Introduction

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Neuronal nAChRs mediate transmission and modulate transmitter release at autonomic and central synapses, are involved in addiction and neurological disease, and participate in key developmental processes (Role and Berg, 1996; Weiland et al., 2000; Dani, 2001; Dani et al., 2001; Rezvani and Levin, 2001; Margiotta and Pugh, 2003). Consistent with their multiple functions, neuronal nAChRs are heterogeneous, assembling as homopentamers of 7 or 8 subunits or as heteropentamers containing 2, 3, 4, or 6 subunits in combination with 5, 2, 3, and/or 4 subunits (Sargent, 1993; Margiotta and Pugh, 2003). Examples of native nAChRs defined by subunit composition are relatively rare, however, and the relationship between nAChR composition and function virtually unknown. Such information would be useful for experimental or therapeutic studies requiring subtype-specific intervention.

To relate receptor composition to channel function, we probed nAChRs on ciliary ganglion neurons with subunit-selective agonists and toxin antagonists. Ciliary ganglion neurons are particularly useful for such studies because they express diverse nAChRs that have known subunit composition (Vernallis et al., 1993; Conroy and Berg, 1995) and channel properties (McNerney et al., 2000). One nAChR type is recognized by -bungarotoxin (Bgt), which targets 7 subunits with high affinity (Couturier et al., 1990) and labels about 10⁶ surface sites per neuron (McNerney et al., 2000). Approximately 95% of Bgt-nAChRs contain only 7 subunits (7-nAChRs); however, 5% lack 7 or any known nAChR subunits, and Bgt-nAChRs comprising this minor subtype are termed T35-nAChRs (Vernallis et al., 1993; Pugh et al., 1995). A second nAChR type (3*-nAChR) is undetected by Bgt but recognized by mAb35, an antibody that detects 3 and 5 subunits (Conroy and Berg, 1998) and labels about 6 × 10⁴ surface sites per neuron (Margiotta and Gurantz, 1989). In immunoprecipitation studies, 3*-nAChRs were found to contain 3, 4, and 5 subunits, with 20% also containing 2 subunits (Vernallis et al., 1993; Conroy and Berg, 1995). Functionally, nAChR agonists induce whole-cell currents featuring rapidly and slowly decaying components (Zhang et al., 1994), which are loosely associated with the four nAChR subtypes defined above and mediated by four discrete nAChR channel conductance classes (McNerney et al., 2000).

Our approach was to activate whole-cell and single-channel nAChR currents with either a pan-specific agonist (nicotine) or agonists selective for 7- (GTS-21) or 3-containing (epibatidine) nAChRs (Gerzanich et al., 1995; Meyer et al., 1997; Papke et al., 2000). The contributions of Bgt- and 3*-nAChR subtypes were then assessed by comparing the currents with those obtained from neurons treated with Bgt, -conotoxin-AuIB (CTx-AuIB), or -conotoxin-MII (CTx-MII). The latter two are "3-selective", recognizing recombinant 34 and 32 nAChRs, respectively, in X. laevis oocytes (Cartier et al., 1996; Luo et al., 1998). The use of subunit selective agonists and toxins combined with whole-cell and single-channel recordings allowed us to relate functional nAChR channels to defined molecular subtypes. Although this strategy relies on available structural information, it avoids uncertainties about subunit assembly and compensatory changes that can make results based on heterologous expression and genetic deletion difficult to interpret (reviewed in Margiotta and Pugh, 2003).

	Materials and Methods

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Neuron and Substrate Preparation

Ciliary ganglion neurons were dissociated from embryonic day 14 (E14) ganglia using collagenase A treatment (0.33 mg/ml 10 min) and mechanical trituration protocols described previously (e.g., Margiotta and Gurantz, 1989; Pardi and Margiotta, 1999; McNerney et al., 2000). Dissociated neurons were suspended in a recording solution (RS) containing 145.0 mM NaCl, 5.3 mM KCl, 0.8 mM MgSO₄, 5.4 mM CaCl₂, 5.6 mM glucose, and 5.0 mM HEPES acid, pH 7.4, that was supplemented with 10% heat-inactivated horse serum (RS^+hs). As substrate, glass coverslips (12-mm diameter; Corning, Palo Alto, CA) were acid-washed and 300-kDa poly-(D-lysine) (2 mg/ml in 0.13 M Borate buffer, pH 8.5) applied to each for 12 to 16 h at 4°C. The coverslips were then washed 10 times with distilled water, air-dried, and suspended neurons were plated at 2 to 3 ganglion equivalents per coverslip. Neurons attached to the substrate within 30 min but were allowed to equilibrate for at 37°C for 1 to 3 h before use.

Electrophysiology

Whole-Cell Current Acquisition and Analysis. Neuron recordings were obtained in RS^+hs at room temperature (21-24°C) using procedures similar to those described previously (Margiotta and Gurantz, 1989; Pardi and Margiotta, 1999; McNerney et al., 2000). Patch pipettes were pulled from Corning 8161 glass tubing, filled with an intracellular solution containing 145.6 mM CsCl, 1.2 mM CaCl₂, 2.0 mM EGTA, 15.4 mM glucose, and 5.0 mM Na-HEPES, pH 7.3, and had tip impedances of 1.5 to 3.0 M when measured in RS^+hs. Whole-cell currents were collected at 70 mV and filtered at 10 kHz using an Axopatch 200B amplifier (Axon Instruments, Union City, CA). The currents were digitized at 2 kHz using a Tl-125 interface controlled by Clampex (pClamp 6.0; Axon Instruments) and stored on a PC-compatible computer (Gateway, Poway, CA). Membrane capacitance compensation was achieved by eliminating the capacitive current transient in response to a 10 mV pulse using the amplifier series resistance (R_s) and capacitance (C_m) controls. To activate nAChRs, agonists [i.e., nicotine hydrogen tartrate (Nic), epibatidine dihydrochloride (Epi), or 3-(2,4-dimethoxybenzylidene)anabaseine (GTS-21)] were dissolved in RS at the desired final concentration from 500× frozen stocks and focally applied to individual neuron somata for 2.5 s. The agonists were delivered by pressure microperfusion activated by a computer-driven valve (Picospritzer II; General Valve Co., Waltham, MA) from patch pipettes (Microhematocrit; VWR Scientific Inc., Westchester PA) using two modifications of described previously protocols (Margiotta and Gurantz, 1989; McNerney et al., 2000). First, to ensure efficient agonist exposure, the neurons selected were small, having soma diameters (distributed around a modal value of 11.0 ± 3.1 µm; N >750) that corresponded to those of the choroid neuron population (McNerney et al., 2000). The present findings are also likely to apply to ciliary neurons, however, because both choroid and ciliary neurons express the same subset of nAChR genes (Corriveau and Berg, 1993) and because the properties of individual nAChR channels on the two neuron populations are indistinguishable (McNerney et al., 2000). Second, the perfusion pipette tips were larger (4-6 µm diameter) and delivered higher pressures (8-10 psi) than those used previously. The increased pipette size and pressure resulted in relatively fast agonist delivery that equilibrated in 20 to 30 ms, as estimated from junction current measurements using open tip pipettes. Although slower than the delivery obtained with fast perfusion driven by piezoelectric switching, pressure microperfusion was simpler and proved sufficient to adequately identify the contributions from Bgt- and 3*-nAChRs. Apart from some slowing of the fastest component of Bgt-nAChR current decay (_f, see below), pressure microperfusion yielded neuronal response parameters (Table 1) that were very similar to those we and others obtained previously using fast piezoelectric switching (Zhang et al., 1994; Pardi and Margiotta, 1999; McNerney et al., 2000). In particular, for neurons of similar small size (C_m  7-12 pF) the peak Bgt-nAChR currents induced by 20 µM Nic applied using piezoelectric switching (3495 ± 400 pA, n = 12; McNerney et al., 2000) or pressure microperfusion (3095 ± 240 pA, n = 29; present study) were indistinguishable (p > 0.1), indicating that the initial fast component amplitudes are similarly resolved using either method.

View this table: [in this window] [in a new window]   TABLE 1 Whole-cell response parameters for three nAChR agonists All values are presented as mean ± S.E.M., and were derived from whole-cell current responses to 20 µM Nic (n = 91), 30 µM GTS-21 (n = 19) or 30 µM Epi (n = 20), all applied for 2.5 s at 70 mV. In each case, specific peak currents (in pA/pF) and time constants () are tabulated for the fast, intermediate, and slow kinetic components, determined as described under Materials and Methods and in Fig. 1.

(1)

Single-Channel Current Acquisition and Analysis. The procedures for single channel data acquisition were similar to those described previously (McNerney et al., 2000). Briefly, outside-out membrane patches were excised from neuron somata in whole-cell mode, and agonists applied by gentle pressure microperfusion (2-5 psi) at holding potentials ranging from 80 to 140 mV. Patch currents were digitized at 50 kHz and filtered at a cutoff frequency (f₁) of 10 kHz using the Bessel filter included in the Axopatch 200B and then at 9.3 kHz (f₂) during data analysis. These settings yielded a final filter frequency (f_c) of 6.8 kHz (f_c² = f₁² + f₂²) allowing channel openings 98 µs (twice the filter rise time, t_r = 0.3321/f_c) to be resolved (Colquhoun and Sigworth, 1995). Single nAChR channel events were detected by visual inspection of transitions from the baseline current and manually selected using Fetchan (pClamp 6.0; Axon Instruments). Only those events exceeding set thresholds for amplitude (2× noise) and duration (100 µs) were accepted for analysis.

Materials. Fertilized white Leghorn chicken eggs were obtained from Hertzfeld Poultry Farms (Waterville, OH) and maintained at 37°C in a forced air draft incubator at 100% humidity. Bgt was obtained from Biotoxins, Inc. (St. Cloud, FL), and GTS-21 was generously provided by Dr. W. Kem (University of Florida College of Medicine, Tampa, FL). Most other reagents were purchased from Sigma (St. Louis, MO).

	Results

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Whole-Cell Studies

Nicotine Activates Both Bgt- and 3*-nAChRs. Whole-cell responses to Nic were used as a baseline for comparing the actions of subunit-specific toxins and agonists on Bgt- and 3*-nAChR populations. Nic applied at 20 µM induces whole-cell currents that activate within 20 ms (I_peak) and then decay because of nAChR desensitization as described by eq. 1 (Fig. 1A). Previous findings indicate that the most rapidly decaying component of this response is attributable to Bgt-nAChRs (Zhang et al., 1994; McNerney et al., 2000) because A_f is absent in neurons treated with Bgt (60 nM, 30-60 min; see, e.g., Fig. 1B). Bgt has also been shown to inhibit a somewhat slower decaying component of the Nic response (Zhang et al., 1994) and in the present study it also reduced A_i by 95% (data not shown). Because both A_f and A_i were blocked by Bgt, they have been combined into a single parameter (I_fast = A_f + A_i) to quantify toxin effects (Fig. 2). Unlike I_fast, only a minor portion of the slow-decaying component (A_s) of the Nic response has been attributed to Bgt-nAChRs (Liu and Berg, 1999). Consistent with these findings, Bgt had a small yet significant effect on A_s, corresponding to a 25% reduction in mean slow current density (A_s/C_m) compared with untreated controls (Figs. 1B and 2). As depicted in Fig. 1, the decay of A_s was somewhat slower in Bgt-treated neurons; however, comparison of _s for Bgt-treated (3890 ± 386 ms, n = 60) and control neurons (Table 1) indicate this trend is not statistically significant (p > 0.1). Treating the neurons with the 7-selective antagonist methyllycaconitine (MLA; 50 nM, 1 h) yielded a pattern of inhibition very similar to that of 60 nM Bgt, causing complete block of A_f/C_m, a 90% reduction in A_i/C_m, and a 30% reduction in A_s/C_m (n = 11; data not shown). These findings, obtained with 7-selective antagonists and supported by results presented below using 7- and 3-selective agonists, indicate that I_fast is mediated solely by 7-containing Bgt-nAChRs (7-nAChRs). Because Bgt does not recognize native 3*-nAChRs (Corriveau and Berg, 1993; Vernallis et al., 1993; Conroy and Berg, 1995; Pugh et al., 1995) and both Bgt and MLA failed to completely block A_s, the results further suggest that, on average, about 25% of the slow-decaying current can also be attributed to 7-nAChRs (see below).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Whole-cell currents induced by nicotine display Bgt-sensitive and -insensitive kinetic components. A, rapid microperfusion with a maximal Nic concentration (20 µM, horizontal bar) induced a peak whole-cell response (Ipeak) of 5700 pA in this neuron (Cm = 12 pF) corresponding to a specific peak response (Ipeak/Cm) of 475 pA/pF. The holding potential for all whole-cell recordings was 70 mV. Inset. The currents decaying from Ipeak for the neuron depicted in A are well described by the sum of three exponential functions (eq. 1). The peak amplitudes of the individual fast (Af), intermediate (Ai), and slow (As) current components (arrows at left) corresponded to specific values of 305, 94, and 75 pA/pF, respectively (compare with values in Table 1). The time-dependent contributions of fast, intermediate, and slow components to the current are shown as dotted, short-dashed, and long-dashed lines, respectively, with associated time constants (f, i, and s; in milliseconds) shown for each. The solid line represents the sum of the three components and is also overlaid on the actual current decay in A. B, response to 20 µM Nic after treating neurons with Bgt (60 nM, 30-60 min). In the neuron depicted (Cm = 11 pF), only the slowly decaying current (s = 3490 ms) remains, corresponding to a specific response of 60 pA/pF. Calibration bars indicate 250 ms, with that shown in B also applying to the main part of A.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Summary of subunit-specific toxin effects on whole-cell responses to 20 µM nicotine. Ifast/Cm () represents the sum of specific fast and intermediate response components [(Af + Ai)/Cm], whereas As/Cm () represents the specific slow component alone. Results were compiled from 10 to 60 recordings from toxin-treated neurons and are expressed as the mean (±S.E.M.) percentage of the indicated response component obtained in similar numbers of control neurons from the same experiments. AuIB and MII represent CTx-AuIB and CTx-MII, respectively. Toxins were applied for 30 to 60 min at concentrations that produced maximal block of the response to 20 µM Nic (60 nM Bgt, 300 nM CTx-MII, 10 µM CTx-AuIB) and were present in both the bath and the agonist-application pipettes. Bars with asterisks above indicate that the parameter values were significantly different (p < 0.05) from those for control neurons; bars without asterisks indicate the values were not detectably different (p > 0.05) from control values.

-Conotoxins Selectively Block 3*-nAChRs. We assessed the contribution of 3*-nAChRs to A_s using Conus spp. toxins expected to target the two 3*-nAChR subtypes present on ciliary ganglion neurons (i.e., 345 and 2345). The first toxin tested was -CTx-AuIB, which blocks recombinant 34 nAChRs expressed in X. laevis oocytes (IC₅₀ = 750 nM) and is >100-fold less potent on recombinant 7-nAChRs and 32-nAChRs, respectively (Luo et al., 1998). With Bgt present to block the contribution from 7-nAChRs, CTx-AuIB applied for 30 to 60 min progressively inhibited the response to 20 µM Nic (IC₅₀ = 350 nM), with a 10 µM dose sufficient to reduce A_s to nearly zero (Figs. 2 and 3, A, B, and D). CTx-AuIB was specific in the sense that the same 10 µM dose applied without Bgt failed to significantly change I_fast (Fig. 2), _f, or _i (not shown). Under the latter conditions, 10 µM CTx-AuIB reduced A_s/C_m by about 85% (Fig. 2), an amount consistent with the 75% contribution of 3*-nAChRs to A_s determined in the tests conducted with and without Bgt. The second Conus spp. toxin tested was CTx-MII, which blocks recombinant rat 32 nAChRs expressed in X. laevis oocytes (IC₅₀ = 0.5-3.5 nM) and is 2 orders of magnitude less potent on recombinant 7- and 34-nAChRs (Cartier et al., 1996; Harvey et al., 1997). Recent studies indicate that CTx-MII also recognizes nAChRs containing 6 subunits in mouse brain (Champtiaux et al., 2002). Although abundant in chick retina, 6 mRNA is absent from the chick peripheral nervous system, including the ciliary ganglion (Fucile et al., 1998), making it unlikely that 6 contributes to nAChRs in this system. Indeed, CTx-MII was previously shown to block slow, Bgt-insensitive synaptic currents in the chick ciliary ganglion (Ullian et al., 1997; Chen et al., 2001), suggesting that it targets 3*-nAChRs on the neurons. With Bgt present to block 7-nAChRs, CTx-MII applied for 30 to 60 min progressively inhibited the neuronal responses to 20 µM Nic, and a 300 nM dose reduced A_s/C_m by 90% (Figs. 2 and 3, A, C, and D). Although CTx-MII was more potent for these native neuronal nAChRs (IC₅₀ = 33 nM) than CTx-AuIB, it was less potent than reported previously for recombinant 32 nAChRs (Cartier et al., 1996; Harvey et al., 1997). The lower potency of CTx-MII seen here could reflect post-translational effects or the presence of 5 and 4 subunits in 3*-nAChRs. As with CTx-AuIB, however, the block of A_s was specific because the same dose had no effect on components attributable to 7-nAChRs. When applied without Bgt, 300 nM CTx-MII failed to detectably alter I_fast (Fig. 2), _f, or _i (data not shown). Once again, under these conditions, A_s/C_m was reduced by about 70%, in accord with a major yet not exclusive contribution of 3*-nAChRs to the slowly decaying current. The residual slow currents seen in the presence of CTx-AuIB or CTx-MII decayed with _s of 1100 and 800 ms, respectively, values that were not statistically different from that obtained for slow currents induced by the 7-nAChR selective agonist, GTS-21 (see below and Table 1). These results indicate that CTx-AuIB and -MII are potent antagonists for native 3*-nAChRs on ciliary ganglion neurons, and support findings obtained with Bgt and MLA suggesting that both 3*- and 7-nAChRs contribute to slowly decaying whole-cell responses induced by 20 µM Nic.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   -Conotoxins block 3*nAChR-mediated slow-decaying currents. A-C, both CTx-AuIB (B, 10 µM) and CTx-MII (C, 300 nM) block the slowly decaying current that persists in the presence of 60 nM Bgt (A). Calibration bars indicate 500 pA and 250 ms. D, dose-inhibition relation for -conotoxin effects on the peak residual slow current. Mean As/Cm values of (± S.E.M.) are depicted for Bgt-treated neurons (; n = 60) or for neurons treated with Bgt and the indicated -conotoxin applied at varying concentrations (filled symbols; n = 7-18). As/Cm values were fit by nonlinear regression analyses using the Hill equation. Arrowheads indicate the fit-derived concentrations of each -conotoxin predicted to produce half-maximal inhibition (IC50) of As/Cm (350 and 33 nM for CTx-AuIB and -MII, receptively).

GTS-21 Selectively Activates 7-nAChRs. To further assess the activation of 7-nAChRs and their contribution to whole-cell currents, we analyzed responses to GTS-21 (Fig. 4, Table 1), an 7-selective agonist also known as DMXB (Kem, 1997; Meyer et al., 1997; Papke et al., 2000). GTS-21 induced inward currents mediated solely by nicotinic nAChRs because they were completely blocked by the classic antagonist d-tubocurarine (dTC, 100 µM, not shown). The currents increased over a range of GTS-21 concentrations (Fig. 4, A and C), with the largest I_peak values attained at about 30 µM (EC₅₀  10-15 µM). When applied at doses higher than 30 µM, GTS-21 induced progressively smaller peak responses, consistent with the ability of agonists to block nAChRs when applied at high concentration. The efficacy of 30 µM GTS-21 in activating 7-nAChRs was comparable with that of 20 µM Nic because values of A_f/C_m were indistinguishable, and those of A_i/C_m were within 25%, for the two agonists (Table 1). The kinetics of fast and intermediate current decay displayed a similar pattern, with values of _f identical, and those of _i within a factor of 2, for the two agonists. In accord with the minor contribution of 7-nAChRs to slow currents determined using Nic, applications of 30 µM GTS-21 yielded mean A_s/C_m values that were only 20% of those obtained with 20 µM Nic. As expected for an 7-selective agonist, the fast, intermediate, and slow whole-cell current components induced by 30 µM GTS-21 were all blocked by 90% or more when neurons were pretreated with Bgt (Fig. 4, B and D). Interestingly, none of the currents induced by GTS-21 application were detectably affected by CTx-AuIB or CTX-MII (Fig. 3D), toxins that recognize and block recombinant 34 and 32 nAChRs, respectively, but have 10- and 400-fold lower potency for recombinant 7-nAChRs (Cartier et al., 1996; Luo et al., 1998). Choline has been shown to activate 7-nAChRs on rat brain neurons; because it is a partial agonist for 34-containing nAChRs on PC12 cells (Alkondon et al., 1997), we were concerned that it would also activate 3*-nAChRs in our system. Indeed, using Bgt or MLA to block the contribution from 7-nAChRs, we found that 1 mM choline still evoked substantial whole-cell currents, as well as long 25- and 40-pS single channel currents indicative of 3*-nAChR (Nai and Margiotta, unpublished observations; see below). These results indicated that choline would not adequately discriminate between 7- and 3*-nAChRs on chick ciliary ganglion neurons. By contrast, whole-cell results obtained with GTS-21 as well as single-channel results presented below indicate this agonist is highly selective for 7-containing Bgt-nAChRs on the neurons. Given these considerations, the whole-cell findings using Nic and GTS-21 support the idea that 7-nAChRs, in addition to underlying fast and intermediate decaying responses, also contribute to a more slowly decaying current. The slow current consistently decayed more rapidly when induced by GTS-21 than Nic (Table 1). Because 7-nAChRs underlie nearly all of the slow-decaying current induced by GTS-21 whereas 3*-nAChRs underlie most of that induced by Nic, this observation seems likely to reflect inherent differences in slow desensitization kinetics of the two receptor types. Considering the dominant contribution of 3*-nAChRs to A_s induced by 20 µM Nic, such differences are consistent with the nominal increase in _s seen after Bgt-treatment (Fig. 1, Table 1).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   GTS-21 selectively activates Bgt-nAChRs. A, increasing concentrations of GTS-21 (1, 3, 10, and 30 µM) induce progressively larger peak inward currents. B, the response to 30 µM GTS-21 is blocked by Bgt. Calibration bars indicate 1 nA and 100 ms. C, dose-response relation for GTS-21. Arrow indicates the approximate GTS-21 concentration required to induce half-maximal Ipeak/Cm (EC50 = 10-15 µM). Responses were obtained from 8 to 20 neurons at each concentration. Note that GTS-21 concentrations higher than 30 µM inhibit the response. D, summary of subunit-specific toxin effects on fast- and slow-component whole-cell responses to 30 µM GTS-21. Toxins were applied and results expressed as described in Fig. 2. Note that Bgt abolished Ifast/Cm and inhibited As/Cm by >90% (n = 8). The nominal changes in Ifast/Cm and As/Cm seen after treatments with CTx-AuIB (n = 5) or CTx-MII (n = 7), however, were not statistically significantly (p > 0.1 for each). Bars with asterisks above indicate that the parameter values were significantly different (p < 0.05) from those for control neurons; bars without asterisks indicate the values were not detectably different (p > 0.05) from control values.

Epibatidine Selectively Activates 3*-nAChRs. Using heterologously expressed nAChRs in X. laevis oocytes, epibatidine (Epi) was previously shown to be 100-fold more potent for chicken 34 and 32 nAChRs than for 7-nAChR homomers (Gerzanich et al., 1995). We therefore examined the ability of Epi to preferentially activate native 3*-nAChRs on ciliary ganglion neurons. Epi induced graded whole-cell currents (Fig. 5A) that were mediated solely by nicotinic nAChRs because they were completely blocked by dTC (not shown). Peak Epi-induced currents increased over a range of concentrations, with the maximal responses attained at about 30 µM (EC₅₀ = 0.56 µM; Fig. 5, A and C). As with Nic and GTS-21, a fast Epi response component (A_f) was apparent but only at relatively high concentrations (3-30 µM). We attribute this fast component to activation of 7-nAChRs having relatively low affinity for Epi for two reasons. First, Bgt blocked A_f and reduced I_peak by a complementary amount in neurons challenged with 30 µM Epi, whereas the toxin had no detectable effect on I_peak in neurons challenged with 0.1 µM Epi (Fig. 5, B and C). Second, the Epi dose-response relation predicts a 15-fold lower apparent affinity for the Bgt-sensitive A_f (EC₅₀ = 6 µM) than for the toxin-insensitive portion of the peak response (I_peak  A_f) (EC₅₀ = 0.4 µM). At concentrations 1 µM, however, Epi seemed selective for 3*-nAChRs. In particular, responses obtained using 0.1 µM Epi were insensitive to Bgt (Fig. 5, B and H) and blocked by CTx (Fig. 5, D, E, and H). These findings are in good agreement with results from expression studies (Gerzanich et al., 1995), and indicate that when applied at low concentration, Epi will preferentially activate 3*- over 7-nAChRs on ciliary ganglion neurons.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Low doses of epibatidine selectively activate 3*-nAChRs. A, Increasing Epi concentrations (0.03, 0.1, 0.3, 3, and 30 µM) evoke progressively larger whole-cell currents. B, responses to 0.1 µM EPI decay slowly and are unaffected by Bgt (60 nM), whereas those induced by 30 µM Epi feature both slow, Bgt-insensitive and fast, Bgt-sensitive decay components (compare with A). C, dose-response relation for Epi. Note that fast decay components () make a negligible contribution to Ipeak () at low Epi concentrations but that this contribution, correlated with Bgt-nAChRs by its sensitivity to Bgt (, ) increases at concentrations above 1 µM. Mean Ipeak/Cm and Af/Cm (±S.E.M.) values depicted were obtained from 9 to 24 neurons at each Epi concentration. D-E, CTx-AuIB (D) and CTx-MII (E) abolish response to 0.1 µM Epi and greatly attenuate the slow-decaying response component induced by 30 µM Epi, indicating that both arise from 3*-nAChRs. F-G, the unmasked fast decay response component induced by 30 µM Epi, is mediated by Bgt-nAChRs because it is blocked when Bgt is coapplied with either -conotoxin. Calibration bars indicate 1 nA and 250 ms. H. Summary of subunit-specific toxin effects on fast and slow component whole-cell responses to 0.1 and 30 µM Epi (n = 6 to 14 neurons for each condition). Results are expressed as described in Fig. 2. Bars with asterisks above indicate that the parameter values were significantly different (p < 0.05) from those for control neurons; bars without asterisks indicate the values were not detectably different (p > 0.05) from control values. Note that 0.1 µM Epi selectively activates Bgt-insensitive, -conotoxin-sensitive 3*-nAChRs, whereas at 30 µM, the agonist also activates Bgt-nAChRs. Toxins were applied in B and D-H as described in Fig. 2.

Combining 3*-nAChR Selective Agonists and Antagonists. The specificity of the two Conus spp. toxins for 3*- over Bgt-nAChRs demonstrated using Nic was tested further using agonists selective for the two nAChR types. We expected that the 3-selective conotoxins would be ineffective in blocking currents induced by the 7-nAChR selective agonist GTS-21 but highly potent in blocking currents induced by the 3*-nAChR selective agonist Epi, and both expectations were borne out. After treating ciliary ganglion neurons with CTx-AuIB or CTx-MII at doses sufficient to block the 3*-nAChR attributable responses to 20 µM Nic (10 µM and 300 nM, respectively) the fast, intermediate, and slow components of whole-cell currents induced by 30 µM GTS21 were unchanged (Fig. 4D). Conversely, the same conotoxin treatments reduced I_peak in response to 0.1 µM Epi by >90%, and reduced A_s in response to 30 µM Epi by about 80% (Fig. 5, D, E, and H). In the presence of either Conus spp. toxin (Fig. 5, D and E), 30 µM Epi induced a large, rapidly-decaying peak current that was indistinguishable from I_peak in control neurons. We attribute this rapidly decaying peak current to 7-nAChRs, usually masked by the somewhat slower-decaying Bgt-insensitive component seen in control neurons. When applied in conjunction with Bgt to block the contribution from 7-nAChRs, CTx-MII further attenuated, and CTx-AuIB abolished, peak responses to 30 µM Epi (Fig. 5, F, G, and H). These findings indicate that Bgt and both conotoxins are useful as specific antagonists for whole-cell currents mediated by Bgt- and 3*-nAChRs, respectively, on ciliary ganglion neurons. The ability of CTx-MII, a presumed 32 selective toxin, to block 90% of the 3*-nAChR mediated slow current seemed at odds with the previous demonstration that only about 20% of surface 3*-nAChRs contain 2 and hence a potential 32 interface. An explanation for this finding may be that, because of differences in channel properties, 3*-nAChRs containing 2 make a greater contribution to whole-cell currents than those lacking 2. This issue is addressed below.

Single-Channel Studies

Nicotine Activates 25-, 40-, 60-, and 80-pS nAChRs That Have Distinct Kinetic Properties. Single-channel studies were performed to correlate effects of the nAChR-specific agonists and toxins, as determined in whole-cell studies, with functional channel types. In outside-out neuron patches held at 100 mV, perfusion with 0.5 µM Nic activated single nAChR channel currents in four distinct amplitude ranges (1-4 in Fig. 6, A and D). The currents reflected nAChR activation because none were observed when the perfusion buffer lacked agonist or when the bath and perfusion buffer contained dTC (Fig. 6, B and C). The four current classes corresponded to nAChR channel conductances of about 25, 40, 60, and 80 pS, with 40-pS events predominating (Fig. 6, D and E). These values are nearly identical to those obtained previously using ACh except that the lowest conductance class scored closer to 30 pS (Margiotta and Gurantz, 1989; McNerney et al., 2000). To investigate nAChR kinetics, open durations corresponding to each conductance class were analyzed in patches in which the total number of events (N_t) exceeded 1000 (Fig. 7, Table 2). In these cases, open duration distributions for both 25- and 40-pS classes were well characterized by three mean open time constants [brief (_b  200 µs), intermediate (200 < _i  1000 µs), and long (_l > 1000 µs)] of approximately 50, 500, and 3000 µs, respectively (Fig. 7, A and B; Table 2). The vast majority of events described by _b (i.e., brief) were <200 µs in duration, and these comprised about 70 and 50% of the 25- and 40-pS events, respectively. By contrast, >90% of the 60-pS events and >99% the 80-pS events were classified as brief, with the open duration distributions dominated by a single component with _b of about 70 and 120 µs, respectively (Fig. 7, C and D; Table 2).

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Nicotine activates heterogeneous single-channel currents. A-C, Nic (0.5 µM) applied to an outside-out patch activated single-channel events at four (1, 2, 3, 4) discrete current levels (A). The currents arise from nAChRs because they were abolished by dTC (B, 100 µM) and absent when the patch perfusate lacked agonist (C). Calibration bars indicate 5 pA and 5 ms. Patches were held at 100 mV, and the currents sampled at 20-µs intervals and filtered at fc = 6.8 kHz, allowing detection of open or closed durations 100 µs (see Materials and Methods). D, single-channel amplitude histogram with four modal peaks reflecting mean current amplitudes corresponding to the four current levels in A. Note that current events of about 3.5 pA (level 2) predominate. E, current-voltage plots reveal four distinct nAChR channel slope conductances (indicated in box) for the patch depicted in A. The currents at each level reversed at about 10 mV. Overall, the conductances associated with channel current classes 1, 2, 3, and 4 were about 25, 40, 60, and 80 pS (means, 26, 42, 61, and 83 pS; n = 26), respectively.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Analysis of open durations for 25- (A), 40- (B), 60- (C), and 80-pS (D) nAChR channels. nAChRs were activated by applying 0.5 µM Nic to outside-out patches held at 100 mV as in Fig. 6. Nic-induced single channel currents were segregated by amplitudes (see Materials and Methods) corresponding to the indicated conductance classes and, in each case, open durations displayed on log duration versus square root axes. Solid lines represent the contributions of brief, intermediate-, and long events, as defined from maximum likelihood fits generated using IntrV (see Materials and Methods). Labeled arrows at individual peaks indicate the derived time constants (in microseconds). Note that brief nAChR openings made a major contribution to the histogram area for events in the 25-, 60-, and 80-pS conductance classes (AB = 69, 89, and 100%, respectively) but not in the 40-pS class (AB = 35%). The insets show the distribution of open durations for the same event classes obtained after treatment with 60 nM Bgt. Note that Bgt blocked nearly all brief events in every conductance class, without markedly affecting intermediate and long 25- and 40-pS events. Data used in main panels were obtained from the patch depicted in Fig. 6.

View this table: [in this window] [in a new window]   TABLE 2 Single-channel parameters for three nAChR agonists All values are presented as mean ± S.E.M. and were obtained from single channel current records obtained at 100 mV using 0.5 µM Nic, 1 µM GTS-21, or 10 nM Epi, each applied for 40 s. Open time constants () and relative histogram areas (A) for brief (B), intermediate (i) and long (L) openings in each conductance class () were derived from patches in which the total number of accepted events (NT) exceeded 1000 for Nic (NT = 1843 ± 287, n = 5 patches), 200 for GTS-21 (NT = 800 ± 264, n = 4), and 800 for Epi (NT = 1264 ± 291, n = 7) as outlined in the text. Because events <98 µs were unresolved, B values should be considered estimates. For all agonists, events with durations 200 µs were classified as brief, and those with durations >200 µs were classified as intermediate or long. The cutoff for long events was 1 ms for Nic and GTS-21 and 2 ms for Epi. Popen values shown were determined empirically from all patches with NT > 200 for Nic and Epi (NT = 749 ± 126, NT = 26 and 707 ± 142, n = 27, respectively) and NT > 100 for GTS-21 (NT = 464 ± 276, n = 8) as described under Materials and Methods. Popen,brief values were compiled from events with durations 200 µs, whereas Popen,long values were compiled from events with durations >200 µs (i.e., intermediate and long events). In the patches subjected to detailed kinetic analysis, Popen values obtained using the relevant  values were indistinguishable from those obtained empirically except for the long 25-pS nAChR events induced by nicotine. In one case, Popen,long derived using L (0.124) was about 2-fold larger than the empirically obtained value.

Bgt Blocks Brief-Duration 25-, 40-, 60-, and 80-pS nAChR Openings. To determine which single nAChR currents represent activation of 7-nAChRs, we first assessed the effects of 60 nM Bgt, a dose that blocked I_fast by >95% in whole-cell experiments (Fig. 2). When patches excised from neurons treated with the toxin were challenged with 0.5 µM Nic, single channel currents corresponding to the all-brief 60- and 80-pS openings were virtually absent, whereas those corresponding to longer duration 25- and 40-pS openings were preserved (Fig. 8A). Inspection of the records and open duration distributions from toxin-treated patches with N_t > 200 further revealed that Bgt also markedly reduced the sizable fraction of brief 25- and 40-pS nAChR openings (Fig. 7, insets). Thus, Bgt seems to target brief, Nic-induced nAChR openings in all four conductance classes. Because the Bgt sensitivity of 25- and 40-pS nAChR events was correlated with open duration, toxin effects were quantified by considering the brief (200 µs) 25-, 40-, 60-, and 80-pS events and the longer (>200 µs) 25- and 40-pS events separately. The relative open-time contribution of each conductance and kinetic class to the total record time (P_open) could then be determined empirically for all patches and the specificity of toxin effects assessed from changes in P_open (Fig. 8D). As indicated by analyses of open duration distributions (Fig. 7) brief openings make significant contributions to the total record time. In fact, the combined P_open values for brief Nic-induced 25-, 40-, 60-, and 80-pS openings is about 40% of that for the long 25- and 40-pS events (Fig. 8D, Table 2). Consistent with results obtained by comparing distributions of nAChR open durations, P_open values obtained with and without Bgt indicate that the toxin selectively targets brief Nic-induced nAChR openings (Fig. 8D). Specifically, Bgt reduced P_open values by >95% for the 60- and 80-pS nAChR events, and by >70% for brief 25- and 40-pS events, but failed to significantly change P_open for long 25- and 40-pS events (p = 0.08 and 0.18, respectively). In an earlier study, Bgt blocked 60- and 80-pS events but failed to detectably alter P_open associated with 25- and 40-pS nAChR openings (McNerney et al., 2000). This is not inconsistent with the present findings because events were previously sorted by conductance alone such that those scoring in 25- and 40-pS classes included long as well as brief openings. Because long 25- and 40-pS openings dominate open times for events in both of these conductance classes (Table 2) and are Bgt-insensitive, the associated P_open values for each will seem unchanged when sorted by conductance alone. Patches excised from six cells treated with MLA (50 nM, 1 h) and then challenged with 0.5 µM Nic yielded results that were indistinguishable from those obtained with Bgt (data not shown). In these cases, MLA reduced P_open values by >95% for the 60- and 80-pS nAChR events, and by 70% and 60% for brief 25- and 40-pS events, respectively, but failed to detectably change P_open for long 25- and 40-pS events (p = 0.84 and 0.08, respectively). Thus results obtained here using two 7-selective antagonists indicate that 7-nAChRs underlie both the majority of brief 25- and 40-pS nAChR events and essentially all 60- and 80-pS events activated by nicotine.

View larger version (23K): [in this window] [in a new window]   Fig. 8.   Subunit-selective toxins target different nAChR channel events. A-C, patches were excised from neurons treated with Bgt (A), CTx-AuIB (B), or CTx-MII (C) and challenged with 0.5 µM Nic plus the indicated toxin. For each toxin treatment, histograms portray the distribution of event amplitudes acquired in individual 40-s recordings, with sample events depicted in the insets (calibration bars indicate 5 pA and 5 ms). Patches were held at 100 mV, and the currents sampled at 20-µs intervals and filtered at fc = 6.8 kHz, allowing detection of open or closed durations 100 µs (see Materials and Methods). As in Fig. 6, the numbers 1, 2, 3, and 4 refer to current events corresponding to nAChR channel conductances of 25, 40, 60, and 80 pS. D, toxin blockade of Nic-induced nAChR openings. nAChR channel events were segregated according to conductance and mean open duration (brief or long), and toxin effects assessed by determining Popen associated with the corresponding events for each treatment condition as described in the text. Note that Bgt reduced Popen for brief open duration events of all four conductances whereas CTx-AuIB and CTx-MII block long 25- and/or 40-pS events, respectively. Apparent reductions in Popen for brief 25-pS events after CTx-AuIB and CTx-MII treatments, and for long 25-pS after Bgt treatments, were not statistically significant (p > 0.1 for each). Results depicted represent mean (±S.E.M.) determinations of Popen obtained from 12 to 26 outside-out patch recordings. Toxins were applied as described in Fig. 2.

-Conotoxins Target Long-Duration 25- and 40-pS nAChR Openings. Based on their selectivities in whole-cell studies, we next compared the effects of Conus spp. toxins with those of Bgt as a means of distinguishing single channel events mediated by 3*- versus 7-nAChRs. Neurons were treated with CTx-AuIB or CTx-MII using doses that maximally inhibited Nic-induced whole-cell 3*-nAChR currents (10 µM and 300 nM, respectively) and patches from treated neurons challenged with 0.5 µM Nic plus Conus spp. toxin. CTx-AuIB, which targets the 3/4 nAChR subunit interface (Luo et al., 1998), inhibited long 25- and 40-pS nAChR events (Fig. 8B), significantly reducing P_open associated with each by 50 and 91%, respectively (Fig. 8D). As in whole-cell studies, CTx-AuIB was specific for 3*-nAChRs in the sense that it failed to detectably change P_open for the brief 25-, 40-, 60-, or 80-pS nAChR events associated with 7-nAChRs. Because P_open for long 40-pS nAChR events far exceeds that of long 25-pS events, the effect of CTx-AuIB are in good agreement with its nearly complete block of 3*-nAChRs seen in whole-cell studies. Because about 20% of 3*-nAChRs on ciliary ganglion neurons contain 2, we next tested CTx-MII, a toxin that targets 3/2 nAChR interfaces (Cartier et al., 1996). Interestingly, CTx-MII was specific for long 40-pS nAChR events, inhibiting their appearance, and therefore reducing their P_open by 90%, without detectably affecting P_open for long 25-pS events (p = 0.75) (Fig. 8, C and D). As with CTx-AuIB, brief 25-, 40-, 60-, and 80-pS events associated with 7-nAChRs were unaffected by CTx-MII. The inability of either CTx-AuIB or -MII to affect P_open for the brief 25-, 40-, 60-, and 80-pS events further supports the attribution of these events to 7-nAChRs. In addition, the ability of CTx-AuIB to reduce P_open for long 25- and 40-pS nAChR currents is consistent with these events arising from 3*-nAChRs. From the known 3*-nAChR subtypes on the neurons (Vernallis et al., 1993; Conroy and Berg, 1995