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1Biology, Animal Physiology, Philipps-University of Marburg, Marburg; and 2Institute of Biology, Animal Physiology, University of Kassel, Kassel, Germany
Submitted 28 December 2008; accepted in final form 3 August 2008
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ABSTRACT |
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INTRODUCTION |
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). Accordingly, pheromone detection in moths is one of the best-studied models of how olfactory systems transduce odor information (Hansson 2002; Hildebrand 1995; Kaissling 2004; Rospars et al. 2007). Previous biochemical (Boekhoff et al. 1990; Breer et al. 1990), electrophysiological (Stengl 1993, 1994; Stengl et al. 1992; Wegener et al. 1997; Zufall and Hatt 1991; Zufall et al. 1991), and molecular genetic studies (Jacquin-Joly et al. 2002; Riesgo-Escovar et al. 1995) extensively characterized the initial steps of the insect olfactory transduction cascade. It is largely unknown, however, how pheromones induce adaptation or sensitization of the insect olfactory transduction cascade. Available data demonstrate that pheromone stimuli induce slow and delayed increases in the cyclic guanosine monophosphate (cGMP) concentration in the olfactory receptor neurons (ORNs) of different moth species (Boekhoff et al. 1993; Ziegelberger et al. 1990). Tip recordings from trichoid sensilla of moths showed that cGMP reduces the action potential frequency on pheromone stimulation and thus may trigger long-term adaptation (Flecke et al. 2006; Redkozubov 2000). The increase of cGMP after adapting pheromone stimuli appears to activate protein kinase G (Boekhoff et al. 1993). In single-channel recordings of Manduca sexta ORNs, millimolar concentrations of cGMP as well as cyclic adenosine monophosphate (cAMP) inhibited a pheromone-activated delayed rectifier potassium channel (Stengl et al. 1992; Zufall et al. 1991). Taken together, these findings suggest that cyclic nucleotides modulate olfactory transduction in moths.
In vertebrates, cyclic nucleotide-gated (CNG) channels have been extensively studied (Hofmann et al. 2005; Kaupp and Seifert 2002; Pifferi et al. 2006). Vertebrate olfactory CNG channels form heterotetrameric complexes composed of two principal CNGA2 subunits, a modulatory CNGA4, and a modulatory CNGB1b subunit (Bonigk et al. 1999; Sautter et al. 1998; Shapiro and Zagotta 1998; Zheng and Zagotta 2004). The subunit composition of vertebrate olfactory CNG channels determines their functional features such as ligand sensitivity, ion selectivity, and gating properties (Bradley et al. 2005; Munger et al. 2001). Both cAMP and cGMP directly activate vertebrate olfactory CNG channels, which nonselectively conduct Ca2+ and monovalent cations (Dzeja et al. 1999; Frings et al. 1992, 1995). Since vertebrate olfactory CNG channels are negatively regulated by Ca2+/calmodulin (CaM), Ca2+ influx through CNG channels probably leads to adaptation (Bradley et al. 2001; Chen and Yau 1994; Munger et al. 2001; Zufall and Leinders-Zufall 2000).
In insects, CNG channels have been characterized in the olfactory system of Drosophila melanogaster (Baumann et al. 1994; Miyazu et al. 2000), Apis mellifera (Gisselmann et al. 2003), and Heliothis virescens (Krieger et al. 1999). Here, we describe cyclic nucleotide-activated currents in the ORNs of M. sexta, which are likely to play a role for olfactory adaptation and sensitization. In whole cell patch-clamp recordings, we found that cAMP and cGMP directly activated currents. Pharmacological and ion exchange experiments demonstrated that at least one cAMP- and two cGMP-activated nonselective cation currents occurred in the ORNs of M. sexta. The cGMP-activated currents carried more Ca2+ and were more strongly influenced by Ca2+/CaM-dependent negative feedback than the cAMP-activated current. Furthermore, the cGMP-activated currents differed in their Ca2+-dependent inhibition. Unlike cGMP, cAMP activated a previously undescribed Ca2+ current that resembled a protein kinase–modulated low-voltage–activated (LVA) Ca2+ current.
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METHODS |
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Insects
M. sexta (Johannson; Lepidoptera: Sphingidae) larvae were reared on an artificial diet (modified after Bell and Joachim 1976). Larvae were maintained under long-day photoperiod (L:D 17:7) at 24–27°C and 40–60% relative humidity. Pupae were staged using external markers (Jindra et al. 1997) and anesthetized by cooling until antennal flagella were dissected.
Primary cell cultures
The cell culture protocol was modified after Stengl and Hildebrand (1990). Briefly, antennae of male M. sexta pupae were dissected in Hanks’ balanced salt solution containing 1% penicillin–streptomycin solution (HBSS/PS). Antennae were incubated for 5 min at room temperature in HBSS/PS containing 1.3 mM EGTA, rinsed in HBSS/PS, and dissociated in two batches for 5 and 3 min at 37°C in HBSS/PS containing 24 mM papain. Dissociation was stopped with Leibovitz L-15 medium supplemented with 10% fetal bovine serum. The cell suspension was centrifuged at 90 to 110 relative centrifugal force for 5 min and pellets were resuspended in HBSS/PS. Dispersed cells were plated on glass-bottom culture dishes, which were coated with concanavalin A and poly-L-lysine, and allowed to settle for 30 min. Then, 1 ml of a 2:1 cell culture medium [two parts of Leibovitz L-15 medium supplemented with 10% fetal bovine serum and one part of Manduca embryonic cell line conditioned medium MRLL-CH1 (Eide et al. 1975) was added. The medium was completely replaced within 24 h after dispersion. Cell cultures were maintained at 20°C and used for electrophysiology from 10 to 
25 days (Stengl and Hildebrand 1990).
Solutions and reagents
Solutions contained reagents at doses commonly used for patch-clamp recordings (Stengl 1993, 1994; Zufall et al. 1991). All solutions were adjusted to pH 7.1–7.2 and 370–380 mOsm for extracellular solutions and 340 mOsm for pipette solution, respectively. During recordings, cells were kept in 2 ml extracellular solution. Standard extracellular solution contained (in mM): NaCl 156; KCl 4; CaCl2 6; glucose 5; and HEPES 10. To inhibit voltage-dependent sodium currents all extracellular solutions included 10–8 M tetrodotoxin. To investigate whether currents depend on the extracellular Ca2+ concentration, CaCl2 in the extracellular solution was either reduced to 10–5 or 10–7 M (buffered with EGTA) or substituted with 6 mM BaCl2. For simplicity, standard extracellular solution is referred to as "6Ca"; solutions with reduced calcium concentration as "low Ca"; and barium solution as "6Ba." To identify voltage-gated channels, extracellular solutions contained 6 mM NiCl2 (6Ni) or 1 mM ZnCl2 (1Zn). The Cl–-reduced extracellular solution (16 mM; "low Cl") was obtained by replacing NaCl with the sodium acetate salt. A gravity-feed perfusion system controlled the exchange of extracellular solutions at a low flow rate. The complete exchange of extracellular solution took <1 min. The ionic composition of the patch pipette solution was identical in all experiments (in mM): CsCl 160; CaCl2 1; EGTA 11; and HEPES 10. Cesium was used to prevent potassium-dependent outward currents.
Reagents were directly pipetted into the extracellular solution. At the beginning of each experiment, extracellular solution was directly pipetted onto the cells as a control. After a delay of 
2 min, cyclic nucleotides were applied. The membrane-permeant cAMP and cGMP analogues 8-bromo cAMP and 8-bromo cGMP were dissolved in extracellular solution and applied at final concentrations ranging from 5 nM to 50 µM (Supplemental Table S1).1 The calmodulin (CaM) antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and the nonspecific inhibitor of Ca2+-permeable cation channels lanthanum (La3+) were dissolved in water and applied at final concentrations of 10 to 50 µM for W-7 (Seno et al. 2005) and 0.05 to 1.1 mM for La3+. The protein kinase inhibitors staurosporine (dissolved in dimethyl sulfoxide) and H7 (dissolved in EtOH) were applied at concentrations of 1 µM for staurosporine and 10 µM for H7 (Stengl 1993). Cell cultures were incubated with the respective protein kinase inhibitor for 15 to 30 min before breaking into whole cell configuration and application of cyclic nucleotides.
Patch-clamp technique and data analysis
Patch-clamp recordings were performed in whole cell configuration at room temperature according to the conventional patch-clamp method (Hamill et al. 1981). Cell cultures were monitored at 600x on an inverted microscope (Axioscope 135; Zeiss, Göttingen, Germany) equipped with phase-contrast optics. ORNs were identified on the basis of their morphology (Stengl and Hildebrand 1990). Patch electrodes were pulled from thick borosilicate glass capillaries (GC150T-10; Clark Electromedical Instruments, Reading, UK) with a micropipette puller (Sutter P97; Sutter Instruments, Novato, CA). Fire-polished patch pipettes with a tip resistance of 2 to 8 M
when filled with pipette solution were used to obtain seals of several gigaohms on the cell membrane. Junction potential was nullified prior to seal formation and capacitance and series resistance of the patch pipette were compensated. For whole cell recordings, the membrane potential was clamped at –60 mV. After breaking into whole cell configuration and a delay of 2 min for the stabilization of outward currents, the experiment was started. Three consecutive voltage-ramp protocols from –100 to +100 mV, with 500 ms each, were applied to establish current–voltage (I–V) relations.
Data acquisition was carried out with an Axopatch 1D amplifier using a Digidata 1200B interface (Molecular Devices, Union City, CA). Data acquisition and analyses were performed with pClamp (version 9; Molecular Devices). Currents recorded during voltage protocols were sampled at 20 kHz and low-pass filtered at 2 kHz. A MiniDigi acquisition device (MiniDigi1A; Molecular Devices) was used to continuously sample currents at 1 kHz and to record the time of drug application on a second acquisition channel. The figures show representative traces corrected for leak currents. The mean current amplitudes were determined at –100 mV. All data were presented as means ± SE. Statistical significance (P < 0.05) was evaluated by means of the two-tailed Student's t-test. Increase of current amplitude was measured at ±100 mV and normalized to the maximum current amplitude obtained in control conditions. Inhibition of current amplitude was measured at ±100 mV and plotted as a percentage of the maximum current amplitude obtained under control conditions.
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RESULTS |
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To characterize cyclic nucleotide-activated currents, we stimulated M. sexta ORNs (n = 188) in whole cell patch-clamp recordings with the membrane-permeable cAMP and cGMP analogues 8-bromo cAMP and 8-bromo cGMP. Since a detailed analysis of all concentrations (5 nM to 50 µM) did not reveal statistically significant differences in the frequency of activation, current amplitude, reversal potential, and latency of activation over the total number of ORNs tested (Supplemental Table S1), we pooled the data.
Cyclic nucleotide application typically induced a stepwise increasing inward current with a delay of several seconds up to several minutes (cAMP 126 ± 9 s, n = 82 ORNs; cGMP 110 ± 10 s, n = 75 ORNs). The amplitude of the inward current typically reached a plateau and did not decline over the course of the recording (Fig. 1, A and B). Both cyclic nucleotides induced a current with a linear I–V relation in standard extracellular solution (6Ca; Tab1e 1; Fig. 1, C and D). The cAMP-activated current had a mean reversal potential of 0.2 ± 1.6 mV and a mean amplitude of 608 ± 114 pA (n = 49 of 56 ORNs; Fig. 1C). The cGMP-activated current did not significantly differ from the cAMP-activated current and had a mean reversal potential of 1.6 ± 1.2 mV and a mean amplitude of 918 ± 208 pA (n = 43 of 52 ORNs; Fig. 1D). Application of La3+ (Table 2), a nonspecific blocker of Ca2+-permeable cation channels, significantly inhibited both cyclic nucleotide-activated currents (P < 0.01; Fig. 1), i.e., 71.6 ± 3.5% of the cAMP-activated current (n = 41 of 49 ORNs) and 68.1 ± 2.6% of the cGMP-activated current (n = 45 of 50 ORNs), respectively. The La3+-induced current inhibition did not significantly differ between cAMP- and cGMP-activated currents. However, La3+ inhibited the inward current of both cyclic nucleotide-activated currents significantly more strongly than the outward current (P < 0.01). The remaining La3+-independent outwardly rectified current had a mean reversal potential of –5.4 ± 2 mV for cAMP- and –1.8 ± 1.5 mV for cGMP-activated currents.
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Cyclic nucleotides activate nonselective cation currents
To investigate whether cAMP- and cGMP-activated currents depend on the extracellular Ca2+ concentration, CaCl2 was reduced in the extracellular solution to either 10–5 or 10–7 M (low Ca) or substituted with 6 mM BaCl2 (6Ba; Table 1). Both cAMP and cGMP induced currents in low-Ca and 6Ba solutions. Subsequent La3+ application inhibited these currents. In low-Ca solution [see Fig. 4, A–C, traces (1)], cAMP induced a linear or inwardly rectified current with a mean reversal potential of –0.5 ± 4.3 mV and a mean amplitude of 1,001 ± 391 pA (n = 13 of 17 ORNs). Similarly, the cGMP-activated current showed a linear I–V relation, a mean reversal potential of 6.8 ± 4.9 mV, and a mean amplitude of 997 ± 280 pA (n = 13 of 16 ORNs). In 6Ba solution, both cyclic nucleotides induced currents with a linear I–V relation. The cAMP-activated current (Fig. 2A) had a mean reversal potential of –0.4 ± 1.6 mV and a mean amplitude of 749 ± 199 pA (n = 6 ORNs). The cGMP-activated current (Fig. 2B) had a mean reversal potential of –3.6 ± 3.1 mV and a mean amplitude of 1,054 ± 719 pA (n = 7 of 8 ORNs). The mean amplitudes or reversal potentials of the cAMP- and cGMP-activated currents did not significantly differ in the various extracellular solutions. In some experiments (n = 8 of 14 ORNs), 6Ba solution induced a small inward current through Ca2+ channels (Fig. 2B).
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Cs+ (1). Cyclic nucleotide-activated currents depend on the extracellular Ca2+ concentration
To investigate the Ca2+ dependence of cyclic nucleotide-activated currents, ORNs were exposed to different extracellular Ca2+ concentrations. In a first set of experiments, ORNs were initially kept in 6Ca solution, stimulated with cyclic nucleotides, and then perfused with low-Ca solution (10–5 M; Table 2). Both the cAMP-activated (n = 8 ORNs; Fig. 3A) and the cGMP-activated (n = 5 of 7 ORNs; Fig. 3B) currents significantly increased in low-Ca solution (P < 0.01; Fig. 3C). Subsequent La3+ application inhibited the cyclic nucleotide-activated currents (Fig. 3). After perfusion with low-Ca solution, both cyclic nucleotide-activated currents expressed a linear I–V relation with a constant mean reversal potential.
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Cyclic nucleotide-activated currents are modulated by Ca2+/CaM
To test whether Ca2+/CaM inhibits cyclic nucleotide-activated currents, the CaM antagonist W-7 was applied. Application of W-7 (10 to 50 µM) alone did not elicit a current response (n = 14 ORNs), but significantly increased the cyclic nucleotide-activated currents (Table 2). Application of W-7 induced an approximately twofold increase of the cAMP-activated current (P < 0.01; n = 8 of 12 ORNs; Fig. 5, A and C) and an almost tenfold increase of the cGMP-activated current (P < 0.05; n = 7 of 14 ORNs; Fig. 5, B and C). Thus W-7 increased the cGMP-activated current significantly more strongly than the cAMP-activated current (P < 0.05; Fig. 5C). Apart from the differing current amplitudes, the properties of the cyclic nucleotide-activated currents remained unchanged, i.e., the currents still showed a linear I–V relation, a mean reversal potential of –0.5 ± 1.7 mV for cAMP- and 1.2 ± 4.3 mV for cGMP-activated currents, and inhibition by La3+.
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Transition metals like Ni2+ and Zn2+ inhibit Ca2+ permeable channels. Thus to further characterize the Ca2+ dependence of cyclic nucleotide-activated currents, ORNs were kept in 6Ca solution, stimulated with cyclic nucleotides, and then exposed to solutions containing 6 mM NiCl2 (6Ni) or 1 mM ZnCl2 (1Zn; Table 2). Both the 6Ni and 1Zn solution significantly reduced the cAMP- and the cGMP-activated currents (P < 0.01). Perfusion with 6Ni solution inhibited 56 ± 7.3% of the cAMP-activated current (n = 7 of 13 ORNs, Fig. 6), whereas 1Zn solution inhibited 31.1 ± 5.4% (n = 5 of 7 ORNs; Fig. 6). Thus the 6Ni solution induced a significantly stronger current decline than the 1Zn solution (P < 0.05). The mean reversal potential of the outwardly rectified currents remained unchanged in both solutions. Similarly, perfusion with 6Ni solution inhibited 57.3 ± 8% of the cGMP-activated current (n = 9 of 14 ORNs, Fig. 6) and 1Zn solution inhibited 40.9 ± 7.6% (n = 6 ORNs; Fig. 6). In contrast to the cAMP-activated current, the cGMP-activated current appeared to be equally inhibited by the 6Ni and 1Zn solutions. The mean reversal potential of the outwardly rectified currents remained unchanged in both solutions. La3+ inhibited 61.5 ± 6.3% of the remaining cyclic nucleotide-activated currents in 6Ni solution (n = 13 of 16 ORNs) and 72.3 ± 7.1% in 1Zn solution (n = 5 of 6 ORNs; data not shown), but did not significantly change the mean reversal potential. In some experiments, however, perfusion with 6Ni solution increased cAMP- (n = 4 of 13 ORNs) or cGMP-activated currents (n = 3 of 14 ORNs; data not shown).
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To investigate whether Cl– currents constitute a fraction of the cyclic nucleotide-activated currents, ORNs were kept in 6Ca solution, stimulated with cyclic nucleotides, and then exposed to an extracellular solution with a reduced Cl– concentration (low Cl; Table 2). In 6Ca solution, cAMP-activated currents had a mean reversal potential of –0.2 ± 3 mV, which significantly shifted to –11.6 ± 4 mV in low-Cl solution (P < 0.05; n = 9 ORNs). Similarly, the mean reversal potential of cGMP-activated currents significantly shifted from 7.1 ± 8 mV in 6Ca solution to –14.3 ± 3.9 mV in low-Cl solution (P < 0.05; n = 7 ORNs). Perfusion with low-Cl solution typically increased both the cAMP- (n = 5 of 9 ORNs; Fig. 7A) and the cGMP-activated currents (n = 5 of 7 ORNs; Fig. 7B). Subsequent La3+ application inhibited 82.9 ± 4% of the cyclic nucleotide-activated currents in low-Cl solution (n = 10 ORNs; Fig. 7), but did not significantly change the mean reversal potential.
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To investigate whether cAMP- and cGMP-activated currents depend on protein kinases, ORNs were preincubated with the protein kinase inhibitors staurosporine (1 µM) or H7 (10 µM) for 15 min and then stimulated with cyclic nucleotides. Both protein kinase inhibitors did not prevent cAMP- and cGMP-activated nonselective cation currents (Table 1). In the presence of staurosporine, both cyclic nucleotide-activated currents showed a linear I–V relation (Fig. 8, A and B). The cAMP-activated current had a mean reversal potential of 5.7 ± 2.3 mV and a mean amplitude of 607 ± 343 pA (n = 7 of 8 ORNs; Fig. 8A). Comparably, the cGMP-activated current had a mean reversal potential of 8 ± 8.7 mV and a mean amplitude of 738 ± 253 pA (n = 6 of 9 ORNs; Fig. 8B). In the presence of H7, both cyclic nucleotides predominantly activated an inwardly rectified current (Fig. 8, C and D). The cAMP-activated current had a mean reversal potential of 5.7 ± 3.6 mV and a mean amplitude of 330 ± 243 pA (n = 6 of 9 ORNs; Fig. 8C). Similarly, the cGMP-activated current had a mean reversal potential of 5.3 ± 3.3 mV and a mean amplitude of 142 ± 55 pA (n = 6 of 7 ORNs; Fig. 8D). Subsequent La3+ application inhibited both cyclic nucleotide-activated currents.
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DISCUSSION |
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Moth ORNs appear to possess CNG channels
In M. sexta ORNs, cyclic nucleotide-activated currents have properties similar to those of currents through vertebrate olfactory CNG channels. Since cGMP has been shown to activate a 55-pS nonselective cation channel in single-channel recordings (Dolzer et al. 2008), our findings suggest that cyclic nucleotide-activated currents depend on prospective M. sexta cyclic nucleotide-gated (MsCNG) channels. Like vertebrate olfactory CNG channels (Hofmann et al. 2005; Kaupp and Seifert 2002; Pifferi et al. 2006), the prospective MsCNG channels are Ca2+ permeable nonselective cation channels that are directly activated by both cAMP and cGMP. Both prospective MsCNG channels and vertebrate olfactory CNG channels show weak voltage dependence and do not desensitize in the continuous presence of cyclic nucleotides. In addition, prospective MsCNG channels and vertebrate olfactory CNG channels are modulated by Ca2+/CaM. Like vertebrate olfactory CNG channels, the prospective MsCNG channels differ in ligand sensitivity, ion selectivity, and gating properties.
Unlike vertebrate olfactory CNG channels, however, prospective MsCNG channels did not change their response to varying concentrations of cAMP and cGMP. This lack of dose dependence was also found for cGMP-activated channels in single-channel recordings of M. sexta ORNs (Dolzer et al. 2008). Since both cAMP and cGMP typically induced a stepwise inward current, prospective MsCNG channels are probably coupled and act coordinately. Accordingly, in single-channel recordings of M. sexta ORNs, cGMP-dependent large-conductance events consisted of several simultaneous subconductance events in the open state (Dolzer et al. 2008). In the rat olfactory system, the principal CNG channel subunit CNGA2 localizes to lipid rafts (Brady et al. 2004) that facilitate the lateral assembly of signaling cascades (Simons and Toomre 2000) and thus likely coordinate gating. Compartmentalization of vertebrate olfactory CNG channels ensures rapid and efficient cyclic nucleotide signaling (Brady et al. 2004). Since prospective MsCNG channels appear to open in a concerted manner, several lipid rafts possibly congregate prospective MsCNG channels in the cell membrane of ORNs and act coordinately.
Prospective MsCNG channels differ in their Ca2+ dependence
Like homomeric olfactory CNG channels (Frings et al. 1995; Seifert et al. 1999), prospective MsCNG channels differ in their sensitivity to extracellular Ca2+. Increases of extracellular Ca2+ inhibited the cAMP-activated currents and one type of the cGMP-activated currents. This is in accordance with the finding that high extracellular Ca2+ concentrations generally inhibit monovalent cation currents and turn vertebrate olfactory CNG channels into "pure" Ca2+ channels (Dzeja et al. 1999; Frings et al. 1995). Importantly, Ca2+ significantly more strongly inhibited cGMP-activated currents than cAMP-activated currents. Since the molecular structure of the channel pore determines the Ca2+ permeability of vertebrate CNG channels (Eismann et al. 1994; Root and MacKinnon 1993; Seifert et al. 1999; Sesti et al. 1995), the prospective cAMP- and cGMP-dependent MsCNG channels are likely to differ in their pore region. Besides the inhibitory effect of Ca2+ on cAMP- and cGMP-activated currents, Ca2+ also increased the current through at least one additional cGMP-dependent prospective MsCNG channel. So far it is unknown which ORNs express which specific prospective MsCNG channel types. Since ORNs do not differ in their morphology in primary cell cultures (Stengl and Hildebrand 1990), we grouped ORNs into different subpopulations according to their electrophysiological properties. Because the increase of external Ca2+ induced opposite effects on cGMP-activated currents, at least two types of prospective cGMP-dependent MsCNG channels appear to be expressed in specific ORNs.
Prospective MsCNG channels differ in their Ca2+/CaM-dependent inhibition
Cyclic nucleotides do not directly inactivate vertebrate olfactory CNG channels, but instead induce adaptation of the respective channels via Ca2+/CaM-dependent negative feedback (Bradley et al. 2005; Chen and Yau 1994; Trudeau and Zagotta 2003). In M. sexta ORNs, the CaM antagonist W-7 increased the amplitude of cyclic nucleotide-activated currents. Thus Ca2+/CaM obviously provides negative feedback to these currents in the continuous presence of cyclic nucleotides. In the vertebrate olfactory system, native CNG channels are composed of two CNGA2, one CNGA4, and one CNGB1b subunit (Bonigk et al. 1999; Sautter et al. 1998; Shapiro and Zagotta 1998; Zheng and Zagotta 2004). The CNGA2 subunit contains a CaM binding site and controls gating (Liu et al. 1994), whereas the modulatory subunits CNGA4 and CNGB1b contain IQ-motifs that regulate the kinetics of Ca2+/CaM-mediated inhibition. Native CNG channels are rapidly inhibited by Ca2+/CaM. However, vertebrate olfactory CNG channels, which consist of only CNGA2 subunits or two differing subunits (CNGA2 + CNGA4/B1b), show a distinctively reduced current decline due to a reduced rate of Ca2+/CaM binding to the CNGA2 subunit (Bradley et al. 2001, 2004; Munger et al. 2001). Since W-7 induced a stronger activation of the cGMP- than the cAMP-activated currents in M. sexta ORNs, the corresponding prospective MsCNG channels may differ in their subunit composition.
Lanthanum and transition metals inhibit prospective MsCNG channels
Several studies used La3+ to inhibit Ca2+-permeable cation channels such as TRP channels (Clapham et al. 2005), Ca2+-activated chloride channels (Tokimasa and North 1996), or store-operated Ca2+ channels (ICRAC; Hoth and Penner 1993). In M. sexta ORNs, La3+ inhibited 83% of cyclic nucleotide-activated currents. Low-La3+ concentrations, however, also potentiated cyclic nucleotide-activated currents through prospective MsCNG channels. Because the structure of prospective MsCNG channels is unknown, and La3+ has not been used as an antagonist on CNG channels before, the dose-dependent effects of La3+ on prospective MsCNG channels remain unclear. Nevertheless, a comparable dose dependence of La3+ has been described for mouse TRPC5 channels. At the extracellular mouth of the TRPC5 channel pore, lanthanides bind to specific glutamate residues, which likely act as "gatekeepers" controlling cation entry (Jung et al. 2003). Remarkably, in M. sexta ORNs, low-La3+ concentrations appear to potentiate more cAMP- than cGMP-activated currents. Thus cAMP- and cGMP-dependent prospective MsCNG channels may differ in their channel pore composition.
Like La3+, transition metals such as Ni2+ and Zn2+ also alter the sensitivity of CNG channels (Gordon and Zagotta 1995a,b; Ildefonse et al. 1992; Karpen et al. 1993). Ni2+, for instance, inhibits the CNGA2 channel in the vertebrate olfactory system (Gordon and Zagotta 1995a). In M. sexta ORNs, both Ni2+ and Zn2+ inhibited cyclic nucleotide-activated currents. This corresponds to the finding that Zn2+ inhibited a cGMP-activated nonselective cation channel of M. sexta ORNs in single-channel recordings (Dolzer et al. 2008). Remarkably, La3+ inhibited the remaining part of the cyclic nucleotide-activated currents. This suggests that transition metals and La3+ bind to different sites of prospective MsCNG channels. However, La3+ typically did not completely inhibit cyclic nucleotide-activated currents. Since the reversal potentials of the cyclic nucleotide-activated currents did not significantly change on La3+ application, the remaining La3+-insensitive current could not be distinguished.
Prospective MsCNG channel permeability depends on the extracellular Cl– concentration
Cyclic nucleotides induce an intracellular Ca2+ increase in vertebrate ORNs that subsequently leads to the activation of Ca2+-activated Cl– channels (Frings et al. 2000). The secondary Cl– efflux forms a large fraction of the current through vertebrate olfactory CNG channels. When Cl– is replaced with larger anions, the corresponding current is eliminated (Kleene 1993). In M. sexta ORNs, however, the replacement of Cl– with acetate typically increased both cAMP- and cGMP-activated currents. Thus Cl– seems to inhibit monovalent cation flux through prospective MsCNG channels. Since the replacement of Cl– with acetate also significantly shifted the reversal potentials of the cyclic nucleotide-activated currents to more negative values, Cl– carries a fraction of the respective currents.
A cAMP-activated Ca2+ inward current depends on protein kinase activity
In the presence of protein kinase inhibitors, an additional cAMP-dependent Ca2+ inward current [ICa(cAMP)] often preceded the cAMP-activated nonselective cation current. In a few recordings, the ICa(cAMP) was not followed by cAMP-activated nonselective cation currents. In other recordings, the ICa(cAMP) was missing and cAMP activated only nonselective cation currents. Thus the ICa(cAMP) does not induce nonselective cation currents through prospective MsCNG channels. The ICa(cAMP) instead shows the typical characteristics of an LVA Ca2+ current (Nilius et al. 2006) with an activation threshold at –60 mV and a peak current at –20 mV. The ICa(cAMP) occurred only in the presence of protein kinase inhibitors on cAMP-, but not cGMP application. Thus the ICa(cAMP) likely depends on cAMP and protein kinase-mediated phosphorylation.
Conclusions
To our knowledge, there are only few reports on CNG channels in moths. In Heliothis virescens, a cAMP- and hyperpolarization-activated CNG channel localized to antennal cells (Krieger et al. 1999). A clone from a cDNA library of Spodoptera littoralis (P. Lucas and E. Jaquin-Joly, personal communication) showed high sequence similarity to invertebrate hyperpolarization-activated CNG channels (Gisselmann et al. 2003; Krieger et al. 1999; Quesneville et al. 2005). In addition, preliminary PCR studies suggested at least two CNG channel types in M. sexta, among them one with sequence similarity to a subunit of the heterotetrameric CNG channels and another one with sequence similarity to hyperpolarization-activated CNG channels (M. Stengl and A. Nighorn, unpublished data).
Our patch-clamp results suggest that M. sexta ORNs express at least one cAMP- and two cGMP-dependent prospective MsCNG channels. These prospective MsCNG channels differ in their Ca2+ dependence and Ca2+/CaM-dependent inhibition. Therefore like vertebrate olfactory CNG channels, prospective MsCNG channels are probably composed of principal and modulatory subunits and may differ in their subunit composition. Besides the prospective MsCNG channels, M. sexta ORNs express at least one cAMP-activated, protein kinase-dependent LVA Ca2+ current. Furthermore, M. sexta ORNs express a delayed rectifier potassium channel of unknown molecular identity, which is inhibited by cyclic nucleotides (Stengl et al. 1992; Zufall et al. 1991). Thus a multitude of cyclic nucleotide-activated channels—nonselective cation, potassium, and calcium channels—are present in ORNs. Since both cAMP and cGMP activated 81% of ORNs, most ORNs appear to coexpress cAMP- and cGMP-dependent prospective MsCNG channels. The prospective MsCNG channels are thus likely to play a prominent role in the modulation of the olfactory transduction cascade. Current molecular cloning studies aim to identify the MsCNG channels that are involved in olfactory transduction.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 The online version of this article contains supplemental data. 
Address for reprint requests and other correspondence: M. Stengl, Biology, Animal Physiology, Philipps-University of Marburg, Karl-von-Frisch-Straβe, Marburg D-35032, Germany (E-mail: stengl{at}uni-kassel.de)
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