| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Millennium Institute for Advanced Studies in Cell Biology and Biotechnology, University of Chile, Santiago, Chile; 2 Department of Biology, Faculty of Sciences, University of Chile, Santiago, Chile; 3 Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, Santiago, Chile
Submitted 24 December 2002; accepted in final form 28 May 2003
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
|---|
; Kleene and Gesteland 1991; Kurahashi and Yau 1993; Lowe and Gold 1993; Nakamura and Gold 1987). Both transduction channels as well as all other transduction proteins so far identified localize to the cilia (Buck and Axel 1991; Jones and Reed 1989; Menco et al. 1992; Pace et al. 1985). The ionic currents through the CNG and the ClCa conductances both contribute to generate a depolarizing receptor potential, responsible for increasing the action potential rate above the spontaneous rate (see Schild and Restrepo 1998).
Odorants can also activate inhibitory responses in olfactory receptor neurons (ORNs), consisting of decreases in their spontaneous spiking activity. Inhibitory responses have been observed in animal species whose ORNs exhibit a relatively high rate of spontaneous action potentials (Dionne 1992; Kang and Caprio 1995; Maue and Dionne 1987; Morales et al. 1994; see Bacigalupo et al. 1997; Getchell 1986). Inhibitory chemotransduction has been proposed to involve a KCa conductance, given that a charybdotoxin (CTx)-sensitive odor-induced KCa current was found both in toad (Caudiverbera caudiverbera) and rat ORNs in whole cell voltage-clamp experiments (Morales et al. 1994, 1995; Sanhueza et al. 2000). This K+ current generates a hyperpolarizing receptor potential, causing the spiking rate to decrease. If KCa channels indeed participate in odor inhibition, they are expected to be present in the chemosensory cilia, along with the other transduction channels. Experiments with focal odorant stimulation provided indirect evidence supporting the localization of these putative transduction K+ channels to the chemosensory cilia (Morales et al. 1997). A survey of the ion channels present in a purified preparation of toad olfactory cilia incorporated into planar lipid bilayers lent additional support to the presence of KCa channels in this compartment (Jorquera et al. 1995). However, this technique does not rule out possible contamination with membranes from other sources and therefore the possibility that the observed K+ channels were not from the cilia. Definitive evidence demonstrating the presence of KCa channels in the cilia is crucial to substantiate a role of such channels in odor transduction. We addressed this issue by means of immunochemical and electrophysiological approaches, to examine directly whether KCa channels are present in the chemosensory cilia.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
|---|
TOAD. Adult C. caudiverbera were cooled in ice, killed, and pithed before dissecting out their olfactory epithelia. Epithelia were cut into small pieces (about 1 mm2) and stored 
24 h at 4°C in a solution containing (in mM): 120 NaCl, 1 CaCl2, 2 MgCl2, 3 KCl, 5 glucose, 10 HEPES, 5 Na-pyruvate, 0.1 IU/ml penicillin, pH 7.5. Dissociated cells were obtained by gently passing the pieces of epithelia through the tip of a fire-polished Pasteur pipette and were then transferred to the experimental chamber containing Ringer solution (in mM): 115 NaCl, 1 CaCl2, 1.5 MgCl2, 2.5 KCl, 3 glucose, 10 HEPES, pH 7.6. RAT. Adult Wistar rats were anesthetized by CO2 inhalation and decapitated. The head capsule was opened by a sagittal section and the olfactory epithelium was removed from the dorsal posterior part of the nasal septum and from the turbinates. The epithelium was cut into pieces of 1 mm2, stored in Leibovitz L-15 medium at 4°C and used only on the day of the preparation. Dissociation was achieved by trituration through a fire-polished glass pipette, without the use of enzymes.
Preparation of ciliary membranes
We modified the protocols utilized by Jorquera et al. (1995) and by Schandar et al. (1998). Six to 8 rats were used each time; after extraction the olfactory epithelia were incubated in normal saline containing (in mM): 120 NaCl, 5 KCl, 1.6 K2HPO4, 25 NaHCO3, 7.5 glucose, pH 7.4, with the following protease inhibitors (in µg/ml): 10 aprotinin A, 10 leupeptin, 10 pepstatin, 0.5 benzamidine, 0.1 PMSF. Once the dissection procedure was finished, the tissues were transferred to a high-Ca2+ saline for deciliation (in mM): 112 NaCl, 3.4 KCl, 10 CaCl2, 2.4 NaHCO3, 2 HEPES, pH 7.0, plus protease inhibitors as in the normal saline; they were gently shaken in this solution for 10 min at 4°C. The ciliary membranes were then segregated from the rest of the epithelial material by centrifugation at 7,700 g for 5 min; the supernatant was loaded on top of a 45% sucrose solution in high-Ca2+ saline solution and centrifuged for 1 h at 100,000 g. The band in the sucrose–supernatant interface was extracted, diluted 
10-fold, and centrifuged for 1 h at 100,000 g. The pellet was resuspended in normal saline solution adjusted to pH 7.0 and supplemented with 2.0 mM EGTA and 0.5 mM sucrose.
Immunochemistry
WESTERN BLOT ANALYSIS. Purified olfactory cilia membranes were separated in 9% SDS–PAGE gels, electrotransferred (Mini-Trans Blot System; Bio-Rad, Hercules, CA) to nitrocellulose membranes (Hybond ECL; Amersham-Pharmacia, Piscataway, NJ), which were incubated with 5% nonfat milk in phosphate-buffered saline (PBS) with 0.05% Tween 20 (PBS-T) for 1 h at room temperature to block unspecific sites. They were afterward incubated with anti-BKCa channel polyclonal antibody (1:350 in 5% nonfat milk PBS-T) kindly donated by L. Toro (UCLA, School of Medicine, Dept. of Anesthesiology, Los Angeles, CA) or a commercial one (Alomone Labs, Jerusalem, Israel), both giving similar results, or with anti-adenylyl cyclase type III antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA). The gels were then developed using a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch Labs, West Grove, PA) and a chemiluminescence assay (ECL Lumigen PS-3 Detection Reagent; Amersham-Pharmacia).
The anti-BKCa channel antibody is directed against residues 883–896 of the human BKCa channel 
-subunit (part of the hydrophilic segment between S9 and S10, which is highly conserved across species), GenBank accession number U11058
[GenBank]
(Song et al. 1999).
For immunocytochemistry in isolated olfactory neurons, small pieces of olfactory epithelium (about 1 mm2) cells were transferred to 4% paraformaldehyde in PBS at 20°C and immediately triturated using a polished Pasteur pipette. Cells were adhered to Pegotin-covered microscope slides and allowed to dry. After rehydration in PBS with 0.2% Triton-X 100 for 10 min, the cells were blocked with 5% BSA for 10 min and incubated in the primary anti-BKCa antiserum diluted 1:100 for 4 h at 20°C. After being washed, cells were incubated with the secondary FITC-coupled antibody diluted 1:300 in PBS with 0.2% Triton-X 100 for 1 h at 20°C, washed, and stored in fluorescence mounting medium (Dako, Carpinteria, CA). Images were taken with a confocal scanning microscope (Carl Zeiss, model Axiovert 135M, Jena, Germany).
Electrical recordings and stimulus application
Electrical recordings were obtained from inside-out patches using the patch-clamp technique, for which we used an Axopatch 1D amplifier (Axon Instruments, Union City, CA). Experimental protocols and data analysis were conducted using pClamp 6.0 (Axon Instruments). Recording pipettes were made from borosilicate glass capillaries with filament (Hilgenberg-GmbH, Postfach, Germany) and drawn with a P 80/PC horizontal puller (Sutter Instruments, Novato, CA) to a tip resistance of 40–50 M
. Pipette solution (in mM): 110 K-acetate, 10 KCl, 1 MgCl2, 1 CaCl2, 10 HEPES, pH 7.6. When indicated, K+ was replaced with Na+ in this solution. The bath solution was exchanged by perfusing the whole chamber (6 chamber volumes in about 30 s). The bath pseudo-intracellular solution used for excised patch experiments contained (in mM): 110 K-acetate, 10 KCl, 1 MgCl2, 10 HEPES, pH 7.6; for adjusting the pCa to values of 6.0 or higher, 1 mM EGTA and CaCl2 were added according to Winmaxc 2 software (http://www.stanford.edu/~cpatton/winmaxc2.html). For lower pCa, EGTA was omitted and Ca2+ was added to the desired final concentration.
A photograph of each ORN recorded was taken before the patch was excised.
REAGENTS. All reagents were purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise indicated.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
|---|
We investigated the localization of the KCa channels involved in inhibitory transduction by Western blots and immunochemistry. Using a specific antibody against human large conductance KCa channels, we examined the distribution of a homologous channel in rat ORNs. Exposure of a membrane preparation of purified rat olfactory cilia to the antibody (Fig. 1A) revealed a reactive protein of about 116 kDa. This band was occasionally observed in Western blots of deciliated epithelium, probably reflecting the presence of a KCa channel in the soma and dendrite of ORNs (see Fig. 2) (Delgado and Labarca 1993; Lynch and Barry 1991). The band was also present in the positive control, a preparation of brain membranes (Fig. 1A), whereas no label was observed in the cilia after preincubation of the antibody with the antigen peptide (Fig. 1B). The purity of the ciliary preparation was tested with an antibody against adenylyl cyclase type III, an enzyme that localizes to the olfactory cilia (Pfeuffer et al. 1989). The antibody marked a single band in the ciliary fraction (Fig. 1C), which was absent in the deciliated tissue fraction (Fig. 1C). In addition, an antibody against the voltage-dependent Na+ channel (Sigma-Aldrich) did not label the ciliary band, whereas it did label the deciliated epithelium (not shown), indicating that contamination of the ciliary membranes preparation with other membrane was undetectable with our methodology.
|
|
To further examine the presence of KCa channels in the chemosensory cilia, we conducted immunocytochemical experiments using anti-BK antibody as in the Western blots. These experiments were done both in rat and toad because previous electrophysiological evidence suggested the presence of the odor-induced KCa conductance in both species (Sanhueza et al. 2000). Isolated ORNs exposed to the antibody revealed the presence of KCa channels in the ciliary region in both species (Fig. 2, A–C for rat, D–F for toad); the evidence is especially clear in the toad, whose cilia are considerably larger than those in the rat. ORNs exhibit some labeling in their cell bodies and dendrites, where the presence of KCa channels is well established (Delgado and Labarca 1993; Maue and Dionne 1987).
Single-channel Ca2+-dependent K+ current recordings from olfactory cilia
Although the abovementioned results strongly support the existence of ciliary KCa channels, we considered that definitive evidence for the presence of such channels in the cilia could be provided only by direct patch-clamp recording from these chemosensory structures. Considering the miniscule dimensions of the cilia, we carried out these experiments in toad olfactory neurons, where the cilia have a larger size and are more resistant against rupture during dissociation than in mammalian ORNs. However, in spite of the more favorable features of the amphibian cilia for conducting these experiments, they were still extremely difficult. Inside-out membrane patches were excised from the cilia, allowing the exposure of their luminal face to solutions containing different Ca2+ concentrations. Figure 3A shows a photograph of the pipette sealed on a cilium before excision. Figure 3B illustrates unitary channel currents under various Ca2+ concentrations. The ciliary membrane patch remained silent in 0.1 µM free Ca2+. Elevation of Ca2+ to 50 µM activated the channel, which was further activated on increasing Ca2+ to 100 µM. The channel closed on returning back to the 0.1 µM Ca2+ solution. Figure 3C displays the channel open probability (Po) as a function of [Ca2+]; the curve determines a K0.5 of approximately 50 µM. The channel is strongly voltage dependent, as depicted in the recordings of Fig. 3D, where Po increased from 0.03 to 0.38 as the transmembrane potential was raised from –10 to +40 mV (under 50 µM Ca2+). The I–V relation (Fig. 3E) yields a slope conductance of approximately 210 pS (obtained under 50 µM Ca2+), in agreement with large conductance Ca2+-dependent K+ channels (Vergara et al. 1998). Substituting Na+ for K+ in the bath caused a shift of the I–V curve that indicates a K+-selective channel.
|
In addition to the 210-pS channel, we observed 3 other KCa channel types. One of them is depicted in Fig. 4A. This channel was exposed to 0.1, 10, and 50 µM Ca2+ (at –40 mV), displaying Po values of 0, 0.05, and 0.97, respectively. Its K0.5 for Ca2+ is in the low micromolar range and its unitary conductance is approximately 62 pS (60 and 64 pS, from 2 patches), as determined from the slope of the I–V curve (Fig. 4B, circles). Replacement of K+ with Na+ in the bath solution shifted the curve by about 80 mV in the direction predicted for a K+-selective channel (Fig. 4B, triangles). The third KCa channel species found in the olfactory cilia is presented in Fig. 4C, under conditions in which the pipette contained Na+ solution and the bath K+ solution. The channel is shown under 0.1, 40, and 100 µM Ca2+, at +20 mV transmembrane potential. The unit conductance of this channel is about 12 pS (10 and 14 pS, from 2 patches; Fig. 4D) and its K for Ca2+ 0.5 is about 53 µM (Fig. 4E). A fourth channel type also exhibits a marked Ca2+ dependency (Fig. 4F). This channel has 2 open conductance states. This is indicated by the observation (made in the 2 patches examined) that transitions between the 2 open levels were observed, whereas events with magnitudes equal to the sum of the 2 open levels were absent, as would be expected if 2 independent channels were present (Fig. 4G). In addition, the amplitude histogram (built from the same recording as in G, but from a longer time period) clearly resolves the 2 conductance levels (Fig. 4I). The unitary conductance values determined by the slope of the I–V curves (Fig. 4H) were approximately 29 and 60 pS (27 and 58 pS, and 31 and 61 pS, from 2 patches). The kinetics of this channel is clearly different from that of the 60-pS channel in Fig. 4A, as illustrated by the fact that the former channel reaches a Po value close to 1 under 50 µM Ca2+ (Vm = –40 mV), whereas the Po of the latter channel (Fig. 4F) is only 0.27 at 100 µM Ca2+ (Vm = –60 mV). In addition, the average open time of the single-open state 60-pS channel is considerably longer (27.9 ± 8.4 ms, mean ± SE) than that of the two-open states of the other channel (7.8 ± 1.6 and 3.5 ± 0.5 ms for the 29-pS and the 60-pS states, respectively; same conditions as for the Po measurements). In all 4 cases, the recording ionic conditions indicate that the only ion that could account for the observed single-channel currents was potassium.
|
The electrophysiological data from ciliary membrane patches are in agreement with our immunochemical results and confirm the presence of KCa channels in the ciliary membrane. Altogether, the evidence presented is consistent with a role of such channels in olfactory transduction.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
|---|
). The major contribution of the present study is the demonstration that toad olfactory cilia contain Ca2+-dependent K+ channels. We arrived at this conclusion by 3 separate experimental approaches, all of which allowed us to directly test for the presence of K+ channel proteins in the cilia.
Earlier electrophysiological studies revealed the existence of an odorant-induced Ca2+-dependent K+ conductance in Caudiverbera. Furthermore, it was shown that in the neurons presenting such conductance the same odorant stimulus induced a membrane hyperpolarization, which was proposed to generate the reduction in the spike rate associated with odor inhibition (Morales et al. 1994, 1995). A similar odor-induced inhibitory effect was reported in the newt Necturus as well as in Xenopus (Dionne 1992; Vogler and Schild 1999), although the underlying ionic conductances were not determined in either case. The odor-dependent KCa current has been studied in some detail by macroscopic current measurements. It depends on the influx of Ca2+ through an unidentified conductance. Its Ca2+ dependency and current–voltage relation, together with its sensitivity to CTx (Morales et al. 1995; Sanhueza et al. 2000) are consistent with a Ca2+-dependent K+ conductance, although CTx also blocks some other K+ channels. This conductance is also sensitive to iberiotoxin (IbTx) (Madrid and Bacigalupo, unpublished observations), a specific blocker for Ca2+-dependent K+ channels of the large conductance type (BKCa) (Vergara et al. 1998). However, a definitive identification of the odor-dependent KCa channels requires an approach involving a study of these channels directly in the cilia.
Planar lipid bilayer studies in purified chemosensory membranes suggested the presence of a BKCa channel in Caudiverbera olfactory cilia (Jorquera et al. 1995). This channel exhibited a comparable slope conductance (240 pS, under symmetrical 200 mM KCl) as the 210-pS channel hereby reported. It also has a CTx sensitivity similar to the odor-dependent KCa conductance (Sanhueza et al. 2000), although IbTx was not tested in the bilayer experiments. However, BKCa in bilayers exhibited some differences with the BKCa recorded in excised patches: the former channel displays much longer open states at high [Ca2+] and a higher maximal Po (about 1) than the channel in excised patches (Po = about 0.38). These dissimilarities may be consequences of the distinct experimental conditions used in both cases; it is also conceivable that the patches may retain relevant regulatory factors or peptides as opposed to the channels of the purified ciliary membrane preparation.
It seems most likely that the protein labeled by the anti-BKCa antibody corresponds to the 210-pS channel observed in excised patches rather than to the 3 smaller conductance channels because of its biophysical resemblance to BKCa channels. If this is the case, our Western blot and immunochemical results indicate that this channel is present in the chemosensory cilia (Figs. 1 and 2); they indicate that it is also located to the dendrite and cell body. This would not be surprising considering that it is well established that ORN cell bodies of all species examined so far, including Caudiverbera and rat, possess a Ca2+-dependent K+ conductance (see Schild and Restrepo 1998). A previous single-channel study conducted in mouse ORN somata and dendrites showed the existence of V-independent 80-pS and V-dependent 130-pS KCa channels, but no indication of the presence of BKCa outside the cilia (Maue and Dionne 1987). Further work is necessary to establish whether the labeled proteins in the nonciliary membrane represent BKCa that have not yet been detected or KCa channels with a common epitope with the ciliary BKCa channel.
Both the 12- and the 60-pS ciliary KCa channels reported in the present work are insensitive to the membrane potential. This characteristic and their unitary conductance values suggest that they belong to the small and intermediate conductance KCa channels, respectively (Jensen et al. 2001; Vergara et al. 1998). Pharmacological data are needed to examine their identity further.
Calcium sensitivity of large conductance KCa channels varies widely, ranging from submicromolar to 100 µM levels (Latorre et al. 2000). Our data indicate K0.5 values in the range of about 1 to 69 µM Ca2+ for the ciliary KCa channel types that we found. This range is higher than the submicromolar K0.5 values reported for the small KCa (Vergara et al. 1998) and apparently somewhat higher than the KCa of intermediate conductance, which is in the low micromolar range (Simoes et al. 2002). The K0.5 value of the frog olfactory cilia Ca2+-dependent Cl– (ClCa) channel has been estimated to range between 5 and 26 µM (Hallani et al. 1998; Kleene and Gesteland 1991). Estimations of resting Ca2+ concentration in the cilia, based on fluorescence measurements, gave a value of about 40 nM, which would increase to a few hundred nanomolar during odorant stimulation (Leinders-Zufall et al. 1998). A close look at the fluorescence Ca2+ measurements during odorant exposure suggests that Ca2+ levels are not uniform along the cilia (Leinders-Zufall et al. 1998), although further and finer measurements are required to verify this highly relevant issue. It is conceivable that Ca2+ may reach much higher concentrations in microdomains in the vicinity of the CNG transduction channels within an olfactory cilium, decaying with distance attributed to the powerful extrusion of Ca2+ by the Na+/Ca2+ exchange system that operates in the olfactory cilia (Reisert and Matthews 1998). In this manner, all 4 KCa channels and the ClCa channel may participate in the transduction events that take place in the cilia, the K+ channel presumably in the inhibitory response and the Cl– channel in the excitatory response. If this is the case, the manner by which the cell manages to discriminate which channels, KCa or ClCa, will open on stimulation by a particular odorant is a fascinating open question.
Alternative interpretations regarding the function of the ciliary KCa cannot be ruled out at the moment. There are conflicting data about the K+ concentration surrounding the cilia, thought to be higher than that in the milieu bathing ORN basolateral membrane in the olfactory epithelium (see Schild and Restrepo 1998). Measurements are complicated because a slight damage to the epithelial cells would sharply raise extraciliary K+ levels. If external K+ were only a few times higher than that in the Ringer solution (2.5 mM), K+ reversal potential across the ciliary membrane would be more positive than resting potential and the opening of K+ channels would depolarize the cell, causing excitation. However, although the cell is depolarized during an ongoing excitatory stimulus, opening of the K+ channels induced by an inhibitory chemical stimulus would tend to reduce spiking in an isolated ORN, thus inhibiting it. It is of interest that an injection of only –1 pA (hyperpolarizing) current to a spontaneously spiking ORN clearly reduces its spiking rate (Bacigalupo et al., unpublished observation), suggesting that the ciliary KCa current would effectively inhibit the cell at membrane potentials near rest if extraciliary K+ is in the low millimolar range.
Our demonstration of the presence of KCa channels in olfactory cilia, together with prior electrophysiological whole cell results, strongly supports a role of these channels in inhibitory chemotransduction, as previously suggested (Morales et al. 1994; Sanhueza et al. 2000). At least 4 different channel species appear to exist in toad olfactory cilia. It seems likely that such channels are responsible for the odorant-induced inhibitory current, although our experiments do not allow us to determine the relative contribution of each channel type to the total inhibitory K+ current. The fact that chemosensory cilia of olfactory receptor neurons of phylogenetically distant vertebrates, such as the toad and the rat, contain KCa channels suggests a conserved function of these channels in olfactory neurons.
|
DISCLOSURES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
|---|
|
FOOTNOTES |
|---|
Address for reprint requests and other correspondence: J. Bacigalupo, Millennium Institute for Advanced Studies in Cell Biology and Biotechnology, University of Chile, P.O. Box 653, Santiago, Chile (E-mail: bacigalu{at}uchile.cl).
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
|---|
Buck LB and Axel R. A novel multigene family may encode odorant receptors: a molecular basis for odor recognition. Cell 65: 175–187, 1991.[Web of Science][Medline]
Delgado R and Labarca P. Properties of whole cell currents in isolated olfactory neurons from the Chilean toad Caudiverbera caudiverbera. Am J Physiol Cell Physiol 264: C1418–C1427, 1993.
Dionne VE. Chemosensory responses in isolated receptor neurons from Necturus maculosus. J Gen Physiol 99: 415–433, 1992.
Firestein S, Zufall F, and Shepherd GM. Single odor-sensitive channels in olfactory receptor neurons are also gated by cyclic nucleotides. J Neurosci 11: 3565–3572, 1991.[Abstract]
Getchell TV. Functional properties of vertebrate olfactory receptor neurons. Physiol Rev 66: 772–818, 1986.
Hallani M, Lynch JW, and Barry PH. Characterization of the calcium-activated chloride channels in patches excised from the dendritic knob of mammalian olfactory receptor neurones. J Membr Biol 161: 163–171, 1998.[Web of Science][Medline]
Jensen BS, Strøbaek D, Olesen SP, and Christophersen P. The Ca2+-activated K+ channel of intermediate conductance: a molecular target for novel treatments. Curr Drug Targets 2: 401–422, 2001.[Web of Science][Medline]
Jones DT and Reed RR. Golf: an olfactory neuron specific G protein involved in odorant signal transduction. Science 244: 790–795, 1989.
Jorquera O, Latorre R, and Labarca P. Ion channel classes in purified olfactory cilia membranes: planar lipid bilayer studies. Am J Physiol Cell Physiol 269: C1235–C1244, 1995.
Kang J and Caprio J. In vivo responses of single olfactory receptor neurons in the channel catfish, Ictalurus punctatus. J Neurophysiol 73: 172–177, 1995.
Kleene SJ and Gasteland RC. Calcium-activated chloride conductance in frog olfactory cilia. J Neurosci 11: 3624–3629, 1991.[Abstract]
Kurahashi T and Yau K-W. Coexistence of cation and chloride components in odorant-induced current of vertebrate olfactory receptor cells. Nature 363: 71–74, 1993.[Medline]
Latorre R, Vergara C, Alvarez O, Stefani E, and Toro L. Voltage-gated calcium-modulated potassium channels of large unitary conductance: structure, diversity and pharmacology. In: Handbook of Experimental Pharmacology. Pharmacology of Ion Channel Functions: Activators and Inhibitors, edited by Endo M. Heidelberg, Germany: Springer-Verlag, 2000, vol. 147, chapt. 8, p. 197–223.
Leinders-Zufall T, Greer CA, Shepherd GM, and Zufall FF. Imaging odor-induced calcium transients in single olfactory cilia: specificity of activation and role in transduction. J Neurosci 18: 5630–5639, 1998.
Lowe G and Gold GH. Nonlinear amplification by calcium-dependent chloride channels in olfactory receptor cells. Nature 366: 283–286, 1993.[Medline]
Lynch JW and Barry PH. Slowly activating K+ currents in rat olfactory receptor neurons. Proc R Soc Lond B Biol Sci 244: 219–225, 1991.[Medline]
Maue RA and Dionne VE. Patch-clamp studies of isolated mouse olfactory receptor neurons. J Gen Physiol 90: 95–125, 1987.
Menco BPM, Bruch RC, Dau B, and Danho W. Ultra-structural localization of olfactory transduction components: the G protein subunit Golf and type III adenylyl cyclase. Neuron 8: 441–453, 1992.[Web of Science][Medline]
Morales B, Labarca P, and Bacigalupo J. A ciliary K+ conductance sensitive to charybdotoxin underlies inhibitory responses in toad olfactory receptor neurons. FEBS Lett 359: 41–44, 1995.[Web of Science][Medline]
Morales B, Madrid R, and Bacigalupo J. Calcium mediates the activation of the inhibitory current induced by odorants in toad olfactory receptor neurons. FEBS Lett 402: 259–264, 1997.[Web of Science][Medline]
Morales B, Ugarte G, Labarca P, and Bacigalupo J. Inhibitory K+ current activated by odorants in toad olfactory neurons. Proc R Soc Lond B Biol Sci 257: 235–242, 1994.[Medline]
Nakamura T and Gold GH. A cyclic-nucleotide gated conductance in olfactory-receptor cilia. Nature 325: 442–444, 1987.[Medline]
Pace U, Hanski E, Salomon Y, and Lancet D. Odorant-sensitive adenylate cyclase may mediate olfactory reception. Nature 316: 255–258, 1985.[Medline]
Pfeuffer E, Mollner S, Lancet D, and Pfeuffer T. Olfactory adenylyl cyclase. Identification and purification of a novel enzyme form. J Biol Chem 264: 18803–18807, 1989.
Reisert J and Mathews HR. Na+ dependent Ca2+ extrusion governs response recovery in frog olfactory receptor cells. J Gen Physiol 112: 529–535, 1998.
Sanhueza M, Schmachtenberg O, and Bacigalupo J. Excitation, inhibition and suppression by odors in isolated toad and rat olfactory receptor neurons. J Am Physiol Cell Physiol 279: C31–C39, 2000.
Schandar M, Laugwitz Kl, Boekhoff I, Kroner C, Gudermann T, Schultz G, and Breer H. Odorants selectively activate distinct G protein subtypes in olfactory cilia. J Biol Chem 273: 16669–16677, 1998.
Schild D and Restrepo D. Transduction mechanisms in vertebrate olfactory receptor cells. Physiol Rev 78: 429–466, 1998.
Simoes M, Garneau L, Klein H, Banderali U, Hobeila F, and Roux B. Cysteine mutagenesis and computer modelling of the S6 region of an intermediate conductance IKCa channel. J Gen Physiol 120: 99–116, 2002.
Song M, Zhu N, Olcese R, Barila B, Toro L, and Stefani E. Hormonal control of protein expression and mRNA levels of the MaxiK channel subunit in myometrium. FEBS Lett 460: 427–432, 1999.[Web of Science][Medline]
Vergara C, Latorre R, Marrion NV, and Adelman JP. Calcium-activated potassium channels. Curr Opin Neurobiol 8: 321–329, 1998.[Web of Science][Medline]
Vogler C and Schild D. Inhibitory and excitatory responses of olfactory receptor neurons of Xenopus to stimulation with amino acids. J Exp Biol 202: 997–1003, 1999.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  S. J. Kleene The Electrochemical Basis of Odor Transduction in Vertebrate Olfactory Cilia Chem Senses, November 1, 2008; 33(9): 839 - 859. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Zhang, C. Yang, and R. J. Delay Urine Stimulation Activates BK Channels in Mouse Vomeronasal Neurons J Neurophysiol, October 1, 2008; 100(4): 1824 - 1834. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Mayer, N. Ungerer, D. Klimmeck, U. Warnken, M. Schnolzer, S. Frings, and F. Mohrlen Proteomic Analysis of a Membrane Preparation from Rat Olfactory Sensory Cilia Chem Senses, February 1, 2008; 33(2): 145 - 162. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Ukhanov, T. Leinders-Zufall, and F. Zufall Patch-Clamp Analysis of Gene-Targeted Vomeronasal Neurons Expressing a Defined V1r or V2r Receptor: Ionic Mechanisms Underlying Persistent Firing J Neurophysiol, October 1, 2007; 98(4): 2357 - 2369. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T.-Y. Chen, H. Takeuchi, and T. Kurahashi Odorant Inhibition of the Olfactory Cyclic Nucleotide-gated Channel with a Native Molecular Assembly J. Gen. Physiol., August 28, 2006; 128(3): 365 - 371. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. K. Raychowdhury, M. McLaughlin, A. J. Ramos, N. Montalbetti, R. Bouley, D. A. Ausiello, and H. F. Cantiello Characterization of Single Channel Currents from Primary Cilia of Renal Epithelial Cells J. Biol. Chem., October 14, 2005; 280(41): 34718 - 34722. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Madrid, R. Delgado, and J. Bacigalupo Cyclic AMP Cascade Mediates the Inhibitory Odor Response of Isolated Toad Olfactory Receptor Neurons J Neurophysiol, September 1, 2005; 94(3): 1781 - 1788. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
), as in D, and after substituting Na+ for K+ in bath (
) (seeMETHODS). Slope conductance: 210 pS; each data point represents measurements of 20 individual events at each potential value, corresponding error bars to SE (n = 1 patch). Voltage polarities: –, hyperpolarizing potential; +, depolarizing potential (opposite to applied pipette polarities).