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Pharmacological Properties and Functional Role of a TRP-Related Ion Channel in Lobster Olfactory Receptor Neurons

¹Whitney Laboratory for Marine Bioscience, ²Departments of Zoology and Neuroscience, Center for Smell and Taste, and McKnight Brain Institute, University of Florida, Gainesville, Florida

Submitted 21 September 2004; accepted in final form 26 October 2004

	ABSTRACT

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Odors activate lobster olfactory receptor neurons (ORNs) through phosphoinositide signaling that appears to target a Na⁺-gated nonselective cation channel. The Na⁺-gated channel is a potential member of the growing family of transient receptor potential (TRP) channels. Here, we test the effect of potential antagonists on the channel in cell-free patches from cultured lobster ORNs. We show that the channel is antagonized by H⁺ and the TRP channel blockers 2-aminoethoxydiphenyl borate, SKF96365 ruthenium red, Al³⁺, Gd³⁺, and La³⁺. We then use this enhanced antagonist profile together with the agonists Na⁺ and Ca²⁺ to implicate the channel in signal amplification in the cells.

	INTRODUCTION

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Phosphoinositide signaling plays an increasingly appreciated role in chemosensory transduction. Vertebrate taste receptor cells for bitter, sweet, and possibly amino acids signal through a common TRP ion channel, TRPM5, and PLC2 (Clapp et al. 2004; Liu and Liman 2003; Margolskee 2002; Pérez et al. 2003; Zhang et al. 2003). The vomeronasal organ (VNO), which plays an essential role in the detection of pheromones (Dulac and Torello 2003; Trinh and Storm 2003) in vertebrates, likely relies on phosphoinositide signaling because the TRPC2 channel gene expressed in all VNO receptor cells (Liman et al. 1999) is essential for VNO function (Leypold et al. 2002; Stowers et al. 2002) and most members of the TRPC subfamily appear to be regulated via the canonical phosphatidylinositol turnover pathway, although details of VNO activation are still being resolved (Brann et al. 2002; Cinelli et al. 2002; Liman 2003; Lucas et al. 2003; Spehr et al. 2002). Whether phosphoinositide signaling plays a role in the main olfactory organ of vertebrates is less clear, although there is emerging evidence that it does (Spehr et al. 2002).

The involvement of phosphoinositide signaling in olfaction is better understood in lobster ORNs. In what could be an interesting parallel to invertebrate phototransduction (e.g., Ranganathan et al. 1995), activation of lobster ORNs is primarily mediated by phosphoinositide signaling (e.g., Fadool and Ache 1992). The outer dendrites of the cells express the major elements of the canonical turnover pathway, including a Gq, PLC, and an inositol 1,4,5 trisphosphate receptor (IP₃R) (McClintock et al. 1997; Munger et al. 2000). An IP₃R and PLC activity can be functionally localized to the outer dendrites (Boekhoff et al. 1994; Hatt and Ache 1994), and PLC associates with G proteins in response to odorants (Xu and McClintock 1999). There is also an emerging, but still to be understood, role of phosphatidylinositide 3-kinase (PI3K)-mediated signaling in these ORNs (Zhainazarov et al. 2001). A potential target of phosphoinositide signaling in lobster ORNs is a sodium-gated nonselective cation (SGC) channel (McClintock and Ache 1990; Zhainazarov and Ache 1995, 1997) that contributes to the generation of a substantial part of the depolarizing receptor potential (Zhainazarov et al. 1998). The channel, a presumptive member of the growing family of TRP channels, can be modulated by exogeneous phosphoinosotides in cell-free patches (Zhainazarov and Ache 1999; Zhainazarov et al. 2001). Establishing this channel as a target of phosphoinositide signaling in situ would provide an interesting link between phosphoinositide signaling in olfaction and that in other chemosensory system where TRP channels are targeted.

Here, we test the effect of potential antagonists of the lobster SGC channel in cell-free patches from cultured lobster ORNs. We show that the channel is antagonized by H⁺ and the TRP channel blockers 2-aminoethoxydiphenyl borate (2-APB), SKF96365 ruthenium red (RR), Al³⁺, Gd³⁺, and La³⁺. We then use this enhanced antagonist profile together with the agonists Na⁺ and Ca²⁺ to implicate the channel in signal amplification in the cells in situ.

	METHODS

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Cell preparations

The lobster SGC channel was studied in three different preparations. First, freshly isolated vesicles of the outer dendritic membrane of lobster ORNs were obtained by incubating the olfactory organ (antennule) for 10–20 min in a solution containing (in mM) 210 NaCl, 696 glucose, 10 HEPES, 0.1 CaCl₂, and 1 EGTA buffered to a free calcium concentration of 10 nM, and cutting the tips of the olfactory sensilla (aesthetascs) into the same solution, as described earlier (Hatt and Ache 1994). Membrane patches were excised from these vesicles. Second, primary cultures of lobster ORNs were prepared as described previously (Fadool et al. 1991). Membrane patches were excised from the soma of cells cultured from 1 to 7 days. Finally, the cells were studied in situ using a modification of the preparation developed earlier (Doolin et al. 2001). Separate perfusion contours washed the ORN somata with Panulirus saline (PS, see Solutions) and the outer dendrites in the olfactory sensilla with either PS or PS containing an odorant or drug. Solution switching times of 5–50 ms were controlled using a nine-channel rapid solution changer (RSC-100/160, Bio-Logic).

Electrophysiology and data analysis

Currents were measured with an Axopatch 200A or 200B patch-clamp amplifier (Axon Instruments) through a digital interface (Digidata 1320A, Axon Instruments), low-pass filtered at 5 kHz, sampled at 20 kHz and digitally filtered at 1–1.4 kHz. Data were collected and analyzed with pCLAMP 8.1/9.0 software (Axon Instruments) in combination with Microcal Origin 6.0 (Microcal Software) and SigmaPlot 5.0/8.02 (SPSS). Channel activity was investigated in steady-state conditions at a holding potential of –70 mV unless otherwise noted. The polarity of the currents is presented conventionally, i.e., relative to intracellular membrane surface, in spite of the membrane patch configuration. Open probabilities in the case of multichannel patch recordings were estimated assuming P_o = I/Ni, where I is integral current, N is the number of channels, and i is the single channel current amplitude. Appropriate corrections for liquid junction potentials were made when necessary. Patch pipettes were fabricated from borosilicate capillary glass (Sutter Instrument, BF150-86-10) using a Flaming-Brown micropipette puller (P-87, Sutter Instrument). Extracellular in situ recordings were conducted using standard glass electrode filled with PS. Odor-evoked activity was examined after 30- to 60-s incubation with the solution(s) of interest. In multi-cell extracellular recordings, the discharge rates of individual cells were estimated using template search procedure provided by pCLAMP 9.0 software. The data are presented as means ± SE of n observations. All recordings were performed at room temperature (21°C). Two modifications of the Hill equation were used to fit the experimental data: F(x) = F_max*x^h/(x_1/2^h + x^h) for activation and F(x) = 1 – F_max*x^h/(x_1/2^h + x^h) for inhibition, where F is the open probability, normalized current or frequency of action potentials, x is the agonist/antagonist concentration, x_1/2 is the half-effective agonist/antagonist concentration, and h is the Hill coefficient. An additional parameter reflecting the basal level of F (Fb) was incorporated when necessary.

Solutions

PS contained (in mM) 458 NaCl, 13.4 KCl, 13.4 Na₂SO₄, 13.6 CaCl₂, 9.8 MgCl₂, 2 glucose, and 10 HEPES, pH 8. In some cases, the Na₂SO₄ in PS was replaced with equimolar NaCl. Low-calcium sodium solution contained (in mM) 210 NaCl, 1 EGTA, 0.1 CaCl₂, 696 glucose, and 10 HEPES, pH 7.8. Low-calcium lithium solution consisted of (in mM) 210 LiCl, 1 EGTA, 0.1 CaCl₂, 696 glucose, and 10 HEPES, pH 7.8. The estimated free calcium concentration ([Ca²⁺]_free) in low-calcium sodium/lithium solutions was 10 nM. Solutions containing >1 µM Ca²⁺/Mg²⁺ were prepared without chelating agents. PS without Ca²⁺ contained: 486 NaCl, 13.4 KCl, 23.4 MgCl₂, 10 HEPES, 0.5 EGTA, pH 8. PS without Na⁺ contained 486 LiCl instead of NaCl. Solutions with pH <7 (adjusted with Tris-HCl) contained 5 mM MES and 5 mM HEPES. An aqueous extract of TetraMarin (TET, Tetra Werke, Melle, Germany), a commercially available fish food, was used as an odorant and prepared as described earlier (Schmiedel-Jacob et al. 1990). All inorganic salts were purchased from Fisher Scientific, except for AlCl₃ and LaCl₃, GdCl₃, which were purchased from Sigma Scientific. All organic compounds were obtained from Sigma except for 2-APB, which was obtained from Calbiochem.

	RESULTS

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General properties of the lobster SGC channel

The SGC channel was studied in patches obtained from both outer dendritic vesicles and cultured lobster ORNs without obvious differences. Although the SGC channel occurs in both Ca²⁺-sensitive and -insensitive forms (Bobkov and Ache 2003), we focused on the Ca²⁺-sensitive form in both preparations because it predominates in the outer dendritic vesicles. We first demonstrate that general properties of the channel studied here are consistent with those previously reported and extend our characterization of those properties. Na⁺ (1–300 mM) applied to the cytosolic side of the patch reversibly activates the channel in a concentration-dependent manner (Fig. 1A and B). At low (10 nM) Ca²⁺ (Fig. 1B, ), the Na⁺ concentration required for a half-maximal effect, [Na⁺]_1/2, is 112.6 ± 6.1 mM, with a cooperativity coefficient, h, of 4.9 ± 1.2, and a maximal open probability, P_max, of 0.44 ± 0.04 (n = 6–10). Higher concentrations of Ca²⁺ (100 µM; Fig. 1B, ) dramatically increase the maximal open probability of the channel activated by Na⁺, altering the degree of cooperativity and decreasing the Na⁺ activation threshold, with P_max = 0.92 ± 0.08, h = 0.94 ± 0.19, and [Na⁺]_1/2 = 28.7 ± 7.6 mM (n = 5–12).

View larger version (42K): [in this window] [in a new window]   FIG. 1. Basic properties of the Ca2+-sensitive form of the lobster sodium-gated channel (SGC). A: original recordings of SGC channel activity in the presence of different [Na+] (as noted below the current traces) and either 10 nM (top) or 100 µM (bottom) Ca2+. B: plot of the dependence of SGC channel open probability on [Na+]i (in range 1–300 mM) in the presence of 10 nM () and 100 µM () Ca2+. —, Hill-equation fit with the following parameters: [Na+]1/2 = 112.6 ± 6.12 mM, h = 4.9 ± 1.2 (n = 6–10) for 10 nM Ca2+ and [Na+]1/2 = 28.7 ± 7.6 mM, h = 0.94 ± 0.19 (n = 5–12) for 100 µM Ca2+. Open probability was estimated assuming Po = I/Ni. Data were obtained in inside-out patch recordings; HP, –70 mV; electrode solution, NaCl 210 mM + Ca2+ 10 nM. Voltage (C) and calcium (D) dependences of the channel. Current-voltage (I-V) characteristics obtained from an inside-out patch by applying a voltage ramp in the presence of different [Ca2+]i (10 nM, 520 nM, 3.3 µM, 10 µM, 50 µM, 100 µM and 1 mM—noted by gray level). Only the negative segment of I-V relations are shown. A 30-ms hyperpolarizing step to –100 mV preceded linear voltage changing (ramp, diagram in C). Ramp duration, 200 ms. Each line is an average of 40 ramps generated in the corresponding [Ca2+]. Corresponding corrections were introduced by successive subtraction of leakage ramp current obtained in conditions without sodium. D: calcium dependence of normalized (I/Imax) current at different voltages. Parameters of the calcium dependent augmentation of the normalized (I/Imax) current at –70 mV ([Ca2+]1/2 = 665 ± 146 nM, h = 1.39 ± 0.4, n = 4) and reduction at –10 mV ([Ca2+]1/2 = 25.8 ± 9 µM, h = 1 ± 0.2, n = 4) were estimated using corresponding Hill equation modifications (see METHODS). Current values were obtained from I-V relations at corresponding voltages (C, · · · ) and represented by gray level.

Mg²⁺ applied to the intracellular side of the patch in the absence of Ca²⁺ (0 Ca²⁺, 50 µM EGTA) reversibly blocked the channel both by decreasing the open probability ([Mg²⁺]_1/2 = 811 ± 90 µM, h = 1.33 ± 0.3, n = 8) and reducing the single channel amplitude (16.2 ± 0.4 pA in control conditions vs. 15.07 ± 0.2 pA in the presence 1 mM Mg²⁺, n = 7, –70 mV, data not shown). The ratio of [Mg²⁺] to [Ca²⁺] and/or presumably [Na⁺] determines the magnitude of the magnesium inhibition constant with the [Mg²⁺]_1/2 increasing with an increase in Na⁺ or Ca²⁺. In 10 µM Ca²⁺, for instance, [Mg²⁺]_1/2 = 3.3 ± 0.12 mM, h = 1.1 ± 0.4. The complex interaction between these ions probably explains the paradox that, otherwise, the channel would be blocked in normal physiological conditions given that sea water and PS contain 53 and 9.8 mM Mg²⁺, respectively (see METHODS). With PS on the extracellular face, the unitary conductance of the channel is 23.4 ± 0.6 pS (n = 5) versus 204 ± 7 pS (n = 7) in low-Ca²⁺ conditions. The activity of the SGC channels in physiologically relevant conditions has been published earlier (Bobkov and Ache 2003).

Effect of pH on the channel

Acidification reversibly inhibits the channel (Fig. 2). Lowering the pH of the solution bathing the extracellular face of outside-out patches in which the channel was continuously activated by Na⁺ (210 mM) in the pipette blocked the channel (Fig. 2A). Reducing the extracellular pH from 8.0 to 7.0 almost completely blocked channel activity (n = 11; Fig. 2B). The proton-mediated inhibition had an apparent inhibition constant, [H⁺]_1/2, of 2.67e-8 ± 1.66e-9 M (corresponding to pH 7.57), with a cooperativity coefficient, h, of 4.0 ± 0.8, in good agreement with what we found previously with inside-out patches (pH_1/2 = 7.3, h = 5) (Bobkov and Ache 2003). The proton effect presumably was not exclusively associated with changing the affinity of the Ca²⁺ binding site of the channel because acidification resulted in full blockade not just blockage of the Ca²⁺-potentiated component. It is possible that protons additionally or alternately competed for the Na⁺ binding site of the channel, although we did not attempt to identify the mechanism of proton-mediated inhibition.

View larger version (22K): [in this window] [in a new window]   FIG. 2. pH dependence of the SGC channel. A: increasing [H+]out blocked the SGC channel. B: open probability of the SGC channel as function of the extracellular pH. Line, approximation by Hill equation ([H+]1/2 = 2.67e-8 (pH,7.57) ±1.66e-9, h = 4.04 ± 0.8, (n = 5–11)]. The open probability was estimated assuming P = I/Ni. The analysis was corrected for changes in single channel amplitude and current baseline shift at increasing [Ca2+]. Experimental conditions: outside-out patch recording; HP, –70 mV; electrode solution, NaCl 210 mM + Ca2+ 10 nM. Application of solutions of different pH is shown by the line over the current trace. LiCl, 210 mM LiCl + 10 nM Ca2+; NaCl, 210 mM NaCl + 10 nM Ca2+.

We then tested the potential effect of antagonists widely used to block TRP channels by applying them to the inside face of patches containing the lobster channel. RR (10–20 µM), which also blocks the ryanodine receptor (RyR), inhibited the activity of the channel (n = 7; Fig. 3A). The effect of RR was reversible (data not shown). RR (10 µM) primarily led to long-term closures, reducing the open probability (P_o) to 0.12 ± 0.04 from 0.42 ± 0.1 in 10 nM Ca²⁺ and from 0.91 ± 0.08 in 10 µM Ca²⁺ (n = 3), but it also induced fast open channel block, as could be seen in the short, burst-like episodes that occasionally interrupted the sustained closures (Fig. 3A, bottom). The latter effect was voltage dependent (data not shown).

View larger version (40K): [in this window] [in a new window]   FIG. 3. Blockade of the SGC channel by cytosolic ruthenium red (A, 10 µM), SKF96365(B, 50 µM) and 2-aminoethoxydiphenyl borate (2-APB, C, 50 µM). Experimental conditions: inside-out patch recordings; HP, –70 mV; electrode solution, NaCl210 mM + Ca2+10 nM. Bar diagrams above current traces in B and C show time course of solution application. Time scales are different in all portions of the figure. LiCl in B and C—LiCl210 mM + Ca2+10 nM.

2-APB (25–200 µM), a structurally unrelated TRP channel blocker, also known to block IP₃Rs, completely inhibited the channel when applied to the inside face of the patch (n = 17; Fig. 3C). The effect was reversible. As with SKF96365 2-APB decreased the open probability of the channel without affecting the single channel conductance, although the effect of 2-APB was more rapid (Fig. 3C).

The trivalent cations lanthanum (La³⁺), gadolinium (Gd³⁺) and aluminum (Al³⁺) also blocked the channel when applied to the intracellular face of cell-free patches containing the channel. We were careful to minimize possible confounding effects due to chelation by EGTA, sulfate, or Tris (Caldwell et al. 1998) by not including these compounds in the solutions. The channel was blocked completely by Gd³⁺ (100–200 µM, n = 12), La³⁺ (100–200 µM, n = 23), and Al³⁺ (200 µM, n = 3, Fig. 4A). The lanthanides and aluminum appeared to block primarily by decreasing the open channel probability, although there was some evidence in the records suggesting that they also altered the single channel conductance. The blocking effect of all three trivalent cations was not reversible itself but could be fully reversed by application of a solution containing EGTA. The blocking effect of the trivalent cations was concentration-dependent in that decreasing [Gd³⁺] or [La³⁺] from 200 to 10 µM slowed the onset of the block, but the extent of the block (full) remained unchanged (n = 30; Fig. 4B). The actual time required for 50% blockade in channel activity varied from patch to patch and experiment to experiment due, for example, to possible differences in the size and geometry of the patch, so we elected not to quantify the concentration-dependent change in the rate of blockade. Lower concentrations (5 µM) of Gd³⁺ and La³⁺ were without effect 3–5 min postapplication (Fig. 4B, light traces). The blocking effect of the trivalent cations was voltage independent. Although positive holding potentials would be expected to accelerate the rate of blockade by positively charged ions, the rate of blockade, if anything, was actually slowed at more positive holding potentials (Fig. 4B). There was no noticeable effect of Ca²⁺ on the effect of the trivalent cations in the range of 10–100 µM, even though one might expect La³⁺ and Gd³⁺ would compete with Ca²⁺ ions to screen negative membrane surface charges and for Ca²⁺-binding sites.

View larger version (37K): [in this window] [in a new window]   FIG. 4. Effect of trivalent cations on the SGC channel. A: La3+ (100 µM), Gd3+ (100 µM), Al3+ (200 µM) fully block the channel. Zero time corresponds to the moment of blocker application. Traces have different current scales. HP was –70 mV. B: different concentrations of Gd3+ (indicated above current traces) fully and irreversibly blocked the channels. Note: superfusion with Gd3+ 5 µM (light traces) for 3–5 min was without noticeable effect. To contrast kinetic parameters, the current traces were truncated and aligned relative to the moment of blocker application (0 time does not correspond to the start of the recordings). Note: blockade with Gd3+ and other trivalent cations is lost after incubation with chelating agents. La3+ and Al3+ had similar effects. Experimental conditions: inside-out recording; HPs, indicated on the plot; electrode solution, NaCl210 mM + Ca2+10 nM; superfusion solution, NaCl210 mM + Ca2+ 10 µM.

Effect of blocking the channel in situ

We used the preceding pharmacological profile to more rigorously implicate the lobster SGC channel in the olfactory transduction cascade in situ. The rate and pattern of spontaneous and evoked discharge of lobster ORNs in situ suggest the presence of several functional subpopulations of cells that we are in the process of characterizing in more detail elsewhere. Here, we focus only on cells that are tonically active and comprise the predominant subpopulation. These cells discharge spontaneously at 2.23 ± 0.36 Hz (minimum = 0.125, maximum = 4.9, n = 48); odors phasically increase the rate of discharge in a concentration-dependent manner (Fig. 5A, top traces and plot).

View larger version (35K): [in this window] [in a new window]   FIG. 5. Effects of removing Ca2+ on the excitability of lobster olfactory receptor neurons (ORNs) in situ. A: representative responses of a single lobster ORN to odorant pulses of increasing intensity in the presence (top and bottom traces and plots) and absence (middle traces and plot) of extracellular Ca2+ [Ca2+-free Panulirus saline (PS)]. Left: raster displays of actual discharge. Stimulus concentration increases from bottom to top, changed by increasing the duration of the odor pulse in 40-ms increments from t1 = 40 ms (lines below traces). Time between successive sweeps, 60 s. Shaded area, 2-s peristimulus interval starting 0.34 s prior to stimulus onset during which mean discharge frequency was determined to generate the dose-response relationships shown in C and D. Right: plots of the instantaneous frequency (f = 1/t, where t = time between adjacent events) for each raster shown on the left. In Ca2+-free PS, Ca2+ was replaced with equimolar Mg2+. Total [Mg2+], 23.4 mM. EGTA (1 mM) added to avoid possible Ca2+ contamination. B: net Ca2+-dependent component of the odor-evoked discharge (bottom) obtained by subtracting the average of 8 responses to the same intensity stimulus (200 ms) in the presence and absence of Ca2+ (top). Bin width, 100 ms. C: activity of the same ORN as in A plotted as a function of stimulus intensity (duration, ms). Fmax = 14.7, S1/2 = 64, h = 3.1 (control conditions, solid line). D: the same type of data as in C collected from 3 different cells. The dose-response relation of each cell was normalized to Fmax and S1/2 in control conditions. Control conditions (light circles, Fc): Fmax = 0.89, S1/2 = 1, and h = 3.32. Ca2+ free conditions (dark circles, Fs): Fmax = 0.56, S1/2 = 3.07, and h = 2.53. Gray curve, gain coefficient (k) calculated as k = (Fc – Fs)/Fs.

Removal of extracellular Na⁺ was shown earlier to reduce the odor-evoked output of these cells (Zhainazarov et al. 1998). To compare the relative effect of removing extracellular Ca²⁺ with that of removing Na⁺, we used the approach described above to quantify the effect of removing extracellular Na⁺. Substituting 486 mM of the Na⁺ bathing the outer dendrites with Li⁺ reduced the response to saturating concentrations of odors by 52.4 ± 2% (n = 11). The cells (n = 4) responded with F_max = 0.84, S_1/2 = 1, and h = 3.25 in control conditions (Fc; Fig. 6A, light circles), but with F_max = 0.45, S_1/2 = 2.78, and h = 2.09 in low Na⁺ conditions (Fs; Fig. 6A, dark circles). Overall, the effect of removing extracellular Na⁺ was similar to the effect of removing extracellular Ca²⁺ (Figs. 6A vs. 5D).

View larger version (35K): [in this window] [in a new window]   FIG. 6. Effects of low sodium PS (Li-PS, A), acidification (low pH-PS, B) and La3+ (La3+ 300 µM, C) on the excitability of lobster ORNs in situ. Left: net component of the odor-evoked discharge after the respective treatments, obtained by subtracting the averaged response to the same intensity stimulus before and after treatment. Bin width, 100 ms. Right: plots of the activity as a function of stimulus concentration (duration) before and after the respective treatments. Data were collected, processed, and normalized using the same experimental paradigm as described above for Ca2+-free PS effects (Fig. 5). Hill equation approximation parameters (left-hand axis): A, the cells (n = 4) responded with Fmax = 0.84, S1/2 = 1, and h = 3.25 in control conditions (Fc, light circles), while with Fmax = 0.45, S1/2 = 2.78, and h = 2.09 in low-Na+ conditions (Fs, dark circles); B, the cells (n = 4) responded with Fmax = 0.85, S1/2 = 1, and h = 3.43 in control conditions (Fc, light circles), while with Fmax = 0.53, S1/2 = 2.08, and h = 2.8 in low-pH conditions (Fs, dark circles); C, the cells (n = 3) responded with Fmax = 0.87, S1/2 = 1, and h = 4.6 in control conditions (Fc, light circles), while with Fmax = 0.5, S1/2 = 2.1, and h = 3.2 in the presence of La3+ (Fs, dark circles). Gray curve in each plot is the gain coefficient estimated as k = (Fc – Fs)/Fs, where Fc is the activity of ORNs in control conditions expressed as relative discharge frequency and Fs is the activity of ORNs after treatment.

La³⁺ (300 µM), tested as a representative trivalent cation, reduced the odor-evoked response to saturating concentrations of odorant by 43.8 ± 3.6% (n = 9). The cells (n = 3) responded with F_max = 0.77, S_1/2 = 1, and h = 5.5 in control conditions (Fc; Fig. 6C, light circles), while with F_max = 0.43, S_1/2 = 2.35, and h = 4.1 in the presence of La³⁺ (Fs; Fig. 6C, dark circles). In contrast to the effect of La³⁺ on the channel, however, lower concentrations of La³⁺ (10, 50, 100 µM) did not have any detectable effect on the odor-evoked activity of the cells in situ and effective concentrations (e.g., 300 µM) of La³⁺ were reversible (n = 17). This finding suggests the outer dendritic compartment may possess some mechanism for chelating or otherwise eliminating trivalent cations, although this possibility was not pursued further. High concentrations of La³⁺ (0.5 mM) irreversibly altered the spontaneous discharge, causing irregular, sporadic but long-lasting bursts (n = 4; data not shown).

The Ca²⁺ dependence of SKF96365made it impractical to test the drug in physiologically relevant conditions, so this antagonist was not tested on the cells in situ. RR and 2-APB are also known to target IP₃Rs, which have been implicated in the activation of lobster ORNs (Munger et al. 2000). These probes, too, were not tested on the cells in situ to avoid possible confounding effects. We found that RR (Fadool and Ache 1992) and 2-APB (data not shown) blocked odor-evoked responses in these cells, and this blockage could potentially be ascribed to the IP₃R.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
The general properties of the lobster SGC channel studied here, specifically the characteristics of the Na⁺, Ca²⁺, and voltage dependencies of its activation, were identical to those reported earlier for the channel (e.g., Bobkov and Ache 2003). We assume, therefore that the present results serve to extend our previous understanding of this channel.

As a nonselective cation channel, the lobster SGC channel potentially is a member of the growing family of TRP channels (review: Minke and Cook 2002). Although there is no specific pharmacological profile that serves to identify TRP channels, the lobster SGC channel was blocked by drugs known to block and commonly used to characterize TRP channels in other systems. Specifically, the lobster SGC channel was blocked by RR, known to block channels of the TRPV and TRPM subfamilies (reviewed by Gunthorpe et al. 2002; Patapoutian et al. 2003); SKF96365 known to block members of TRPC subfamily (Halaszovich et al. 2000; Inoue et al. 2001; Zhu et al. 1998); and 2-APB, known to block store operated/TRP channels (Bootman et al. 2002; Clapham et al. 2001; Lucas et al. 2003; Ma et al. 2001). The effect of common organic blockers of known TRP channels on the lobster SGC channel is consistent with the latter being a member of the TRP family.

The lack of specific organic blockers for TRP channels has precipitated the use of differential sensitivity to the lanthanides, La³⁺ and Gd³⁺, to discriminate TRP channels (rev. Minke and Cook 2002; Zitt et al. 2002), even though lanthanides are known to nonspecifically block other (e.g., mechanosensitive) (Hamill and McBride 1996; Yang and Sachs 1989) channels and have other confounding effects (Caldwell et al. 1998). The ability of La³⁺ and Gd³⁺ to block the lobster SGC channel therefore further supports the interpretation that the lobster channel is a member of the TRP family. Two characteristics of the blockade of the lobster channel by lanthanides, however, to our knowledge have not been reported previously. The slow, irreversible onset, building over as much as 30 s in a concentration-dependent manner, although unusual, may reflect the gradual adsorption of lanthanides to high-affinity binding sites on the channel itself or other membrane constituents, including possibly membrane lipids. Lanthanide binding to phospholipids alters various physical properties of lipid bilayers, which presumably would alter channel behavior (Awayda et al. 2004; Ermakov et al. 2001). Alternately, lanthanide binding could sequester lipids essential for channel function because phosphoinositides activate the SGC channel (Zhainazarov and Ache 1999). A similar interpretation has been proposed to explain the interaction between polyvalent cations, including lanthanides, and membrane or membrane-associated proteins in other systems (Hilgemann and Ball 1996; McDonald and Mamrack 1995; Verstraeten and Oteiza 2002). Indeed, the effects of lanthanides on ion channels could be predictive of sensitivity to anionic polyphosphoinositides. Second, the effect "stepped" from nothing to full blockade over <5 µM Gd³⁺, suggesting a steep dependency of steady-state channel activity (P_o) on trivalent cation concentration, with a potential Hill coefficient in excess of 8. Although such a high level of cooperativity might seem to be unusual, and to our knowledge has not been determined previously for TRP channels, Gd³⁺-induced inhibition of a stretch-activated nonselective cation channel in Xenopus oocytes shows a similarly steep dose-dependency (Yang and Sachs 1989).

The antagonistic effect of lowering the extracellular pH from 8.0 to 7.4 on the lobster SGC channel is particularly interesting. The effect was relatively dramatic, and to our knowledge, a pH change in this range is not known to effectively block other types of ion channels in lobster ORNs. Thus proton blockade in this range could potentially serve as a selective probe for the SGC channel. If indeed the lobster SGC channel proves to be a member of the TRP family, proton blockage in this range could potentially serve as a selective probe for one or more classes of TRP channels. Although the present findings do not allow us to tentatively assign the lobster SGC channel to a particular class of TRP channels, the blockade by lanthanides together with its Ca²⁺-dependent activation and its involvement in chemosensory transduction would be consistent with the lobster channel being a member of the TRPC/M family of TRP channels (Liu and Liman 2003; Lucas et al. 2003; Perez et al. 2002; Prawitt et al. 2003; Reuss et al. 1997). It will be interesting to see if TRPC/M channels are subject to proton blockade in this range.

Because all the antagonists also acted from the extracellular face of the membrane, we were able to use them in conjunction with the agonists Na⁺ and Ca²⁺ to more rigorously implicate the channel in the transduction cascade of the cells in situ with the exception of those noted earlier to have potential confounding effects by their ability to also target InsP₃Rs. All the antagonists tested, as well as removing extracellular Na⁺ and Ca²⁺, had essentially the same effect on both the magnitude and time course of the odor-evoked response; they decreased the maximum response and increased the stimulus intensity needed for half-maximal response. In particular, they had the same effect on the Ca²⁺-dependent component of the odor-evoked response as determined by our subtractive protocol. The similarity of their collective effect on the odor-evoked response argues strongly that the pharmacological probes are targeting the same effector, the SGC channel. Their similar collective action cannot be deemed to be definitive proof because we cannot eliminate the possibility that some or all of the probes had nonspecific effects on other channels and/or elements of the signaling cascade that resulted in a similar overall effect. Nonetheless, the similarity of the collective effect of the various probes on the cells is compelling.

The effect of blocking the channel in situ supports the earlier proposal based on the replacement of extracellular Na⁺ that the channel serves to amplify the receptor current (Zhainazarov et al. 1998). If the lobster SGC serves to amplify the receptor current, its role in the phosphoinositide-signaling cascade in the lobster cells may be functionally similar to that proposed for the Ca⁺-activated Cl^– channel in the cyclic nucleotide signaling cascade that mediates olfactory transduction in vertebrate olfactory receptor cells. In the latter case, Ca⁺ entering the cell through the primary effector, the cyclic nucleotide-gated cation channel, is thought to secondarily activate a Ca²⁺-activated Cl channel (Kleene and Gesteland 1991; Kurahashi and Yau 1993), which serves to amplify the primary signal (Lowe and Gold 1993; Reisert et al. 2003). This analogy raises the question of what channel would function as the primary effector in lobster ORNs. One possibility might be the IP₃R, which these cells express in the plasma membrane of the outer dendrite (Munger et al. 2000). Activation of the IP₃R presumably would allow Ca²⁺ to enter the cell, in this case from extracellular "stores" because there are no known intracellular stores in the fractional micrometer-diameter olfactory outer dendrites (Grunert and Ache 1988). The fact that the antagonists tested had the same effect on the Ca²⁺-dependent component of the odor-evoked response would be consistent with this interpretation. Other scenarios certainly are possible, however, and currently we are exploring the detailed sequence of events leading to activation of the lobster SGC channel in the intact cell.

	GRANTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
This work was supported by National Institute of Deafness and Other Communication Disorders Grant DC-01655.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
We thank J. Tulsian for preparation of the cultured cells and L. Milstead for assistance with the illustrations.

	FOOTNOTES

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

Address for reprint requests and other correspondence: Y. V. Bobkov, Whitney Laboratory for Marine Bioscience, University of Florida, 9505 Ocean Shore Blvd, St. Augustine, FL 32080-8610 (E-mail: bobkov{at}whitney.ufl.edu)

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