the de-scribed dual mechanism for channel activation could produceboth a short-lived and a sustained reaction.The evidence summarized above strongly suggests that

insect sex pheromone transduction differs from transductionin vertebrate olfactory cilia by using protein phosphorylationrather than direct gating of ion channels by cyclic nucleotidesto initiate an electrical response. However, our results oninsect olfactory transduction provide some analogy to inver-tebrate photoreception, where both inositol trisphosphateand cGMP seem to be involved in the transduction process(35-37). In this respect, a report of a Drosophila proteinkinase C gene that is specifically expressed in photoreceptorcells (38) is most interesting.

We thank Dr. K.-E. Kaissling for stimulating our interest in thedendritic preparation; Drs. J. Dudel, J. Daut, U. B. Kaupp, andG. M. Shepherd for valuable comments on the manuscript; Dr. Ch.Franke for help with computer evaluation; and Miss B. Preibisch forexcellent technical assistance. This work was supported by theDeutsche Forschungsgemeinschaft.

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Proc. Natl. Acad. Sci. USA 88 (1991)

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