INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

Neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric complexes (Anand et al., 1991, Cooper et al., 1991) and share a considerable number of structural and functional features with muscle-type nAChRs. Structural similarities of individual subunits include a large amino-terminal extracellular domain followed by four putative transmembrane segments with a large intracellular domain between transmembrane domains three and four (for review, see Changeux et al., 1992; Karlin and Akabas, 1995). Whereas muscle-type nAChRs are composed of four distinct proteins (1, 1,  or , and  in the ratio of 2:1:1:1), functional neuronal nAChRs can be composed of as few as two (- heteromers) or, in some cases, one class of subunit (-subunit homomers). Eight different genes encoding putative neuronal -subunits (2-9) and three different genes (2-4) coding for putative -subunits have been cloned to date. Pairwise injection of the neuronal -subunit genes 2, 3, 4, and, in some instances, 6 with the -subunit genes 2 or 4 provides for the expression of receptors with pharmacologically and physiologically distinct activation profiles in Xenopus oocytes (Gerzanich et al., 1997; Luetje and Patrick, 1991). This situation both simplifies the study of heterologously expressed receptor subtypes and complicates efforts to assign specific functional correlations between heterologously expressed receptor subtypes and the receptor subtypes expressed in vivo. Studies examining the subunit composition of peripheral or ganglionic neuronal nAChRs have shown that receptors may include the 3, 5, 4, and/or 2 subunits (Conroy and Berg, 1995). It has been demonstrated that inclusion or exclusion of particular subunits will confer particular functional roles and pharmacological sensitivities on nAChRs (for review, see Papke, 1993; Sargent, 1993). In keeping with this idea, certain classes of noncompetitive inhibitors known as ganglionic blockers show selectivity for the inhibition of neuronal nAChRs.

The ganglion blocking activity of two such compounds, 2,2,6,6-tetramethylpiperidine (TMP) and 1,2,2,6,6-penta-methylpiperidine or pempidine (PMP), has been well documented in the literature (Lee et al., 1958; Spinks and Young, 1958). More recently, it was reported that neuronal nAChRs exhibit more prolonged inhibition in response to co-application of ACh with a bifunctional analog of TMP, bis(2,2,6,6-tetramethyl-4-piperidinyl sebacate (bis-TMP-10 or BTMPS), whereas muscle-type nAChRs recover completely from inhibition within 5 min (Papke et al., 1994). Furthermore, inhibition of neuronal nAChRs by bis-TMP-10 was demonstrated to be use dependent and have an approximate IC50 of 200 nM under experimental conditions of high p_open. In addition, the time course of recovery from inhibition was shown to be dependent on both the class of -subunit present (1 or 4/2) and, for the and subunit combinations, the presence or absence of the -subunit (Francis and Papke, 1996; Papke et al., 1994). Bis-TMP-10 is a member of the bis-TMP-n series of compounds, which share a common structure consisting of a symmetrical diester of methylated piperidinol rings linked by an aliphatic diacid chain containing n carbons. Because of the bifunctional nature of bis-TMP-10, it was hypothesized that long-term inhibition arises as a result of the potential for binding of the piperidinol rings to distinct sites.

In the case of muscle-type nAChRs, a number of investigators have used noncompetitive inhibitors in conjunction with site-directed mutagenesis or photoaffinity labeling as probes for structure. These studies have localized the binding of noncompetitive blockers of muscle-type nAChRs to the putative pore-lining second transmembrane domain (tm2) or the short extracellular loop (ecl) region between tm2 and transmembrane domain 3 (Charnet et al., 1990; DiPaola et al., 1990; Giraudat et al., 1986; Giraudat et al., 1987; Leonard et al., 1988; Pedersen et al., 1992; White and Cohen, 1992). Recently, more detailed insights regarding structure-function relationships have been obtained either by examination of the accessibility of substituted cysteines to covalent modification or by examination of the functional effects of unnatural amino acid incorporation (Akabas et al., 1992, 1994; Kearney et al., 1996).

The experiments described in the present study seek to extend the analysis of structure-function relationships to neuronal nAChRs by examining subunit-specific determinants of sensitivity to use-dependent inhibition. To characterize the mechanism for the selective long-term inhibition of neuronal nAChRs, we have created a pair of chimeric -subunits that exchange eight amino acids of tm2 between muscle (1) and neuronal -subunits (4 in this case) and a third chimeric -subunit in which eight amino acids of the 4 subunit ecl region is replaced with the homologous sequence from 1.

Long-term inhibition of neuronal nAChRs by bis-TMP-10 is shown to be dependent upon the -subunit tm2 region, although apparently independent of binding to elements of tm2 located within the membrane electric field or accessible to open-channel blockers. Because the inhibition is use dependent, this observation implies binding to sites for which availability depends both upon channel opening and interactions with specific sequence elements in tm2. These findings highlight the dynamic nature of channel gating and emphasize the importance of interactions between tm2 and surrounding structural elements with channel activation.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Chemicals and synthesis

Fresh acetylcholine (Sigma Chemical Co., St. Louis, MO) stock solutions were made daily in Ringer's solution and diluted. Bis-TMP-10 was obtained from Ciba-Geigy (Hawthorne, NY), and TMP was obtained from Aldrich Chemical Co. (Milwaukee, WI). Bis(2,2,6,6,-tetramethyl-4-piperidinyl) succinate (bis-TMP-4) was synthesized by Ciba-Geigy and obtained from Dr. H. Glossmann (Glossmann et al., 1993). All other chemicals for electrophysiology were purchased from Sigma. Chemicals used for the synthesis were purchased from Aldrich. A general synthetic procedure and the characterization of the synthesized compounds are described below.

General synthetic procedure

Bis-TMP-n compounds

Bis(2,2,6,6-tetramethyl-4-piperidinyl) hexanedioate (bis-TMP-6)

Bis(2,2,6,6-tetramethyl-4-piperidinyl) octanedioate (bis-TMP-8)

Bis(2,2,6,6-tetramethyl-4-piperidinyl) dodecanedioate (bis-TMP-12)

Preparation of RNA and expression in Xenopus oocytes

The preparation of in vitro synthesized cRNA transcripts and oocyte injection have been described previously (Boulter et al., 1987). Briefly, in vitro cRNA transcripts were prepared using the appropriate mMessage mMachine kit from Ambion (Austin, TX) after linearization and purification of cloned cDNAs. Two to three ovarian lobes were surgically removed and then cut open to expose the oocytes. The ovarian tissue was then treated with collagenase from Worthington Biochemical Corp. (Freehold, NJ) for 2 h at room temperature (in calcium-free Barth's solution: 88 mM NaCl, 10 mM HEPES, pH 7.6, 0.33 mM MgSO₄, 0.1 mg/ml gentamicin sulfate). Subsequently, stage 5 oocytes were isolated and injected with 50 nl each of a mixture of the appropriate subunit cRNAs after harvest. Recordings were made 2-7 days after injection depending on the cRNAs being tested.

Electrophysiology

Initial recordings were made on a Warner Instruments (Hamden, CT) OC-725C oocyte amplifier and RC-8 recording chamber interfaced to a Macintosh IIcx personal computer, although the majority of experiments employed a Gene Clamp 500 amplifier (Axon Instruments, Foster City, CA) interfaced to a Gateway 2000 (North Sioux City, SD) P5-75 personal computer. Comparable results were obtained on both sets of equipment. Initial experiments were performed in a configuration such that a 2-ml bolus of drug was applied after loading of a loop at the terminus of the drug delivery system, whereas subsequent experiments were conducted in a configuration where drug application was electronically controlled and regulated by duration rather than volume, permitting more rapid solution exchange without stoppage of flow through the chamber. Oocytes were placed in a Lexan recording chamber with a total volume of ~0.6 ml and perfused at room temperature by frog Ringers (115 mM NaCl, 2.5 mM KCl, 10 mM HEPES, pH 7.3, 1.8 mM CaCl₂) containing 1 µM atropine to block potential muscarinic responses. A Mariotte flask filled with Ringers was used to maintain a constant hydrostatic pressure for drug deliveries and washes. Drugs were diluted in perfusion solution and applied from a reservoir for 10 s using a two-way electronic valve. Data were acquired using Axoscope 1.1 software (Axon Instruments) at a 20-Hz sample rate and filtered at a rate of 10 Hz using either a CyberAmp 320 external filter (Axon Instruments) or the filter in the amplifier. The rate of drug application was 6 ml/min in all cases. Current electrodes were filled with a solution containing 250 mM CsCl, 250 mM CsF, and 100 mM EGTA and had resistances of 0.5-2 M. Voltage electrodes were filled with 3 M KCl and had resistances of 1-3 M. Oocytes with resting membrane potentials more positive than 30 mV were not used.

Experimental protocol and data analysis

Current responses to drug application were studied under two-electrode voltage clamp at a holding potential of 50 mV unless otherwise noted. Holding currents immediately before agonist application were subtracted from measurements of the peak response to agonist. All drug applications were separated by a wash period for a length of time as noted. At the start of recording, all oocytes received an initial control application of ACh to which subsequent drug applications were normalized to control for the level of channel expression in each oocyte. An experimental application of ACh with inhibitor was followed by an application of ACh alone 10 min after the control ACh application used for normalization. In some receptor subtypes (e.g., 34), rundown was observed to stabilize after a second application of ACh. For these subtypes, responses were normalized to the second of two initial control ACh applications. Means and standard errors (SEMs) were calculated from the normalized responses of at least four oocytes for each experimental concentration.

For all experiments involving use-dependent inhibitors, a concentration of ACh was selected sufficient to stimulate the receptors to a level representing a reasonably high value of p_open at the peak of the response, while minimizing rundown with successive ACh applications. For potent use-dependent inhibitors, we have found that this concentration is adequate to achieve maximal inhibition (Francis and Papke, 1996; Papke et al., 1994). Specific concentrations for each receptor subtype are as noted.

For concentration-response relations, data were plotted using Kaleidagraph 3.0.2 (Abelbeck Software, Reading, PA), and curves were generated using the following modified Hill equation (Luetje and Patrick, 1991):

(1)

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, and the r values of the fits were all >0.96 (the average r value = 0.97).

For use-dependent inhibitors, measurements of peak response at the time of co-application of agonist with inhibitor underestimate steady-state inhibition in our system. Therefore, in experiments assessing rate of recovery from use-dependent inhibition (see Fig. 2), inhibitor alone was pre-applied for a length of time sufficient to achieve maximal concentration in the chamber before application of agonist. This pre-application protocol maximized the probability of block upon channel activation as a function of inhibitor concentration and permitted the use of peak current as a more accurate measure of steady-state inhibition for applications of ACh in the presence of the TMP compounds. After normalization to the control response (as described above), total inhibition can be calculated by subtracting the normalized value from 1. In this manner, we confirm almost total inhibition at the time of co-application of agonist with inhibitor and are able to generate a recovery rate from this point in time. Agonist concentrations were selected to minimize rundown with successive ACh applications while still providing a high enough probability of channel activation during the time course of a response to achieve close to 100% inhibition and are noted in the figure legend. Recovery rate data were fitted by the following equation:

(2)

which describes a first-order process where I₀ represents the inhibition at time t = 0 and  is the time constant for recovery. The r values of the displayed fits were all >0.96 (the average r value = 0.98).

For experiments assessing voltage dependence of inhibition, oocytes were initially voltage clamped at a holding potential of 50 mV and a control application of ACh alone was delivered. The holding potential was stepped to +20 mV for 30-60 s before co-application of either ACh with bis-TMP-10 or ACh alone. Thirty to sixty seconds after the peak of the co-application response, voltage was stepped back down to 50 mV, and residual inhibition was evaluated with two subsequent applications of ACh alone separated by 5 min.

For experiments assessing protection from long-term inhibition by application of a short-term inhibitor, a saturating concentration of the short-term inhibitor (either QX-314 (lidocaine N-ethyl bromide) or TMP) was applied for 30-60 s before the application of ACh and the long-term inhibitor (bis-TMP-10). The application of the short-term inhibitor continued throughout the time course of the co-application of ACh with long-term inhibitor until at least 30 s after the co-application. The concentration of inhibitor and period of application was selected to maximize the potential for protection effects. Recovery from inhibition was evaluated in 3-min intervals after the application of inhibitor in the case of 11(4tm2) receptors and 10 min after application of inhibitor in the case of 34 receptors.

Production of tm2 chimeras and sequencing

Chimeric genes were constructed by the method of overlap extension polymerase chain reaction (PCR) (Horton et al., 1989). In brief, the genes 1 and 4 were cloned into p-Bluescript SK(). Specific PCR primers were designed to generate mutants exchanging just the bases necessary to code the tm2 or ecl region. Each primer contained 27 bases of the sequence flanking the tm2 or ecl sequence on one side and 24 bases that coded for the tm2 or ecl region to be exchanged. Oligonucleotides were designed to contain a unique silent restriction site in the mutant region for future screening. Separate PCR reactions consisting of the appropriate PCR primer with template and either T3 or T7 primer selectively amplified the upstream and downstream portions of the gene of interest with overhanging chimeric sequence. These two products were then put together in a second PCR reaction with T3 and T7 primers. The region of chimeric sequence overlap formed double-stranded DNA that primed elongation in both directions, and the full-length product was amplified using T3 and T7 primers. The region coding for the mutant sequence was then cut out with restriction enzymes and cloned back into the original plasmid, reducing the amount of PCR-generated sequence in the final constructs. Clones were evaluated by both restriction analysis and sequencing through the PCR-generated region by the dideoxy chain termination method (Sanger et al., 1977) using the Sequenase 2.0 kit from United States Biochemical Corp. (Cleveland, OH).

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Concentration-response relations of chimeric nAChRs

Chimeric DNAs exchanging sequence coding for eight amino acids of the tm2 region (underlined below) between a neuronal -subunit (4) and the muscle -subunit (1) were constructed by overlap extension PCR. Following the terminology of Miller (1989) and later Charnet et al. (1991), the tm2 chimeric region extends from position 4' to position 11', including the position homologous to the inner polar site of Leonard et al. (1990) at which the charged amino group of the local anesthetic QX-222 has been hypothesized to bind in muscle-type nAChRs (position 6'). The amino acid sequences of the membrane-spanning region, from the intracellular to the extracellular side, are as follows:

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These chimeric -subunits were then expressed with the other muscle subunits (1,,) to produce 11(4tm2) and 14(1tm2) receptors. The effects of these exchanges were initially evaluated in the muscle receptor because only a single -subunit is included per receptor, whereas neuronal nAChRs include multiple -subunits. The presence of a single neuronal -subunit in combination with the other muscle subunits has been previously shown to be sufficient for achieving long-term inhibition after co-application of bis-TMP-10 with ACh (Papke et al., 1994).

Co-injection of chimeric or wild-type -subunit RNA with RNA coding for the other muscle subunits provides for the expression of functional 11, 14, 11(4tm2), and 14(1tm2) receptors with activation profiles typical of nAChRs. To interpret data comparing the magnitude of use-dependent inhibition across receptor subtypes, it is necessary to first define a relationship between the experimental concentration of agonist applied and the EC50 for each receptor subtype. Although the EC50s for each of the receptor subtypes differ somewhat, the Hill coefficients for all of the receptor subtypes are in the range of 1-2, typical of nAChRs. Although 11, 14, and 14(1tm2) receptors show comparable EC50s (in the range of 3-8 µM), 11(4tm2) receptors require approximately a fivefold higher concentration of ACh (30 µM) for 50% activation. The concentration of ACh used in specific experiments ranges between 5 and 30 µM depending on receptor type or experimental design and is noted in the figure legends.

Dependence of long-term inhibition by bis-TMP-10 on sequence in the tm2 region

The time course of recovery from inhibition by bis-TMP-10 was examined for a number of different subunit combinations (Fig. 1). Although normal muscle-type receptors consistently recover from inhibition within 5 min after co-application of 30 µM ACh and 2 µM inhibitor, 11(4tm2) chimeric receptors show more prolonged inhibition (Fig. 1 A). To demonstrate a reciprocal dependence of this effect, the time course of recovery from inhibition of 14 receptors after co-application of 2 µM bis-TMP-10 and 30 µM ACh was compared with that of 14(1tm2) receptors (Fig. 1 B). Whereas 14 receptors remain ~73% inhibited after 5 min, 14(1tm2) receptors recover to near control levels. Additionally, coexpression of the neuronal 3 subunit with the chimeric 4(1tm2) subunit produces receptors that recover completely from inhibition within 5 min, whereas normal 34 receptors are still ~93% inhibited (Fig. 1 C). It should also be noted that continuous application of 2 µM bis-TMP-10 to 14(1tm2) receptors for up to 1 min in duration without co-application of agonist does not produce any significant inhibitory effects, indicating the use dependence of inhibition (data not shown).

View larger version (38K): [in this window] [in a new window]   FIGURE 1   -Subunit tm2 exchange has reciprocal effects on the duration of inhibition by bis-TMP-10. In each panel, traces representative of the mean data are shown on the left. The trace marked a is the response to a co-application of ACh with inhibitor, and the trace marked b is the response to ACh alone 5 min later and is used as a measure of recovery from inhibition. To generate mean data, the co-application (a) and recovery (b) responses are normalized to the trace marked control. Mean data are displayed in the bar graphs on the right. In each graph, the pair of bars on the left (a) represent the normalized response to a co-application of ACh with 2 µM bis-TMP-10 and correspond to the trace marked a at left. The pair of bars on the right (b) represent the normalized response to an application of ACh alone 5 min after the co-application response and correspond to the trace marked b at left. Each column represents the mean response ± SEM of at least four oocytes injected with RNA coding for either: (A) 11 ( ) or 1(4tm2) ( ); (B) 14 ( ) or 14(1tm2) ( ); (C) 34 ( ) or 34(1tm2) ( ) nAChRs. The concentration of ACh in each experiment is either 30 µM (A and B) or 100 µM (C). All responses are separated by 5-min wash intervals.from left to right: 1) application of ACh alone during a voltage step to +20 mV, 2) co-application of ACh with bis-TMP-10 during a voltage step to +20 mV, or 3) co-application of ACh with bis-TMP-10 at a constant holding potential of 50 mV.

The rapid recovery of 34(1tm2) receptors from inhibition by bis-TMP-10 indicates that the neuronal 3 subunit is insensitive to long-term inhibition after application of bis-TMP-10. The sensitivities of the neuronal 2 and 4 subunits to inhibition by bis-TMP-10 were also evaluated. Although attempts to get expression of 44(1tm2) receptors were unsuccessful, 24(1tm2) receptors recover completely within 5 min from the inhibition elicited with co-application of 30 µM ACh and 2 µM bis-TMP-10 (data not shown). As the 4 and 2 subunits are identical within the tm2 domains, it is hypothesized that -subunits do not play a role in determining sensitivity to long-term inhibition by bis-TMP-10.

A chimeric -subunit in which a sequence from the putative ecl region between tm2 and tm3 of a neuronal 4 subunit was replaced with the homologous sequence from the muscle 1 subunit (shown in bold above) was also constructed. The bis-TMP-10 sensitivity of receptors resulting from coexpression of the 4(1ecl) subunit with either the other muscle subunits (, , and ) or the neuronal 3 subunit was evaluated. The substitution of the 1 ecl sequence does not reverse the long-term inhibition normally observed with co-application of bis-TMP-10 and ACh to either the 14 or 34 subunit combinations. Five minutes after co-application of 2 µM bis-TMP-10 with an appropriate concentration of ACh (10 or 100 µM, respectively), the responses of 14(1ecl) (n = 3) and 34(1ecl) receptors (n = 4) recover to only 13 ± 02% and 7 ± 01% of initial control responses to ACh alone (data not shown).

To examine the rate of recovery from inhibition for individual receptor subtypes, ACh was applied at time points beyond 5 min after co-application of ACh with bis-TMP-10 and residual inhibition was evaluated (Fig. 2). For these experiments, 2 µM bis-TMP-10 alone was applied continuously for 15-20 s before application of ACh in the continued presence of 2 µM bis-TMP-10. The ACh concentrations used were either 10 µM for the 11, 14, 11(4tm2), or 14(1tm2) subunit combinations or 100 µM ACh for 34 receptors. Muscle-type receptors show the most rapid recovery from inhibition with a time constant of recovery (_r) of ~3 min, whereas chimeric 11(4tm2) receptors exhibit the most prolonged inhibition (_r = 81 min). As expected, 34 receptors also show prolonged inhibition with a time constant of recovery of ~70 min. The rate of recovery of 14(1tm2) receptors (_r = 6 min) is most comparable to that of muscle-type receptors, whereas the recovery rate of 14 receptors falls in an intermediate range (_r = 16 min).

View larger version (22K): [in this window] [in a new window]   FIGURE 2   Recovery from inhibition by bis-TMP-10 as a function of time. The data points at t = 0 min represent a measure of total inhibition observed after co-application of ACh with 2 µM bis-TMP-10 (see Materials and Methods). Data points at t = 5, t = 10, or t = 15 min represent mean values of residual inhibition measured after application of ACh alone. For 11 receptors, full recovery was effectively achieved after only 10 min of wash, so the data at the 15-min time point for this receptor subtype is omitted for clarity. All drug applications are separated by 5-min wash periods. The concentration of ACh in each experiment is either 10 µM (11, 1(4tm2), 14, and 14(1tm2)) or 100 µM (34 and 34(1tm2). All data points represent the mean responses of at least four oocytes and are fit with an equation describing exponential recovery (see Materials and Methods). In some cases (e.g., 11), the recovery process appears to contain two components. However, the normalized responses at later time points reflect the minimal contribution of response rundown over time.

Inhibition is independent of voltage

As the residence time of the previously characterized open-channel blockers QX-222 and QX-314 has been shown to be dependent on membrane voltage (Leonard et al., 1988; Neher and Steinbach, 1978) and block by a variety of bis-ammonium compounds has also been shown to be voltage-dependent (Ascher et al., 1979; Bertrand et al., 1990; Zhorov et al., 1991), we hypothesized that inhibition by bis-TMP-10 should also show voltage dependence if inhibition occurs via binding to the chimeric tm2 region directly.

As 11(4tm2) receptors resemble neuronal nAChRs in their time course of recovery from inhibition but maintain the linear current-voltage relationship typical of muscle-type nAChRs (Fig. 3 A, bottom), it is possible to examine the effects of voltage on sensitivity to inhibition independent of voltage effects on channel gating. A voltage step to +20 mV for the duration of the co-application of 30 µM ACh with 2 µM bis-TMP-10 does not increase the rate of recovery of 11(4tm2) receptors from inhibition or significantly reduce the relative magnitude of inhibition from that observed with a steady holding potential of 50 mV (Fig. 3, A and C). Thus, the binding of bis-TMP-10 to its activation-sensitive site appears to be independent of membrane voltage in 11(4tm2) receptors.

View larger version (25K): [in this window] [in a new window]   FIGURE 3   Long-term inhibition of either 11(4tm2) (A) or 34 (B) receptors by bis-TMP-10 is independent of voltage. In the representative traces of A and B, an initial control application of either 30 µM (A) or 100 µM (B) ACh is followed by either successive applications of ACh alone (upper trace) or by a single co-application of ACh with 2 µM bis-TMP-10 followed by subsequent application of ACh alone (lower trace). The timing of the voltage step from 50 mV to +20 mV is represented by   and begins ~30 s before and ends ~30 s after the peak of the co-application response in each case. Five-minute wash periods separate each response. Representative current-voltage relationships for each of these receptor subtypes during the plateau phase of the response to extended application of either 30 or 100 µM ACh are also shown. As measured from the I-V relationships, the mean reversal potential for 11(4tm2) receptors is 4.0 ± 2.1 mV (n = 3) and for 34 receptors is 11.0 ± 2.3 mV (n = 4). Holding currents during ramps in the absence of agonist were point to point subtracted. In C, mean normalized data for the 5-min time point (corresponding to traces at far right in A and B above) are shown. Each pair of bars represents the normalized response of either 11(4tm2) ( ) or 34 ( ) receptors to application of either 30 µM (11(4tm2)) or 100 µM (34) ACh alone at a holding potential of 50 mV 5 min after one of three experimental conditions,

The same paradigm was used to examine the dependence of inhibition on membrane voltage for the 34 receptor subtype (Fig. 3 B). However, as neuronal receptors show pronounced inward rectification (Fig. 3 B, bottom), a lack of inhibition at positive potentials may result from either a voltage dependence of inhibition itself or a voltage dependence for channel opening. Although neuronal nAChRs pass very little outward current at depolarized potentials, a co-application of 100 µM ACh with 2 µM bis-TMP-10 during a voltage step to +20 mV produces ~75% residual inhibition as assessed with application of ACh alone at a holding potential of 50 mV 5 min after co-application of inhibitor with ACh (Fig. 3 B, lower trace, and Fig. 3 C). This inhibition is clearly independent of the minimal rundown observed with control applications of ACh alone (Fig. 3 B, upper trace).

Open-channel blockers do not protect nAChRs from long-term inhibition by bis-TMP-10

Although the -subunit tm2 chimeras demonstrate a dependence of inhibition on sequence in the tm2 region, the lack of voltage dependence for this inhibition suggests an indirect effect of the tm2 region rather than binding of the inhibitor directly to this region. We hypothesized that, if bis-TMP-10 binds to the tm2 region directly, a pre-application of the previously characterized open-channel blocker lidocaine N-ethyl bromide (QX-314) should protect from long-term inhibition. To evaluate this hypothesis for chimeric 11(4tm2) receptors, we applied 100 µM QX-314 alone continuously for 1 min before and throughout a 10-s co-application of 2 µM bis-TMP-10 with 5 µM ACh until 1 min after the co-application of agonist and long-term inhibitor (Fig. 4 A). Residual inhibition was then evaluated with applications of ACh alone at 3 and 6 min after co-application of ACh with inhibitor (Fig. 4, A and C). Although this receptor subtype consistently recovers within 3 min from inhibition elicited using the same application paradigm without the bis-TMP-10 application (middle trace), co-application of bis-TMP-10 in the presence of QX-314 consistently produces long-term inhibition (~90% at t = 3 min) that is not significantly different from control applications of bis-TMP-10 with ACh (compare upper and lower traces). These results demonstrate a lack of effect of QX-314 on inhibition by bis-TMP-10. However, because of the potential for multiple intraburst blocking and unblocking events during the time course of agonist application in the presence of the short-term inhibitor, we also repeated the experiment at higher concentrations of QX-314 (up to 500 µM) and at more negative potentials. No protection from long-term inhibition was observed with voltage steps to 80 mV or applications of 500 µM QX-314 (n = 3; data not shown).

View larger version (19K): [in this window] [in a new window]   FIGURE 4   Effects of presence of short-term inhibitors on long-term inhibition of 11(4tm2) receptors. In the representative traces of A and B, the thick lines above the traces indicate the duration of agonist and/or inhibitor application. In each set of three traces of A, the first trace (from left) represents the response to a 10-s application of 5 µM ACh ( ), whereas the second trace (from left) represents the response to either a co-application of 5 µM ACh with 2 µM bis-TMP-10 ( , upper), an application of 5 µM ACh in the continued presence of 100 µM QX-314 ( , middle), or 5 µM ACh co-applied with 2 µM bis-TMP-10 in the continued presence of 100 µM QX-314 (lower). In each set of three traces of B, the first trace (from left) represents the response to 5 µM ACh, whereas the second trace (from left) represents the response to either an application of 5 µM ACh in the continued presence of 30 µM TMP ( , upper) or a co-application of 5 µM ACh with 2 µM bis-TMP-10 in the continued presence of 30 µM TMP (lower). In all cases, the third trace (from left) represents the response to ACh alone applied after a 3-min wash period. The scatter plot in C displays the mean normalized responses ± SEM of at least four oocytes as a function of time. The points at t = 0 min correspond to the second trace (from left) in each of the sets of traces above: bis-TMP-10 with ACh (), QX-314 with ACh (), ACh with QX-314 and bis-TMP-10 (), TMP with ACh (), and ACh with TMP and bis-TMP-10 (); , the response to successive applications of ACh alone. The data points at 3 and 6 min show the mean responses to ACh alone after washout of inhibitor and are used as measures of recovery from inhibition. Overlapping plot symbols were shifted by ~30 s to aid in the viewing of all data points.

The effects of the monofunctional inhibitor TMP (30 µM) on long-term inhibition of 11(4tm2) receptors were also evaluated using the same application paradigm (Fig. 4 B). TMP is able to produce ~30% protection from long-term inhibition. These results are summarized in Fig. 4 C.

A similar set of experiments was conducted on the 34 receptor subtype. The co-application of short- and long-term inhibitors was conducted in the same manner as described above. Application of 500 µM QX-314 produces a small amount of protection (~18%) from long-term inhibition by bis-TMP-10 (Fig. 5 A), whereas application of 4 µM TMP with bis-TMP-10 (Fig. 5 B) limits long-term inhibition to a level that is not significantly different from that observed with application of only the short-term inhibitor TMP. These results are summarized in Fig. 5 C.

View larger version (22K): [in this window] [in a new window]   FIGURE 5   Effects of presence of short-term inhibitors on long-term inhibition of 34 receptors. In the representative traces of A and B, the thick lines above the traces indicate the duration of agonist and/or inhibitor application. In each set of three traces of A, the first trace (from left) represents the response to a 10-s application of 100 µM ACh ( ), whereas the second trace (from left) represents the response to either a co-application of 100 µM ACh with 2 µM bis-TMP-10 ( , upper), an application of 100 µM ACh in the continued presence of 500 µM QX-314 ( , middle), or 100 µM ACh co-applied with 2 µM bis-TMP-10 in the continued presence of 500 µM QX-314 (lower). In each set of three traces of B, the first trace (from left) represents the response to 100 µM ACh, whereas the second trace (from left) represents the response to either an application of 100 µM ACh in the continued presence of 4 µM TMP ( , upper trace) or a co-application of 100 µM ACh with 2 µM bis-TMP-10 in the continued presence of 4 µM TMP (lower trace). In all cases, the third trace (from left) represents the response to an application of 100 µM ACh alone after a 10-min wash period. The scatter plot in C displays the mean normalized responses ± SEM of at least four oocytes as a function of time. The points at t = 0 min correspond to the second trace (from left) in each of the sets of traces above: bis-TMP-10 with ACh ([), QX-314 with ACh ([), ACh with QX-314 and bis-TMP-10 (), TMP with ACh (), and ACh with TMP and bis-TMP-10 (); , the response to successive applications of ACh alone. The data points at 10 min show the mean responses to ACh alone after washout of inhibitor and are used as measures of recovery from inhibition. Overlapping plot symbols at t = 0 min were shifted in time to aid in the viewing of all data points.

Long-term inhibition is dependent upon compound length

If binding of both TMP moieties of the bifunctional compounds is necessary for long-term inhibition, we reasoned that the distance between TMP moieties might represent a constraint on inhibitory activity. To test this hypothesis, the amount of inhibition remaining at time points 5 and 10 min after co-application of inhibitor with ACh to either chimeric 11(4tm2) or neuronal 34 receptors was assessed for bifunctional TMP molecules differing only in the length of their carbon linker. TMP, bis-TMP-4, bis-TMP-6, bis-TMP-8, bis-TMP-10, and bis-TMP-12 were tested for their inhibitory effects (Fig. 6). For both receptor subtypes, all of the compounds, including the monofunctional inhibitor TMP, show some inhibitory activity at the time of co-application with ACh. However, no significant residual inhibition of 11(4tm2) receptors results from co-application with ACh of compounds with linkers of eight carbons or less whereas pronounced residual inhibition is present after application of inhibitors with linkers of 10 or more carbons. In contrast, for neuronal nAChRs (34), all bifunctional compounds show some degree of residual inhibitory activity at time points 5 and 10 min after the time of co-application with ACh (Fig. 6, bottom), and the magnitude of residual inhibition increases with increasing compound length. These results are consistent with binding of each of the piperidinyl groups to distinct sites separated by a characteristic length specific to each subunit combination.

View larger version (26K): [in this window] [in a new window]   FIGURE 6   The effects of compound length on long-term inhibition of 11(4tm2) and 34 nAChRs. All data points represent the mean peak response ± SEM of at least four oocytes normalized to an initial control response to ACh alone. In each graph, the data point at t = 0 min represents the normalized response to co-application of either a 4 µM concentration of the monofunctional compound (TMP, left) or a 2 µM concentration of the bifunctional compounds with 30 µM ACh. The data points at t = 5 and t = 10 min represent the normalized responses to 30 µM ACh alone after washout of inhibitor and are used as measures of recovery from inhibition. It should be noted that, in these experiments, measures of relative inhibition of peak current at t = 0 min do not quantitate steady-state inhibition (see Materials and Methods) but serve to demonstrate the fundamental sensitivity of both receptor subtypes to the TMP compounds. For this reason, the data points at t = 0 min are connected to the 5-min data points by a dashed line.

Based on previous work demonstrating that the -subunit of muscle-type nAChRs is sensitive to the TMP moiety (Francis and Papke, 1996), we reasoned that distinct sites might be represented on separate subunits. To demonstrate a requirement for contributions by at least two TMP-sensitive subunits for long-term inhibition, it was necessary to examine the time course of recovery from inhibition of receptors containing only a single sensitive subunit. 11(4tm2) receptors show no measurable residual inhibition 5 min after co-application of 30 µM ACh with 2 µM bis-TMP-8 (n = 4) or 2 µM bis-TMP-10 (n = 6; data not shown).

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

A great deal of research has employed the use of noncompetitive inhibitors to explore the relationship between structure and function in ion channels. The muscle-type/Torpedo nAChR is the prototype system for these studies both because of its ready availability in purifiable quantities and the rigorous characterization of its functional role at the neuromuscular junction. By examining the mechanism of inhibition of a class of drugs that show selectivity for the long-term inhibition of neuronal nAChRs, we attempt to extend the analysis of structure-function relationships to neuronal nAChRs. Interestingly, our data demonstrate that long-term inhibition is strictly use dependent within the time frame of our application of inhibitor but apparently not associated with direct binding to site(s) situated within the portion of the ion channel pore influenced by the membrane electric field. The most parsimonious interpretation of these data allows for interactions upon channel activation between residues located within the tm2 region of neuronal -subunits and sequence elements situated outside of the membrane electric field. It is possible that these interactions, in turn, result in the exposure of the use-dependent binding site(s) for bis-TMP-10.

TM2 determines kinetics of long-term inhibition

Our studies examining the time course of inhibition of receptors containing chimeric -subunits localize the major structural determinant of sensitivity to long-term inhibition to an eight-amino-acid stretch of neuronal -subunits. All receptor subtypes tested that incorporate the neuronal -subunit tm2 region paired with either a second neuronal -subunit (neuronal nAChRs) or a -subunit (neuronal-muscle chimeras) exhibit a significant degree of residual inhibition as measured at time points 5 min or more after the co-application of agonist with inhibitor. The reversal of long-term inhibition upon substitution of the 1 subunit tm2 region for the 4 subunit tm2 region in 14(1tm2) and 34(1tm2) receptors in conjunction with the observation that substitution of the 1 ecl region does not reverse long-term inhibition demonstrates this effect to be specific for the tm2 region. Although 14 receptors exhibit prolonged inhibition compared with 14(1tm2) or muscle-type receptors (Fig. 2), they recover from inhibition more rapidly than the 11(4tm2) or 14(1ecl) receptor subtypes. Taken together, these observations perhaps suggest that, when associated with the other muscle subunits in 14 receptors, regions of the 4 subunit not contained within the tm2 domain may affect the binding of inhibitor directly or otherwise allow for increased recovery rate. The 4 subunit has been shown to confer the property of prolonged burst kinetics upon the neuronal subunits with which it is expressed (Papke and Heinemann, 1991). It may be the case that activation properties such as burst duration or channel open time, which may be specific to individual receptor subtypes, can also influence recovery rate or the probability of block as a function of peak current.

Significance of voltage-independent inhibition

In contrast to studies on the open-channel blockers QX-222 and QX-314 (Neher and Steinbach, 1978) and studies on the symmetrical bis-ammonium series of ganglionic blockers (Ascher et al., 1979) in which block has been shown to be dependent on membrane potential, the inhibition by bis-TMP-10 seems to be independent of voltage. The lack of voltage dependence for inhibition brings to mind two possibilities: either the noncompetitive binding site is outside of the membrane electric field or bis-TMP-10 is uncharged at physiological pH. Although direct evaluation of the pK_a of bis-TMP-10 has not been possible because of solubility limitations, it is known that the monofunctional inhibitor TMP has a pKa in the range of 10-11 (Perrin, 1965). The pKa of simple bis-amino compounds are reduced if the amines are separated by short (two to four) carbon chains. However, when the amines are separated by a longer (e.g., eight) carbon chain, the pKa values of the two ionizable groups both approach that of the monofunctional amine. Therefore, with the pKa of the monofunctional piperidine in the range of 10-11, it is unlikely that the lower of the 2 pKa values of the bis compound would be under 9-10. Thus, at physiological pH, both functional groups should be predominantly charged.

The voltage-independent long-term inhibition of 34 receptors is particularly intriguing. As shown in Fig. 3 C, the inhibition observed 5 min after the co-application of agonist with inhibitor at +20 mV (~75%) is slightly less than that typically observed 5 min after co-applications of ACh with bis-TMP-10 at 50 mV (~90%). This effect may represent a voltage-dependent component of long-term inhibition but more likely represents an effect of the voltage dependence of channel gating independent of any voltage dependence for inhibition. In either case, at this potential, no outward current was measured in response to control applications of ACh alone, implying that the activation-dependent state associated with inhibition is distinct from the conducting state. The inward rectification of neuronal nAChRs is due, at least in part, to a magnesium block dependent upon the sequence at the cytosolic mouth of the channel (Ifune and Steinbach, 1992; Imoto et al., 1988; Sands and Barish, 1992). Our data suggest that a gating conformation may still be present at depolarized potentials and are consistent with channel block by magnesium from the intracellular side. Thus, bis-TMP-10 appears to be able to produce long-term inhibition of neuronal nAChRs that are gated (or activated) but nonconducting as a result of block from the intracellular side. Alternatively, it may be the case that a high-affinity desensitized state is favored at depolarized potentials in the presence of agonist. Our data would suggest that a gated conformation is associated with this state as well.

We also tested the hypothesis that bis-TMP-10 can produce long-term inhibition in nAChRs that are nonconducting as a result of blockade from the extracellular side. We chose to use the open-channel blocker QX-314 in these experiments based upon three criteria: 1) the QX-314 binding site is believed to be located approximately three-fourths of the way across the membrane electric field, and thus inhibition by this compound is voltage dependent (Neher and Steinbach, 1978); 2) QX-314 has been shown to interact with residues at homologous positions to those contained within the region of our -subunit tm2 chimeras (Pascual and Karlin, 1997); and 3) QX-314 has a longer residence time in the pore than the structurally related local anesthetic QX-222 (Neher and Steinbach, 1978). In addition, studies in our laboratory have shown that the inhibition of chimeric 11(4tm2) receptors by QX-314 is voltage dependent, indicating that, for this subunit combination also, the QX-314 binding site is located within the membrane electric field (M.M. Francis and R.L. Papke, unpublished observations).

For 11(4tm2) receptors, the lack of an effect of even very high concentrations of QX-314 (500 µM) on the magnitude of residual inhibition after co-application of bis-TMP-10 with ACh is consistent with the observed voltage independence of inhibition by bis TMP-10. For 34 receptors, QX-314 does produce a detectable reduction in the magnitude of residual inhibition (Fig. 5 C). This observation suggests that in this subunit combination the sites of action for bis-TMP-10 and QX-314 are not totally independent. From our data, it is difficult to ascertain whether the partial protection we observe represents interactions of both drugs at a single site or, alternatively, an allosteric interaction between two distinct sites, such that the binding of QX-314 decreases the gating-dependent changes at the site of TMP binding.

For both receptor subtypes, the short-term inhibitor TMP produces a significantly greater degree of protection from long-term inhibition than was observed with QX-314. This finding demonstrates a degree of overlap in the sites of action between the mono- and bifunctional compounds. The lack of complete protection may suggest a contribution of the hydrophobic linker region in stabilizing bis-TMP-10 binding or may indicate that a different subset of binding sites are available to the smaller TMP compound. The latter hypothesis is supported by the observation of a slight voltage dependence for inhibition by TMP (Papke et al., 1994).

Significance of compound length requirement for long-term inhibition

We have previously shown that receptors are sensitive to long-term inhibition by bis-TMP-10. Based on these data, we hypothesized that a TMP binding site is present on the -subunit of muscle-type nAChRs and that long-term inhibition occurs via interactions at multiple sites (Francis and Papke, 1996). Consistent with this hypothesis, we now present data that suggest that substitution of the muscle -subunit tm2 sequence with the neuronal -subunit tm2 sequence results in the exposure of a second TMP binding site located outside of the membrane electric field. Furthermore, because long-term inhibition is dependent upon the sequence in tm2 for both chimeric muscle and chimeric neuronal receptors, it seems reasonable that the inhibitor binding site may represent a structural