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The Effects of Subunit Composition on the Inhibition of Nicotinic Receptors by the Amphipathic Blocker 2,2,6,6-Tetramethylpiperidin-4-yl Heptanoate

Department of Pharmacology and Therapeutics, College of Medicine, University of Florida, Gainesville, Florida (R.L.P., J.D.B., M.M.F., J.S.T.); and Department of Chemistry, University of Florida, Gainesville, Florida (K.I.C., N.A.H.)

Received February 1, 2005; accepted March 10, 2005

	Abstract

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The therapeutic targeting of nicotinic receptors in the brain will benefit from the identification of drugs that may be selective for their ability to activate or inhibit a limited range of nicotine acetylcholine receptor subtypes. In the present study, we describe the effects of 2,2,6,6-tetramethylpiperidin-4-yl heptanoate (TMPH), a novel compound that is a potent inhibitor of neuronal nicotinic receptors. Evaluation of nicotinic acetylcholine receptor (nAChR) subunits expressed in Xenopus laevis oocytes indicated that TMPH can produce a potent and long-lasting inhibition of neuronal nAChR formed by the pairwise combination of the most abundant neuronal (i.e., 3 and 4) and subunits (2 and 4), with relatively little effect, because of rapid reversibility of inhibition, on muscle-type (11) or 7 receptors. However, the inhibition of neuronal subunit-containing receptors was also decreased if any of the nonessential subunits 5, 6, or 3 were coexpressed. This decrease in inhibition is shown to be associated with a single amino acid present in the second transmembrane domain of these subunits. Our data indicate great potential utility for TMPH to help relate the diverse central nervous system effects to specific nAChR subtypes.

Simple models for nAChR subtypes are provided by pairwise combinations of and subunits expressed in Xenopus laevis oocytes. However, there is a growing appreciation that ancillary subunits such as 5, 6, and 3 that work poorly in pairwise combinations with other single subunits, but nonetheless contribute to functionally important receptor subtypes in vivo. We show that for the commonly used nAChR subunit pairwise combinations inhibition by TMPH is only very slowly reversible. However, the incorporation of these additional subunits results in receptors that recover more rapidly and would thus show lower equilibrium inhibition in vivo. Therefore, based on the characterization of this agent's effects on specific nAChR subtypes, TMPH may identify the particular molecular substrates that underlie the multiple effects of nicotine in the brain.

	Materials and Methods

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Synthesis
Chemicals used for the synthesis were purchased from Aldrich Chemical Co. (Milwaukee, WI). Compounds were characterized by ¹H NMR and fast atom bombardment-mass spectrometry.

TMPH Synthesis. To a mixture of 2,2,6,6-tetramethyl-4-piperidinol (472 mg; 3.0 mmol) and methyl heptanoate (476 mg; 3.3 mmol) in 3.0 ml of dimethyl formamide was added 250 mg of powdered potassium carbonate. The resulting mixture was heated at 145 to 155°C for 64 h under a gentle stream of N₂. After cooling, the reaction mixture was partitioned between water and hexanes. The organic layer was separated, washed with water (two times) and brine, and then dried over anhydrous MgSO₄ and evaporated to afford the crude product as an oil. The oil was dissolved in MeOH and was then treated with 2 equivalents of concentrated HCl. The solvent was removed in vacuo, and the residue was then treated with diethyl ether. The resulting solids were removed by filtration. The ethereal filtrate was concentrated in vacuo and triturated with hexane to afford 380 mg (41%) of TMPH hydrochloride. It was recrystallized from boiling ethyl acetate/hexane to afford short colorless needles, melting point 113 to 115°C. Fast atom bombardment-high resolution mass spectrometer: calculated(C₁₆H₃₂NO₂): 270.2433 found: 270.2435.

Expression in X. laevis Oocytes
Mature (>9 cm) female X. laevis African frogs (Nasco, Ft. Atkinson, WI) were used as a source of oocytes. Before surgery, frogs were anesthetized by placing the animal in a 1.5 g/l solution of 3-aminobenzoic acid ethyl ester for 30 min. Oocytes were removed from an incision made in the abdomen.

To remove the follicular cell layer, harvested oocytes were treated with 1.25 mg/ml type 1 collagenase (Worthington Biochemicals, Freehold, NJ) for 2 h at room temperature in calcium-free Barth's solution (88 mM NaCl, 1 mM KCl, 0.33 mM MgSO₄, 2.4 mM NaHCO₃, 10 mM HEPES, pH 7.6, and 50 mg/l gentamicin sulfate). Thereafter, stage 5 oocytes were isolated and injected with 50 nl (5–20 ng) each of the appropriate subunit cRNAs. Recordings were made 2 to 15 days after injection.

Preparation of RNA. Rat neuronal nAChR clones and mouse muscle nAChR cDNA clones were used. The wild-type clones were obtained from Dr. Jim Boulter (UCLA, Los Angeles, CA). The rat 6/3 (Dowell et al., 2003) clone was obtained from Michael McIntosh (University of Utah, Salt Lake City, UT) and expressed in X. laevis oocytes in combinations with rat 2 and 3. The original 6/3 construct provided was sequenced and was found to have a mutation in the second transmembrane domain (TM2) sequence, which exchanged a valine for an alanine in the 7' position (TM2 numbering scheme; Miller, 1989). The TM2 domain is understood to line the pore of the channel, with helix structure. The 7' position may actually be directed away from the actual pore lining, but this residue is highly conserved in all of the nAChRs. It is valine in all of them except 9 (isoleucine) and 1, where it is alanine. The TM2 mutation in the 6/3 chimera was corrected by using QuikChange (Stratagene, La Jolla, CA) according to their protocols. The corrected 6/3 chimera sequence was confirmed by restriction diagnostics and automated fluorescent sequencing (University of Florida core facility). The corrected clone was expressed as above in X. laevis oocytes with 2 and 3 and compared with the wild-type 3 coexpressed with 2 and 3. After linearization and purification of cloned cDNAs, RNA transcripts were prepared in vitro using the appropriate mMessage mMachine kit from Ambion (Austin, TX).

Electrophysiology. The majority of experiments were conducted using OpusXpress 6000A (Axon Instruments Inc., Union City, CA). OpusXpress is an integrated system that provides automated impalement and voltage clamp of up to eight oocytes in parallel. Cells were automatically perfused with bath solution, and agonist solutions were delivered from a 96-well plate. Both the voltage and current electrodes were filled with 3 M KCl. The agonist solutions were applied via disposable tips, which eliminated any possibility of cross-contamination. Drug applications alternated between ACh controls and experimental applications. Flow rates were set at 2 ml/min for experiments with 7 receptors and 4 ml/min for other subtypes. Cells were voltage-clamped at a holding potential of –60 mV. Data were collected at 50 Hz and filtered at 20 Hz. Agonist applications were 12 s in duration followed by 181-s washout periods for 7 receptors and 8 s with 241-s wash periods for other subtypes. For some experiments, particularly under conditions where residual inhibition precluded making repeated measurements from single cells (see below), manual oocyte recordings were made as described previously (Papke and Papke, 2002). In brief, Warner Instrument (Hamden, CT) OC-725C oocyte amplifiers were used, and data were acquired with a MiniDigi or Digidata 1200A with pClamp9 software (Axon Instruments Inc.). Sampling rates were between 10 and 20 Hz and the data were filtered at 6 Hz. Cells were voltage clamped at a holding potential of –50 mV. Data obtained with these methods were comparable with those obtained with OpusXpress.

Experimental Protocols and Data Analysis. Each oocyte received two initial control applications of ACh, an experimental drug application (or coapplication of ACh and TMPH), and then follow-up control application(s) of ACh. The control ACh concentrations for 11, 34, 42, 32, 325, 323, 6/323 643, and 7, receptors were 30, 100, 10, 30, 1, 100, 100, 100, and 300 µM, respectively. These concentrations were selected because they gave large responses with relatively little desensitization so that the same oocyte could be stimulated repeatedly with little decline in the amplitude of the ACh responses. This allowed us to separate out the inhibitory effects of the antagonist from possible cumulative desensitization.

Responses to experimental drug applications were calculated relative to the preceding ACh control responses to normalize the data, compensating for the varying levels of channel expression among the oocytes. Responses were characterized based on both their peak amplitudes and the net charge (Papke and Papke, 2002). In brief, for net charge measurement a 90-s segment of data beginning 2 s before drug application was analyzed from each response. Data were first adjusted to account for any baseline offset by subtracting the average value of 5-s period of baseline before drug application from all succeeding data points. When necessary, baseline reference was also corrected for drift using Clampfit 9.0 (Axon Instruments Inc.). After baseline correction, net charge was then calculated by taking the sum of all the adjusted points. The normalized net charge values were calculated by dividing the net charge value of the experimental response by the net charge value calculated for the preceding ACh control response. Means and S.E.M. were calculated from the normalized responses of at least four oocytes for each experimental concentration. To measure the residual inhibitory effects, this subsequent control response was compared with the preapplication control ACh response.

For concentration-response relations, data derived from net charge analyses were plotted using Kaleidagraph 3.0.2 (Abelbeck/Synergy, Reading, PA). Curves were generated from the Hill equation as follows:

where I_max denotes the maximal response for a particular agonist/subunit combination, and n represents the Hill coefficient. I_max, n, and the EC₅₀ were all unconstrained for the fitting procedures. Negative Hill slopes were applied for the calculation of IC₅₀ values.

View larger version (18K): [in this window] [in a new window]   Fig. 1. TMPH produces selective long-term inhibition of neuronal ganglionic type 34 receptors. A, chemical structure of TMPH. B, top, raw data traces obtained from an oocyte expressing mouse muscle-type 11 subunits to the application of either 10 µM ACh alone () or the coapplication of 10 µM ACh and 300 nM TMPH (arrow). Bottom, averaged normalized data (±S.E.M.; n 4) from oocytes expressing 11 subunits to the coapplication 10 µM ACh and a range of TMPH concentrations. C, top, raw data traces obtained from an oocyte expressing rat ganglionic-type 34 subunits to the application of either 100 µM ACh alone () or the coapplication of 100 µM ACh and 300 nM TMPH (arrow). Bottom, averaged normalized data (±S.E.M.; n 4) from oocytes expressing 34 subunits to the coapplication 100 µM ACh and a range of TMPH concentrations. Three values are plotted in each of the concentration-response curves: the peak current amplitude of the coapplication response, normalized to the peak amplitude of the previous ACh control (); the net charge of the coapplication response, normalized to the net charge of the previous ACh control () (Papke and Papke, 2002); and the peak current amplitude of the ACh control response obtained after the TMPH/ACh coapplication, normalized to the peak amplitude of the previous ACh control ().

	Results

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TMPH Inhibition of Acetylcholine Receptor Subtypes. TMPH (Fig. 1) was initially tested on mouse muscletype (11) nAChR, three different pairwise combinations of rat neuronal and subunits (34, 42, and 32), and 7 homomeric neuronal nAChR. In addition, combinations of three subunits and an 6/3 chimera were tested. The results are summarized in Table 1. As shown in Fig. 1, both muscle-type and 34 receptors (a minimal model for ganglionic-type receptors) were inhibited during the coapplication of ACh and TMPH. Although the inhibition of muscle-type receptors was readily reversible after a 5-min wash, the inhibition of 34 receptors persisted after the wash. The data also indicate that the inhibition of 34 receptors became progressively greater during the coapplication response so that the inhibition of net charge was greater than the inhibition of peak current. This was not the case for the inhibition of muscle-type receptors. The other neuronal - subunit pairs tested, 42 and 32, were blocked in a manner similar to 34 receptors (Fig. 2), with a larger inhibition of net charge than peak current and virtually no recovery after a 5-min wash. In contrast, 7 receptors, like muscle-type receptors, showed little difference between the inhibition of peak currents and net charge and showed significant recovery after a 5-min wash (Fig. 2). These differences are reflected in the IC₅₀ values presented in Table 1. Note that receptors that rapidly equilibrate inhibition and recover readily (e.g., muscle-type receptors and 7) have ratios of the IC₅₀ for net charge to the IC₅₀ for peak currents of close to 1 (Table 2). In contrast, for receptor types that show progressively more inhibition during the coapplication and have slow recovery, the ratio of the IC₅₀ for net charge to the IC₅₀ for peak currents is much less than 1. Therefore, for receptors that show progressively more inhibition during the coapplication IC₅₀ values estimated from the inhibition of net charge are similar to those that can be derived from persistent inhibition measured after a 5-min wash (i.e., from the recovery data; Table 2).

View larger version (13K): [in this window] [in a new window]   Fig. 2. TMPH inhibition of responses obtained from oocytes expressing rat neuronal nAChR subunits. Shown are the averaged normalized data (±S.E.M.; n 4) from oocytes expressing 42, 32, and 7 subunits (from top to bottom, respectively) to the coapplication ACh and a range of TMPH concentrations. Three values are plotted in each of the concentration-response curves: the peak current amplitude of the coapplication response, normalized to the peak amplitude of the previous ACh control (); the net charge of the coapplication response, normalized to the net charge of the previous ACh control (); and the peak current amplitude of the ACh control response obtained after the TMPH/ACh coapplication, normalized to the peak amplitude of the previous ACh control (). The control ACh concentrations used were 10, 30, and 300 µM for 42, 32, and 7, respectively.

View larger version (17K): [in this window] [in a new window]   Fig. 9. TMPH inhibition of responses obtained from oocytes expressing 4, 3, and either 3 (A) or 6 subunits (B). Shown are the averaged normalized data (±S.E.M.; n 4) to the coapplication 100 µM ACh and a range of TMPH concentrations. Three values are plotted in each of the concentration-response curves: the peak current amplitude of the coapplication response, normalized to the peak amplitude of the previous ACh control (); the net charge of the coapplication response, normalized to the net charge of the previous ACh control (); and the peak current amplitude of the ACh control response obtained after the TMPH/ACh coapplication, normalized to the peak amplitude of the previous ACh control (). Note that the recovery data for oocytes expressing 3, 4, and 3 could not be fit to a one site model. The curve fit show is for a two-site model with approximately 15% fit to an IC50 value of 1 µM and 85% fit to an IC50 value of g30 µM.

Neuronal nAChR Recovery Rates. Our initial experiments evaluated recovery after only a single 5-min wash. To evaluate the actual rates at which 7 and the various nAChR pairwise subunit combinations recovered from TMPH-induced inhibition, we made repeated applications of ACh alone after a single coapplication of ACh and TMPH. As shown in Fig. 3, the rat 42, 32, 34 receptors showed virtually no detectable recovery over a period of 30 min, whereas 7 receptors were fully recovered after approximately 15 min of wash.

View larger version (20K): [in this window] [in a new window]   Fig. 3. TMPH produces long-term inhibition of neuronal beta subunit-containing nAChR but not 7 homomeric receptors. ACh and TMPH were coapplied at time 0 to oocytes expressing rat 34, 42, 32, or 7 subunits. Thereafter, control ACh applications were made at 5-min intervals to measure recovery. The control ACh concentrations used were 100, 10, 30, and 300 µM for 34, 42, 32, and 7, respectively. The initial inhibition was produced by the coapplication of ACh at the control concentration and 30 µM TMPH, except in the case of the 32 receptors, which were inhibited by the coapplication of ACh and 3 µM TMPH.

View larger version (10K): [in this window] [in a new window]   Fig. 4. Varying amounts of use-independent inhibition of neuronal subunit-containing receptors by TMPH. A, representative data showing the effects of the application of 1 µM TMPH alone to 42- and 32-expressing oocytes (open arrows), compared with the application of ACh alone () either before or after the application of TMPH. The control ACh concentrations used were 10 and 30 µM for 42 and 32, respectively. B, comparison of the use-dependent (1 µM TMPH + ACh) and use-independent (1 µM TMPH alone) inhibition of 34, 42, and 32 receptors by TMPH. The data are calculated from the peak amplitudes of control ACh responses obtained after the application of TMPH ± ACh, expressed relative to the peak current amplitude of ACh control responses before the application of TMPH.

Progressive Inhibition of 42 Receptors by Repeated Coapplications of ACh and TMPH below Its IC₅₀ Value. The IC₅₀ values presented in Table 1 were based on the inhibition produced by single coapplications (20 s in duration) of ACh and TMPH. Because for the neuronal subunit-containing receptors the onset of inhibition is apparently much faster than the reversibility of inhibition, measurements based on single applications of TMPH are likely to underestimate what equilibrium IC₅₀ values would be. To test the hypothesis that repeated applications of TMPH would produce an accumulated inhibition that would be greater than the inhibition produced by a single application, we made repeated coapplications of ACh and 100 nM TMPH to oocytes expressing 42 receptors. Coapplications of TMPH and ACh were alternated with applications of ACh alone. As shown in Fig. 5, repeated coapplications of ACh with 100 nM TMPH (IC₅₀ in single-dose experiments) produced 90% inhibition after three applications at 10-min intervals. Further applications did not produce additional inhibition. Making a corresponding shift in the 42 net charge inhibition curve in Fig. 3 (i.e., so that 100 nM is the IC₉₀ rather than the IC₅₀) suggests that the equilibrium IC₅₀ would be approximately 10 nM.

View larger version (13K): [in this window] [in a new window]   Fig. 5. Cumulative inhibition by repeated application of TMPH. A, responses of an oocyte expressing 42 receptors to alternating applications of 30 µM ACh alone () or 30 µM ACh plus 100 nM TMPH (arrows). The total inhibition increased during the first 3 TMPH/ACh coapplications. Note that the TMPH/ACh coapplications differ in kinetics from the ACh controls, showing a greater inhibition of net charge than of peak current. B, normalized average responses of oocytes expressing 42 receptors (±S.E.M.; n 4) to alternating applications of ACh alone or ACh plus TMPH, as in A. Both peak currents and net charge values are plotted, normalized to the ACh control response recorded before the first ACh/TMPH coapplication (t = –5 min). Note that the traces in A are shown on the same time scale as the x-axis in B.

Effect of 5 Coexpression with 32 Subunits on the Sensitivity to TMPH. As noted above, efforts to connect data obtained from oocyte studies with in vivo data can be complicated by the fact that nAChR in vivo may have more complex subunit composition than the simple pairwise / subunit combinations most readily tested in oocytes. One such subunit that contributes to the complexity of acetylcholine receptors in vivo is 5, which is not required to coassemble with other subunits for them to function but is likely to be present in some receptor subtypes in vivo (Wang et al., 1996; Gerzanich et al., 1998). We tested the hypothesis that the presence of the 5 subunit could modulate the sensitivity of a neuronal nAChR subunit to TMPH. For these experiments, we used human 3 and 2 subunits, which readily form receptors with or without the coexpression of the human 5 subunit. We chose to use these subunits because the successful inclusion of the 5 subunit produces an easily detectable change in receptor pharmacology, increasing the potency of ACh (Gerzanich et al., 1998). All batches of oocytes used for these experiments were confirmed to have this predicted effect of 5 expression.

As shown in Fig. 6, ACh responses of oocytes expressing human 32 and human 325 showed similar sensitivity to TMPH during the initial coapplication. (The IC₅₀ values based on net charge analysis were 460 ± 170 and 430 ± 180 nM, respectively.) However, as shown in Fig. 7, oocytes expressing 5 along with 3 and 2 showed much faster recovery than those expressing 3 and 2 alone. The responses of oocytes expressing 325 had a half-time of recovery of approximately 15 min, whereas those expressing human 32 receptors showed no significant recovery over a period of 40 min, similar to the oocytes expressing rat 32 receptors (Fig. 3).

View larger version (15K): [in this window] [in a new window]   Fig. 6. TMPH inhibition of responses obtained from oocytes expressing human neuronal nAChR subunits. Shown are the averaged normalized data (±S.E.M.; n 4) from oocytes expressing human 32 or 325 subunits (top and bottom, respectively) to the coapplication ACh and a range of TMPH concentrations. Three values are plotted in each of the concentration-response curves: the peak current amplitude of the coapplication response, normalized to the peak amplitude of the previous ACh control (); the net charge of the coapplication response, normalized to the net charge of the previous ACh control (); and the peak current amplitude of the ACh control response obtained after the TMPH/ACh coapplication, normalized to the peak amplitude of the previous ACh control (). The control ACh concentrations used were 30 and 1 µM for 32 and 325 expressing oocytes, respectively.

View larger version (13K): [in this window] [in a new window]   Fig. 7. Recovery of human 5-containing 32 receptors from TMPH-produced inhibition is rapid compared with the recovery of receptors formed with 32 subunits alone. A, representative traces obtained from oocytes expressing human 32 or 325 subunits. After an initial application of ACh alone (), a single coapplication was made of ACh and 1 µM TMPH (arrow). Recovery was evaluated by making repeated control ACh applications at 5-min intervals (open circles). B, normalized average responses of oocytes expressing 32 or 325 receptors (±S.E.M.; n 4) to repeated applications of ACh alone, after a single application of ACh plus TMPH (as in A). Peak currents are plotted, normalized to the ACh control response recorded before the ACh/TMPH coapplication. Note that the traces in A are shown on the same time scale as the x-axis in B.

The addition of the 3 subunit to the 3 and 2 subunits had the effect of decreasing sensitivity to an initial application of TMPH (Fig. 8A) such that the IC₅₀ for inhibiting 323 receptors was at least an order of magnitude higher than for the inhibition of 32 without 3 (Figs. 2 and 8; Table 1). The receptors containing the 6/3 chimera in combination with 2 and 3 were not significantly different in their sensitivity to TMPH from those made up of the 323 wild-type subunits (Fig. 8B; Table 1). The recovery of 3-containing receptors from TMPH was relatively complex. There was approximately 50% recovery in the first 10 min, but no further recovery after that. This was similar for both 323 and 6/323 (Fig. 8C). One possible explanation for this would be if the coexpression of these subunits resulted in mixed populations of receptors, some containing 3 subunits and showing rapid recovery, and others formed without 3 and showing the nearly irreversible block seen when 3 and 2 are expressed as a pair. This is certainly a likely scenario for the combination containing wild-type 3 and may also be the case for the combination containing the chimera, because in our experience there is an increase of approximately 2-fold in currents when 3 is coexpressed with 2 and the 6/3 chimera compared with 6/3 and 2 alone (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 8. TMPH inhibition of responses obtained from oocytes expressing 3 and chimeric 6/3 subunits. Shown are the averaged normalized data (±S.E.M.; n 4) from oocytes expressing human 323or 6/323 subunits (A and B, respectively) to the coapplication ACh and a range of TMPH concentrations. Three values are plotted in each of the concentration-response curves: the peak current amplitude of the coapplication response, normalized to the peak amplitude of the previous ACh control (); the net charge of the coapplication response, normalized to the net charge of the previous ACh control (); and the peak current amplitude of the ACh control response obtained after the TMPH/ACh coapplication, normalized to the peak amplitude of the previous ACh control (). The control ACh concentration used was 100 µM. C, progressive recovery of oocytes expressing human 323 or 6/323 subunits. Data at t = 0 represents the average inhibition of peak currents during the coapplication of 100 µM ACh and 10 µM TMPH. Following points represent the responses to subsequent applications of 100 µM ACh alone at 5-min intervals. Points represent the average (±S.E.M.) of at least four oocytes, and the data were normalized to the peak responses obtained 5 min before the ACh/TMPH coapplication.

The responses of oocytes expressing 643 showed essentially full recovery after only a single wash period (Fig. 9B). However, as with the oocytes expressing 3, 2, and 3, it is likely that the cells injected with 3, 4, and 3 had a mixed population of receptors because the recovery data were best fit with a two-site model (Fig. 9A).

Sequence in the Pore-Forming TM2 Domain Regulates TMPH Inhibition. Although receptors containing pairwise combinations with either 2 or 4 showed nearly irreversible inhibition by TMPH, inclusion of any of the common ancillary subunits (5, 6, or 3) resulted in more readily reversible inhibition. Sequence in the pore-forming second transmembrane domain (TM2) is important for regulating sensitivity to other noncompetitive inhibitors, including mecamylamine and BTMPS, which is structurally related to TMPH (Francis et al., 1998; Webster et al., 1999). As shown in Fig. 10A, one point of sequence difference that stands out between 2 and 4 compared with the other neuronal nAChR is at the 10' position (i.e., the 10th residue within the second putative transmembrane domain; Miller, 1989). In particular, there is an alanine in 2 and 4 at this site and either a serine or threonine in subunits associated with reduced sensitivity to TMPH.

View larger version (22K): [in this window] [in a new window]   Fig. 10. Amino acid sequence in the pore-forming transmembrane domain regulates TMPH inhibition. A, sequence of TM2 domains of the nAChR subunits in this study. The sequences are aligned and numbered as proposed by Miller (1989). The 10' site is highlighted. B, representative responses of an oocyte coexpressing 3 and a 4 mutant in which a threonine was substituted for the alanine at the 10' position. Shown are responses to 100 µM ACh alone, 100 µM ACh coapplied with 30 µM TMPH, and again 100 µM ACh alone. Responses were recorded from the same oocyte at 5-min intervals. During the coapplication, peak currents were reduced on average (n = 8) to 9.8 ± 1.0% of the control and the net charge during the coapplication was reduced to 7.7 ± 1.2% of the controls. C, progressive recovery of oocytes expressing 3 and the 4 10'T mutant. Data at t = 0 represents the average inhibition of peak currents during the coapplication of ACh and TMPH. The following points represent the responses to subsequent applications of ACh alone at 5-min intervals. Points represent the average (±S.E.M.) of at least four oocytes, and the data were normalized to the peak responses obtained 5 min before the ACh/TMPH coapplication. Data for the 34 wild type (from Fig. 3) are provided for comparison. Time constants were estimated from exponential functions fit to the data (not shown).

We coexpressed 3 and a previously published 4 mutant (Webster et al., 1999) that contains a threonine substitution at the 10' site. Although these receptors were strongly inhibited during a coapplication of 100 µM ACh and 30 µM TMPH, there was significant recovery after a single 5-min washout (Fig. 10B). The 3410'T receptors recovered from TMPH inhibition with an estimated time constants of 25 ± 3 min, whereas for 34 wild-types the estimated time constant for recovery was 450 ± 50 min (Fig. 10C, wild-type data from Fig. 3 shown for comparison).

Selective Inhibition by TMPH of Mixed Receptor Responses. Oocytes injected with a combination of 7 RNA and 3 plus 4 RNA show responses that exhibit both fast and slow components (Fig. 11A), hypothetically representing two populations of receptors. When these receptors are expressed separately, 7 receptors show only transient block by TMPH, whereas 34 receptors show prolonged blockade. When TMPH and ACh are coapplied to an oocyte injected with all three subunits, the rapid component of the response, presumably corresponding to the 7 receptors is resistant, as can be seen after a 5-min washout. In contrast, most of the slow component of the mixed response was eliminated by the ACh/TMPH coapplication.

View larger version (10K): [in this window] [in a new window]   Fig. 11. Long-term inhibition of the slow components of mixed nicotinic receptor mediated responses. A, responses of an oocyte injected with RNA coding for 7 as well as 3 and 4. The first response was evoked by the application of 1 mM ACh (closed bar). The second response was evoked by the application of 1 mM ACh plus 30 µM TMPH (open bar). The subsequent responses shown were to additional applications of 1 mM ACh alone at 5-min intervals. The expanded traces below show the two kinetic components in the ACh-evoked responses before (*) and after (**) the TMPH/ACh coapplication. In the traces on the left, an estimate of the slow component is shaded out, to illustrate the relative sparing of the fast 7-like component. B, responses of an oocyte expressing both 34 and 7-type receptors. Before the coapplication-evoked response 30 µM TMPH was preapplied for 30 s to ensure that the full concentration of TMPH was present during the early 7-mediated phase of the mixed receptor response. Even though transient inhibition was increased with this protocol compared with the simple preapplication protocol shown in A, there was good recovery of a fast component of the mixed response after only a 5-min wash. C, responses from a patch-clamped neuron in a rat brain slice of the septum. A double-barreled picospritzer pressure application system was used with one barrel containing 1 mM ACh and the other containing 1 mM ACh and 300 µM TMPH. Three initial responses to ACh alone were obtained at 30-s intervals and the averaged response is shown (*). Two applications separated by 30 s were then made from the barrel containing both TMPH and ACh, and the average of those responses is shown. After the ACh/TMP applications, ACh alone was repeatedly applied at 30-s intervals. Shown are the averages of two traces obtained after either 60- and 90-s washout or 150- and 180-s washout (**). As shown in the overlay at the bottom, after washout the peak current amplitudes of the average response (gray) were essentially the same as the average response before the TMPH (black) application, but the late phase of the current is largely absent. Initial responses of the neuron to ACh (*) were well fit with two-exponential decay rates, with two time constants of 218 and 12 ms representing 21 and 79% of the area, respectively. After 2.5 to 3 min of recovery (**), the current decay was best fit with two time constants of 100 and 10 ms, representing 13 and 87% of the area, respectively.

Neurons in the rat septum vary in their nAChR expression in ways that are correlated to their physiological and neurotransmitter phenotypes. Septal neurons that have fast firing rates, likely to be GABAergic, often have both fast and slow components to their ACh-evoked responses, with the fast component blockable by methyllycaconitine (Thinschmidt et al., 2004; Henderson et al., 2005). Figure 11C shows the ACh-evoked response from a septal neuron recording in a fresh brain slice. The coapplication of TMPH and ACh evoked a relatively small response, suggesting that TMPH was an effective inhibitor of the neuronal nAChR in this ex vivo preparation. During the coapplication peaks were reduced to 17 ± 3% of the original average response (n = 2) and the net charge of the coapplication responses were likewise reduced to 15 ± 10%. After 3 min of washout after the TMPH application, there was a differential recovery of peak and net charge. Peaks amplitude had returned to 95% ± 1% of the control amplitude, whereas there was still a 43 ± 9% inhibition of the net charge. As shown in the figure inset, most of that net charge was associated with the 7-like component of the current.

	Discussion

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Our experiments show that TMPH is a potent noncompetitive antagonist of neuronal nAChR. Its properties are in many ways similar to its bis analog BTMPS (Papke et al., 1994), except that TMPH can produce both use-dependent and for some receptor subtypes (notably, the abundant 42 subtype), inhibition, that does not require prior channel activation. Such use-independent inhibition, coupled with slow reversibility suggests that relatively low steady concentrations of TMPH could profoundly down-regulate the function of sensitive nAChR subtypes of the brain, beyond the levels that might be achieved by activity-driven inhibition.

Although experimental agonists and antagonists are the most basic tools of pharmacology, agents with known selectivity for specific receptor subtypes are markedly better tools for understanding the molecular substrates of brain function and potential therapeutic targets. TMPH may be just such a tool for helping us understand brain nicotine receptors. However, neither BTMPS nor the prototypical ganglionic blocker mecamylamine has been tested on the same array of defined subunit combinations as were used in the current study, so the challenge remains to better understand the activity of these inhibitors.

In recent years, the nomenclature for brain nAChR has been changing in such a way as to reflect our acknowledged uncertainty about the exact subunit composition of brain nAChR. What were previously referred to as 42 receptors are now often 42* or even just 4* receptors (Tapper et al., 2004), where the asterisk indicates the uncertain presence or absence of such ancillary subunits that we show determine the reversibility of TMPH inhibition. The present studies show the potential utility of TMPH as an experimental tool that may allow us in some cases to remove the asterisk and in other cases to confirm its appropriateness.

The data obtained with the 4 10' mutant point to the importance of the neuronal 2 and 4 subunit TM2 sequence for defining the specificity of ganglionic blockers, and suggests at least some mechanistic similarities between TMPH inhibition and inhibition by mecamylamine (Webster et al., 1999). The apparent requirement for an alanine at that level of the pore suggests that a hydrophobic interaction may be the basis for long-lived inhibition by TMPH. Although it is a pharmacological convenience to have ganglionic blocking drugs that selectively inhibit neuronal receptors based on the subunit sequence, presumably this crucial sequence element plays some important, albeit unknown, functional role in vivo. Moreover, just as this site makes a crucial distinction between ganglionic and muscle-type receptors, it seems likely that it is of functional importance for those nAChR of the brain that incorporate the serine-containing ancillary subunits.

Based on its selectivity for long-lived inhibition of some nAChR subunit combinations and relative sparing of others, TMPH will be useful in sorting out the effects associated with complex receptor subtypes with in vitro preparations such as fresh brain slices (Fig. 11C). It may also show selectivity for blocking effects of nicotine in vivo (M. I. Damaj and R. L. Papke, manuscript submitted for publication). TMPH also may be of interest from the perspective of therapeutics because nicotinic antagonists have been proposed to be possible adjunct therapies for both Tourette's syndrome (Sanberg et al., 1998) and smoking cessation (Rose et al., 1994). The prototypical antagonist mecamylamine was used for these initial studies and the characterization of selective antagonists such as TMPH may lead the way to the development of better therapies for these, and potentially other, neuropsychiatric indications based on a more limited profile of side effects. Although the sensitivity of 34 receptors to TMPH might suggest a high liability for peripheral side effects, this may not be the case because 5 is likely to be present in ganglionic receptors (Vernallis et al., 1993).

In conclusion, the results we report suggest that drug therapies for the inhibition of central nervous system nicotinic receptors may be developed with greater selectivity than previously appreciated. Although more selective antagonists such as methyllycaconitine are known, these generally work poorly with systemic administration. In addition to the potential therapeutic significance of TMPH, this drug may also prove to be a valuable tool to combine with selective agonists and knockout animals to further unravel the mystery of how neuronal nicotinic receptors play a role in brain function.

	Acknowledgements

 
We thank Julia Porter Papke for technical assistance. We are very grateful to Axon Instruments Inc. for the use of an OpusXpress 6000A and pClamp9. We particularly thank Dr. Cathy Smith-Maxwell for support and help with OpusXpress.

	Footnotes

 
This work was supported by National Institute on Drug Abuse grant DA017548; National Institutes of Health Grants NS32888, P01-AG10485, and MH11258; and the University of Florida College Incentive fund.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.105.011676.

ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; BTMPS, bis-(2,2,6,6-tetramethyl-4-piperidinyl)-sebacate; TMPH, 2,2,6,6-tetramethylpiperidin-4-yl heptanoate; TM, transmembrane; ACh, acetylcholine.

¹ Current address: Department of Biology, University of Utah, Salt Lake City, UT 84112.

² Current address: Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul, 136-791, Korea.

Address correspondence to: Dr. Roger L. Papke, 100267 JHMHSC, 1600 SW Archer Rd., College of Medicine, University of Florida, Gainesville, FL 32610. E-mail: rlpapke{at}ufl.edu

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