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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.)
Department of Chemistry, University of Florida, Gainesville, Florida (K.I.C., N.A.H.)
Abstract
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.
There are multiple types of nicotine acetylcholine receptors (nAChR) in the brain associated with synaptic function, signal processing or cell survival. 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 these receptor subtypes. Mecamylamine is a ganglionic blocker developed many years ago as an antihypertensive and more recently suggested to be useful as a component in the pharmacotherapy for Tourette's syndrome (Sanberg et al., 1998) and smoking cessation (Rose et al., 1994). However, electrophysiological characterization of mecamylamine has shown it to be relatively nonselective (Papke et al., 2001), consistent with the observation that it effectively blocks all of the peripheral and central nervous system effects of nicotine (Martin et al., 1993). We previously identified a family of bis-tetramethylpiperidine compounds as inhibitors of neuronal type nicotinic receptors (Francis et al., 1998). The prototype compound in this series is BTMPS [bis-(2,2,6,6-tetramethyl-4-piperidinyl)-sebacate], which produces a readily reversible block of muscle-type nAChR and a nearly irreversible use-dependent, voltage-independent block of neuronal nAChR. The tetramethyl-piperidine groups of BTMPS are sufficient to produce block of nAChR, and the conjugation of two such groups with a long aliphatic chain accounts for both the selectivity and slow reversibility of BTMPS inhibition of neuronal nAChR (Francis et al., 1998). In the present study, we describe the effects of 2,2,6,6-tetramethylpiperidin-4-yl heptanoate (TMPH), a novel compound that has a single tetramethyl-piperidine group and an aliphatic chain similar to that of BTMPS. TMPH is also a potent inhibitor of neuronal nicotinic receptors.
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
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 (5eC20 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 eC60 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 eC50 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 e, 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 Imax denotes the maximal response for a particular agonist/subunit combination, and n represents the Hill coefficient. Imax, n, and the EC50 were all unconstrained for the fitting procedures. Negative Hill slopes were applied for the calculation of IC50 values.
Brain Slice Recording. Male Sprague-Dawley rats (p11-p20) were anesthetized with Halothane (Halocarbon Laboratories, River Edge, NJ) and swiftly decapitated. Transverse (300 mm) whole brain slices were prepared using a Vibratome and a high Mg+/low Ca2+ ice-cold artificial cerebral spinal fluid containing 124 mM NaCl, 2.5 mM KCl, 1.2 mM NaH2PO4, 2.5 mM MgSO4,10mM D-glucose, 1 mM CaCl2, and 25.9 mM NaHCO3 saturated with 95% O2,5%CO2. Slices were incubated at 30°C for 30 min and then left at room temperature until they were transferred to a submersion chamber for recording. During experiments slices were perfused (2 ml/min) with normal artificial cerebral spinal fluid containing 126 mM NaCl, 3 mM KCl, 1.2 mM NaH2PO4, 1.5 mM MgSO4,11mM D-glucose, 2.4 mM CaCl2, 25.9 mM NaHCO3, and 0.008 mM atropine sulfate saturated with 95% O2, 5% CO2 at 30°C. Medial septum/diagonal band cells were visualized with infrared differential interference contrast microscopy using a Nikon E600FN microscope. Whole cell patch-clamp recordings were made with glass pipettes (3eC5 M) containing an internal solution of 125 mM potassium gluconate, 1 mM KCl, 0.1 mM CaCl2, 2 mM MgCl2, 1 mM EGTA, 2 mM MgATP, 0.3 mM Na3GTP, and 10 mM HEPES. Cells were held at eC70 mV, and a eC10 mV/10-ms test pulse was used to determine series and input resistances. Cells with series resistances >60 M or those requiring holding currents >200 pA were not included in the final analyses. Local somatic application of 1 mM ACh and 1 mM ACh + 300 e TMPH was performed using double barrel glass pipettes attached to a picospritzer (General Valve, Fairfield, NJ) with Teflon tubing (10eC20 psi for 5eC30 ms). For each cell, two baseline evoked responses to ACh were recorded followed by two evoked responses to ACh + TMPH (interstimulus interval of 30 s). ACh was then applied every 30 s for the remainder of the experiment. Signals were digitized using an Axon Digidata 1200A and sampled at 20kHz using Clampex version 9. Data analysis was done with Clampfit version 9.
Results
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).
IC50 values were calculated based on either the decrease in peak current amplitudes or the decrease in the net charge of the ACh response when coapplied with TMPH. For many of the subunit combinations tested, there was no detectable recovery after a 5-min wash, so IC50 values were also calculated based on the inhibition still present at the 5-min time point (recovery). Note that these IC50 values are based on standard single application protocol and are likely to underestimate the IC50 for steady-state conditions.
The value IC50 recovery reflects the residual inhibition of the ACh control response measured after a 5-min wash. If IC50 recovery > IC50 area, then there was significant recovery (i.e. readily reversible inhibition). Likewise, if IC50 area < IC50 peak, then there was significant buildup of inhibition throughout the agonist/antagonist coapplication.
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.
Use Dependence of Inhibition by TMPH. We evaluated the degree to which inhibition by TMPH was use-dependent by applying 1 e TMPH alone and comparing the response to a subsequent control ACh application to that obtained after 1 e TMPH was coapplied with ACh. As shown in Fig. 4, the ability of TMPH to inhibit neuronal nAChR when applied in the absence of agonist varied significantly among the pairwise subunit combinations tested, but in all cases was less than when TMPH was coapplied with agonist. It is interesting that although TMPH alone applied to 42 receptors was almost as effective as when coapplied with ACh, TMPH alone applied to 32 receptors had no detectable effect after the washout period.
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.
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).
Inhibition of Receptors Containing 3 and 6 Subunits. It has been suggested that in vivo 3 subunits may coassemble with 6 and possibly 4 and 2 to make receptors that regulate dopamine release (Champtiaux et al., 2003). However, the 6 subunit expresses poorly in oocytes when used in pairwise combinations with beta subunits (Kuryatov et al., 2000). One approach to get around this problem has been to use a chimera of 6 and 3 that has improved expression properties. Because of the presence of the 6 extracellular domain, receptors formed with this chimera are sensitive to 6-selective competitive antagonists (Dowell et al., 2003). We compared the effects of TMPH on oocytes expressing 323 and a chimera of 6 and 3 subunits, 6/3 (Dowell et al., 2003) along with 2 and 3. This approach allowed us to systematically evaluate first the effects of the 3 subunits (by comparing oocytes injected with 323 to those expressing 32 alone) and second the effects of the 6 extracellular domain (by comparing 6/323 with 323).
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).
The data in Fig. 8 suggest that the 3 subunit imparts some resistance to inhibition by TMPH and also that the extracellular domain of 6 has relatively little effect. We therefore tested oocytes expressing the complete wild-type 6 subunit. The 6 subunit was coexpressed with 4 and 3 because this is the only 6 combination we have found to work with any consistency. For these experiments, as a control we compared the oocytes injected with 643 to oocytes injected with 343. As shown in Fig. 9, the oocytes expressing 643 showed relatively weak inhibition by TMPH during the coapplication of TMPH and ACh, with an IC50 value for the inhibition of net charge nearly an order of magnitude higher than for any other receptor subunit combination tested (Table 1). This reduced sensitivity to TMPH was most probably caused by both the 6 and the 3 subunits because 343-injected oocytes were much less sensitive than those expressing 34 alone.
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.
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 e ACh and 30 e 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.
Note that with a simple coapplication protocol, a large transient current is evoked that we would hypothesize to be caused, at least in part, by activation of both 7 and non-7 receptors before the use-dependent effects of TMPH. However, because 7 receptors activate on the leading edge of the solution exchange (Papke and Thinschmidt, 1998; Papke and Papke, 2002), when the TMPH concentration would still be relatively low, they would be somewhat protected from full TMPH inhibition during coapplication. Therefore, we also tested an alternative protocol which preapplied TMPH alone at 30 e before the coapplication of 30 e TMPH and 1 mM ACh. With this protocol (Fig. 11B), the response was very effectively inhibited during the ACh/TMPH coapplication. There was significant recovery after a 5-min washout, and the recovered response had the rapid kinetics of a pure 7 response, indicating prolonged inhibition of the 34 receptors selectively.
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
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.
doi:10.1124/mol.105.011676.
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.
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