Cilium-attached and excised patch-clamp recordings of odourant-activated Ca-dependent K channels from chemosensory cilia of olfactory receptor neurons

Cilium-attached and excised patch-clamp recordingsof odourant-activated Ca-dependent K channels fromchemosensory cilia of olfactory receptor neurons

Ricardo Delgado1 and Juan Bacigalupo1,2

1Millennium Institute for Advanced Studies in Cell Biology and Biotechnology, University of Chile, PO Box 653, Santiago, Chile2Department of Biology, Faculty of Sciences, University of Chile, Santiago, Chile

Keywords: Caudiverbera caudiverbera, ion channels, odour transduction

Abstract

It has previously been proposed that a Ca2+-dependent K+ conductance is implicated in the inhibitory odourant response in ratand toad olfactory receptor neurons. Previous whole-cell and single-channel measurements on inside-out excised patches, inaddition to immunochemical evidence, indicated the presence of Ca2+-dependent K+ channels in olfactory cilia, the transducingstructures of these sensory cells. Ca2+-dependent K+ channels opened in ‘on-cilium’ membrane patches from C. caudiverberaupon odourant stimulation. Furthermore, after excision in the inside-out configuration, the channel could be opened bymicromolar Ca2+, in a Ca2+-dependent fashion, but it was unresponsive to cyclic AMP. We estimated that the Ca2+ concentrationin the proximity of a Ca2+-dependent K+ channel within the cilia reaches at least 100 lm during the odour response. The K+

channel displayed a higher selectivity for K+ than for Na+. Our results support a role for this Ca2+-dependent K+ channel inchemotransduction.

Introduction

Odourants excite olfactory receptor neurons by triggering a cyclicAMP transduction cascade that leads to the activation of anonselective cationic cyclic nucleotide-gated channel (Nakamura &Gold, 1987) and a Ca2+-dependent Cl– channel (Kleene & Gasteland,1991), both of which underlie the depolarizing receptor potential thatgives rise to an excitatory response (Kurahashi & Yau, 1993; Lowe& Gold, 1993). Additionally, there is a growing body of evidencedemonstrating that the olfactory sensory cells from of a variety ofvertebrate species also generate inhibitory responses (Dionne, 1992;Morales et al., 1994; Kang & Caprio, 1995; Vogler & Schild, 1999;Duchamp-Viret et al., 2000), resulting from a hyperpolarizingreceptor potential (Dionne, 1992; Morales et al., 1994; Vogler &Schild, 1999). In toad and rat this receptor potential appears to bedue to the activation of a Ca2+-dependent K+ conductance, becausean odourant-triggered, Ca2+-dependent and charybdotoxin-sensitiveK+ current has been recorded in both animals (Morales et al., 1995;Sanhueza et al., 2000). Ca2+-dependent K+ channels are present inthe chemosensory cilia, as shown immunochemically as well as bysingle-channel recordings from inside-out membrane patches excisedfrom olfactory cilia. Indeed, four Ca2+-dependent K+ channel typeswere found in olfactory cilia (Delgado et al., 2003). We studied onesuch channel during odour stimulation by recording from on-ciliummembrane patches, followed by recording from the same patchesafter excision.

Materials and methods

Experiments were conducted on olfactory receptor neurons mechan-ically dissociated from the Chilean toad Caudiverbera caudiverbera(Morales et al., 1994). The animals were anaesthetized by cooling inice, after which they were killed and pithed before dissecting out theirolfactory epithelia. Extracted olfactory epithelia were cut into smallpieces (� 1 mm2) and kept at 4 �C for at most 48 h in a solutioncontaining (in mm) NaCl, 120; CaCl2, 1; MgCl2, 2; KCl, 3; glucose, 5;HEPES, 10; and Na-pyruvate, 5; with 0.1 IU mL)1 penicillin; pH 7.5.Dissociation was accomplished by gently passing the pieces ofepithelia through the tip of a fire-polished Pasteur pipette; the cellswere then transferred to the experimental chamber, which containedRinger’s solution (in mm): NaCl, 115; KCl, 2.5; CaCl2, 1; MgCl2, 1.5;glucose, 3; and HEPES, 10; pH 7.6. Prior to each experiment, thebath solution was exchanged for intracellular K+ solution (in mm):K-acetate, 110; KCl, 10; MgCl2, 1; and HEPES, 10; pH 7.6; this wasdone to prevent exposure of the intracellular face of the patch toexternal solution during the subsequent excision. For the ionselectivity experiments, an internal solution in which K-acetate wasreplaced by equimolar Na-acetate was used. To adjust the pCa ofinternal solutions to values of 6.0 or higher, 1 mm EGTA and CaCl2were added according to Winmaxc 2 software (http://www.stanford.edu/�cpatton/winmaxc2.html). To lower pCa, EGTAwas omitted andCa2+ was supplemented to the desired final concentration. The wholechamber was perfused for exchanging bath solutions (six chambervolumes in � 30 s).For the electrical recordings we used the patch-clamp technique in

its cell-attached and excised patch (inside-out) modes, making use ofan Axopatch 1D amplifier (Axon Instruments, Inc., Union City, CA,USA). Recording pipettes were made of borosilicate glass capillaries

Correspondence: Dr Juan Bacigalupo, 1Millennium Institute for Advanced Studies in CellBiology and Biotechnology, as above.E-mail: [email protected]

Received 30 June 2004, revised 18 September 2004, accepted 21 September 2004

European Journal of Neuroscience, Vol. 20, pp. 2975–2980, 2004 ª Federation of European Neuroscience Societies

doi:10.1111/j.1460-9568.2004.03778.x

with filament (Hilgenberg-GmbH, Postfach, Germany) and drawnwith a horizontal puller (P-97, Sutter Instruments Co., Novato, CA,USA) to tip resistances of 40–50 MW. Seal resistances were 10 GW orhigher. Odour stimuli were applied with a puffer pipette with tips of� 1 lm, located � 10 lm from the cilium and filled with a mixture ofisovaleric acid and cadaverine, 100 lm each, in K+ internal solution.Controlled pressure pulses were exerted on the puffer pipettes througha computer-operated picospritzer. Experimental protocols and dataanalysis were carried out with pCLAMP 6.0 (Axon Instruments, Inc.,Union City, CA, USA). Every olfactory neuron recorded wassystematically photographed before the patch was excised from thecilium.Reagents were purchased from Sigma-Aldrich, Inc. (St Louis, MO,

USA) unless otherwise indicated.

Results

It has previously been shown by a variety of methods, includinginside-out patch clamp recording from olfactory cilia, that thesetransduction organelles contained Ca2+-dependent K+ channels(Delgado et al., 2003). However, evidence indicating that thesechannels activate upon odour stimulation was missing. To investigatethe participation of Ca2+-dependent K+ channels in odour transduc-

tion, we conducted experiments on cilium-attached membranepatches.Figure 1 shows an on-cilium patch-clamp experiment, in which the

recording pipette was sealed onto a cilium and an odourant puff wasapplied to it from a short distance (Fig. 1A). In order to simplify theinterpretation of the recordings and to reduce the cell electricalactivity, the bath contained K+-internal solution. The pipette potentialwas set to 30 mV, determining a driving force for K+ that favoured aK+ influx across the membrane patch. As shown in Fig. 1B, channelactivity prior to odourant exposure was very low. Inward currentevents developed shortly after the stimulus onset, and graduallydeclined back to basal level after the end of odour stimulation. Thestimulus was repeated, with similar results (not shown). No increase inchannel activity was detected upon depolarizing the patch in theabsence of odourant (not shown), indicating that channel activationwas triggered by the chemical stimulus rather than being a result ofodour-induced depolarization. A similar observation was made in twoother patches of a total of nine where this protocol could be applied.According to the experimental ionic conditions, the only cation thatcould account for the observed single-channel currents was K+.Figure 2 depicts the experiment in Fig. 1 in further detail. The top

traces were recorded from on-cilium patches in the absence ofchemical stimulation (Fig. 2A). The probability of the channel being

Fig. 1. Single-channel activity induced by odour stimulation in a chemosensory cilium. (A) Photograph under DIC optics showing a recording pipette patched ona cilium, and a nearby puffer pipette. (B) Current through the ciliary membrane patch before, during and after the application of an odour stimulus: bandwidth,1 kHz; Vp, 30 mV. The line above the record indicates the timing of the stimulus. The traces below correspond to segments of the top trace displayed at an expandedtime scale. The neuron was bathed in internal solution and the patch pipette was filled with the same solution (Materials and methods). Scale bar, 5 lm (A).

2976 R. Delgado and J. Bacigalupo

ª 2004 Federation of European Neuroscience Societies, European Journal of Neuroscience, 20, 2975–2980

Fig. 2. On-cilium odour-induced channel activity was activated by Ca2+ but not by cAMP, after patch excision. (A) On-cilium recording under odourant-freeconditions. (B) Channel activity during odourant stimulation. (C–E) Channel activity after the patch was excised (inside-out) and subsequently exposed to internalsolution containing 0.5, 10 and 100 lm free Ca2+ Vpipette, 30 mV. (F) Activity of the same channel in a different excised patch as the inner face of the membranewas exposed to 50 lm cAMP in 0.5 lm Ca2+. (G) The same patch as in F under 100 lm Ca2+, no cAMP; Vpipette, 60 mV.

Odours activate Ca2+-dependent K+ channels in olfactory cilia 2977


open (Po) was 0.01. During odourant application, the channel activitygreatly increased, reaching a Po of 0.26 (Fig. 2B). The patch wassubsequently excised in the inside-out configuration and its intracel-lular face was exposed to internal K+-solution. We tested three Ca2+

concentrations, 0.5, 10 and 100 lm, at which the channel exhibited Povalues of 0.01, 0.06 and 0.20, respectively (Fig. 2C–E), indicating itsCa2+-dependence. This particular patch broke before we could try

cAMP on it. However, we tested 50 lm cAMP (in the presence of0.5 lm Ca2+) in two other patches in which the same K+ channel hadbeen previously activated by 100 lm Ca2+ (in the absence of thecyclic nucleotide); in none of these patches did cAMP induce channelactivation (Fig. 2F and G).Twenty excised ciliary membrane patches were used in our study. In

six of them we could make some measurements. Although eight otherpatches showed channel activity, we could not obtain enough datafrom them for analysis. The remaining six patches were silent.The Ca2+-dependence of the K+ channel from three separate patches

is depicted in Fig. 3A. The data were fitted to a Hill equation, with aK0.5 for [Ca

2+] of � 25 lm and a Hill coefficient (n) of 2.7. The I–Vrelationship for this channel was previously reported to have twosubstates, of unitary values of � 27 and � 58 pS (Delgado et al.,2003). Figure 3B shows the I–V relationship for the low conductancelevel of the channel under symmetrical K+. The I–V curve shifted� 28 mV to the right after K+ was entirely replaced by Na+, undersymmetrical Cl–. If the shift in the I–V curve is calculated using theextrapolated value to zero current of the curve with Na+ in the bathand K+ in the pipette, a permeability ratio PK ⁄ PNa of 2.7 is obtained,indicating that the channel is moderately selective for K+ over Na+.We explored a range of voltages from )40 to +40 mV and could notdetermine any obvious voltage dependence of Po. The Po of thechannel typically fluctuated spontaneously over long time periodsunder steady conditions, suggesting an apparent change in the channelgating mode (not shown). A similar feature has been previouslynoticed for the large conductance Ca2+-dependent K+ channel inskeletal muscle cells (McManus & Magleby, 1988).

Fig. 3. Ca2+ dependence and K+ selectivity of the odourant-dependent ciliarychannel. (A) Po vs. [Ca2+] plot containing the data from three differentpatches. The numbers above each experimental point indicate the number ofpatches from which each one was obtained. The line represents the fit to theHill equation, using {A ⁄ [1 + (K0.5 ⁄ [Ca2+]n)]}, where K0.5 ¼ 25 lm andn ¼ 2.7. (B) I–V relationship for the channel under K+ (d) and Na+ (m)intracellular solutions in the bath.

Fig. 4. Kinetic parameters of the Ca2+-dependent K+ channel. (A) Dwell time plot of the open state (left) and of the closed time (right) in the presence of 0.5 lmCa2+. (B) Dwell time plot of the open state (left) and closed state (right) in the presence of 100 lm Ca2+. Kinetic analysis was made over a recording time of 50 sfor A and 12 s for B. The data were fitted to exponential functions.



The kinetic properties of this ciliary Ca2+-dependent K+ channelwere further analyzed under two different Ca2+ concentrations, 0.5 and100 lm. The channel exhibits one mean open (0.9 ms) and threeclosed times (9, 35 and 127 ms) in low Ca2+ (Fig. 4A) whereas in highCa2+ it has two mean open times (1.9 and 5.4 ms) and three meanclosed times (2, 12 and 80 ms) (Fig. 4B). In addition, the channeltransition rate from closed to open increased from 20 s)1 at 0.5 lm to128 s)1 at 100 lm. Our analysis excluded the long closed times(> 1 s). Altogether, these observations indicate that the increase in thechannel Po on raising Ca2+ from 10 to 100 lm reflect both an increasein its mean open times and a reduction in its mean closed times.

Discussion

The contribution of the present work is to provide direct evidence thatodourant stimulation can open a Ca2+-dependent K+ channel in thechemosensory cilia of olfactory receptor neurons. This study presentsthe first single-channel recordings from an olfactory cilium showingunitary channel activation by the sensory stimulus. Analogousevidence was previously obtained from the native sensory membraneof invertebrate (Bacigalupo & Lisman, 1983; Nasi & Gomez, 1992;Gomez & Nasi, 1994; Haab et al., 2000) and vertebrate (Mathews,1987) photoreceptors. The present evidence supports the notion that aK+ conductance plays a role in odour transduction. In agreement withprevious reports, the observed conductance might be involved in theinhibitory response to odourants in isolated toad and rat (Moraleset al., 1994; Sanhueza et al., 2000; Delgado et al., 2003) and perhapsin other vertebrate species as well.

Previous whole-cell recordings documented the existence of odour-triggered Ca2+-dependent K+ channels in olfactory neurons andsuggested, by means of focal odour stimulation, that such channelsare localized in the olfactory cilia (Morales et al., 1995). Suchevidence supports the view that this conductance underlies inhibitoryodour responses in isolated olfactory neurons. Immunohistochemicalstudies indicated that Ca2+-dependent K+ channels are present in theolfactory cilia. A study on membrane patches excised from such ciliaconfirms such evidence (Delgado et al., 2003) and agrees withprevious evidence from a study of olfactory cilia membranesreconstituted in planar lipid bilayers (Jorquera et al., 1995). Fourdifferent Ca2+-dependent K+ channel types were resolved in theexcised patch-clamp recordings. One such channel was selected inthe present study. To rule out the possibility that the location of theK+ channels in the cilia might be artifactual, resulting from thediffusion of somatic ionic channels into the cilia after the celldissociation, Western blots from purified olfactory neuron ciliarymembranes revealed the presence of a substantial quantity of K+

channels in the cilia. Immunocytochemical studies supported thisevidence (Delgado et al., 2003). In addition, some of the recordingswere obtained shortly (minutes) after cell dissociation and were notobviously different than those obtained from cells at longer timesafter dissociation.

Olfactory cilia are known to contain cyclic nucleotide-gatedchannels (Nakamura & Gold, 1987), Ca2+-dependent Cl– channels(Kleene & Gasteland, 1991) and Ca2+-dependent K+ channels(Morales et al., 1994; Sanhueza et al., 2000; Delgado et al.,2003), all involved in transduction. It is reasonable to think that thechemosensory cilia, as other sensory transduction organelles, aredevoid of ion channels not directly involved in transduction(Delgado et al., 2003). The possibility that the channels generatingthe channel activity of the on-cilium patches observed duringodourant exposure correspond to the cyclic-nucleotide-gated channel,which is nonselective for cations, is unlikely for the following

reasons: (i) the channel events measured from on-cilium recordingsappear to be indistinguishable from those from the same patch afterexcision, with regard to their mean open time and unitary currentsize; (ii) under excised conditions the channel required Ca2+ but notcAMP to activate and, furthermore, channel activity was graded withCa2+ concentration; (iii) in contrast to the effect of Ca2+ on the K+

channel, the cyclic-nucleotide-gated channel is blocked by submil-limolar divalent cations (Zufall & Firestein, 1993), and the pipettecontained millimolar levels of Ca2+ and Mg2+; moreover, the cyclic-nucleotide-gated channel is negatively modulated by Ca2+ (Kramer& Siegelbaum, 1992; Chen & Yau, 1994); and (iv) the channel thatwe studied is at least 2.7-fold more selective for K+ than for Na+,whereas the cyclic-nucleotide-gated channel is virtually nonselectivefor cations (Frings et al., 1992). The possibility that the observedchannels were Cl–-selective is ruled out because the shift of the I–Vcurve upon replacement of Na+ for K+ occurred under symmetricalCl– concentrations (Fig. 3B). On the other hand, if the unitaryconductance of the Ca2+-dependent Cl– channel is � 0.5 pS (Kleene,1997), the size of the events would be undetectable under ourexperimental conditions.An earlier study of olfactory cilia channels reconstituted in planar

lipid bilayers (Jorquera et al., 1995) reported a voltage-independentK+ channel that in several aspects closely resembles the channelcharacterized here, with two subconductance states and with remark-ably similar kinetic behaviour. However, the conductance values of thebilayer channel substates is nearly double those measured by us,perhaps due to the different ionic conditions used. The Ca2+-dependence of the channel was not thoroughly investigated in thatearly work. Recent results from our laboratory, also conducted inbilayers, confirm our finding that this K+ channel is Ca2+-dependent(K. Castillo, J. Bacigalupo and D. Wolff, unpublished observations).The Ca2+-dependence of the K+ channel characterized here is

similar to that previously reported for two other ciliary Ca2+-dependent K+ channels (Delgado et al., 2003). The Hill coefficientof this channel, 2.7, suggests that more than one Ca2+ ion is necessaryto activate it and that Ca2+ binding may be cooperative. The K0.5 forCa2+ of the present channel is 25 lm, while the K0.5 values measuredfor the 210- and the 14-pS channels previously reported were 53 and69 lm, respectively. On the other hand, the K0.5 value for the Ca2+-dependent K+ channel studied herein is similar to that estimated for theCa2+-dependent Cl– transduction channel, 5–26 lm (Kleene &Gasteland, 1991; Hallani et al., 1998). Calcium within the cilianormally increases during odour stimulation due to its influx throughthe cyclic-nucleotide-gated channels (Nakamura & Gold, 1987).However, because the cells were kept in low (micromolar) externalCa2+ during the experiment and the cilia are devoid of internalmembrane systems that could store Ca2+, the only source of Ca2+ forthe activation of the K+ channels present in the membrane patch musthave been the pipette solution. We believe that the cyclic-nucleotide-gated channels of the patch allowed an influx of the divalent cation,but its unitary currents were not observed presumably due to Ca2+

blockade (Zufall & Firestein, 1993).It has been estimated, by means of Ca2+ indicators, that free luminal

Ca2+ concentration during odour responses increase from a restinglevel of 40 nm to 300 nm (Leinders-Zufall et al., 1998). Such valuesrepresent global estimates for the ciliary lumen, giving no informationabout the Ca2+ concentration in the proximity of the ciliary membrane.Single-channel measurements such as those reported here can be usedto obtain local Ca2+ concentrations near the membrane. The Ca2+

concentration that the channel is sensing is reflected in its Po and cantherefore be determined from the Po vs. [Ca2+] plot obtained frominside-out excised patches. For example, when the channel exhibits a

Odours activate Ca2+-dependent K+ channels in olfactory cilia 2979


Po of 0.1, the local Ca2+ concentration near the channel is � 26 lm(Fig. 3A). Ca2+ concentrations in the proximity of the channel mayreach at least � 100 lm at highest odourant levels, more than twoorders of magnitude higher than the global concentration valueestimated by fluorescence measurements (Leinders-Zufall et al.,1998). The values reported here are similar to those estimated forthe Ca2+ levels in the vicinity of Ca2+-dependent K+ channels incerebral smooth muscle cells (Perez et al., 2001), but smaller than inthe soma of hair cells (Roberts et al., 1990).The current recordings from on-cilium membrane patches exhibited

a fluctuating baseline during odourant exposure; such fluctuationsceased after excision. Although this is commonly observed in patch-clamp experiments, the baseline difference was particularly marked inthe present case. This may be partly due to the activity of cyclic-nucleotide-gated channels present in the patch, which would openduring the chemical stimulation even though their unitary currentscannot be resolved under our experimental conditions because ofdivalent blockade (Zufall & Firestein, 1993). Such channels shouldclose after excision because cAMP is no longer present.Although previous results and the evidence reported here argue in

favour of a role of the ciliary K+ conductance in odour inhibition, aninvolvement of this conductance in odour excitation cannot be ruleout. Depending on the real K+ concentration in the mucus surroundingthe cilia, which has not been determined with certainty, and themembrane potential of the cell at a given moment, this channel couldin principle have an inhibitory or an excitatory effect. If extracellularK+ were in the low millimolar range or below, a value that might beexpected in an external milieu (specially in a fresh water animal suchas Caudiverbera), it would give origin to a hyperpolarizing receptorpotential; in contrast, at higher concentrations it would generate anexcitatory receptor potential. Thus, this K+ channel may have one roleor the other, depending on the ionic composition of the mucus and onthe membrane potential.

Acknowledgements

We are indebted to Cecilia Vergara for critical reading of the manuscript.Supported by MIDEPLAN ICM P99-031-F and Fondecyt 1020964.

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Cilium-attached and excised patch-clamp recordings of odourant-activated Ca-dependent K channels from chemosensory cilia of olfactory receptor neurons