Introduction

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Spermine, a polyamine found in cells at concentrations into the millimolar range (Schoemaker et al., 1994), plays important roles in cell growth and differentiation. It is of specific interest to neuroscience because it binds to modulatory sites on NMDAR, either potentiating and/or antagonizing these important signaling proteins (Brackley et al., 1990; Usherwood and Blagbrough, 1991; Williams et al., 1991; Rock and Macdonald, 1992a, b, 1995; Reynolds, 1990b). The interactions of polyamines with NMDAR were first reported by Ransom and Stec (1988), who showed that micromolar concentrations of spermine and spermidine increase the affinity of NMDAR for [³H]MK-801, possibly by binding to an excitatory modulatory site on the receptor. This change in [³H]MK-801 affinity is inhibited by arcaine through competitive antagonism at the polyamine binding site on NMDAR (Reynolds, 1990a, b). At concentrations greater than ~10 µM, spermine inhibits the binding of [³H]MK-801 to NMDAR. Romano and Williams (1994) suggested that this results from binding of the polyamine to an inhibitory modulatory site, a proposal that is in accordance with the results of electrophysiological studies on these receptors (Brackley et al., 1990; Usherwood and Blagbrough, 1991). The excitatory and inhibitory modulatory sites for polyamines on NMDAR have been extensively reviewed (e.g., Carter, 1994; Johnson, 1996).

As with NMDAR, nAChRs are ligand-gated cation channels found peripherally and centrally in the nervous systems of mammals and their pharmacological properties have been extensively studied. Usherwood (1987) suggested that at physiological pH, polyamines would be expected to inhibit ion fluxes through the cation-selective channels gated by these receptors and experimental evidence to support this suggestion was obtained by Hsu (1994). The latter studies of nAChR of frog muscle also showed that spermine potentiates responses of these receptors. The TE671 cell line expresses human muscle type nAChR (Schoepfer et al., 1988; Luther et al., 1989; Yamamoto et al., 1991) and has been used extensively in biochemical, physiological, pharmacological and immunological studies (McAllister, 1977; Dranoff et al., 1985; Luther et al., 1989; Bencherif and Lukas, 1991; Grassi et al., 1993). The experiments described herein were initially undertaken to determine whether spermine antagonizes nAChR, but during the course of the studies it became clear that its actions were more complex than this.

	Methods

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Cell culture. TE671 cells originating from the American Type Culture Collection (Bethesda, MD) were maintained in Dulbecco's Modified Eagle's Medium (DMEM) (4.5 g liter¹ glucose) supplemented with 10% fetal calf serum, 1 mM pyruvic acid, 4 mM glutamine, 10 IU ml¹ penicillin and 10 µg ml¹ streptomycin (Gibco, Grand Island, NY), and incubated at 37°C in a 5% CO₂ atmosphere. Cultures were divided 1:10 when the cells were ~75% confluent and grown on pieces of glass coverslip (~5 × 20 mm) in 35-mm petri dishes (Nunc, Roskilde; Denmark). For electrophysiological recordings, the coverslips were transferred to a perfusion bath mounted on the stage of an inverted microscope.

Electrophysiology. Whole-cell recordings and single channel recordings from excised outside-out patches (Hamill et al., 1981) were used to investigate the effects of spermine on nAChR. Fire-polished patch pipettes were fabricated from borosilicate glass (GC150-10; Clark Electromedical Instruments, Reading, UK) using a DMZ (Zeitz) or Sutter (P-97) programmable puller. Pipettes were filled with 140 mM CsCl, 1 mM MgCl₂, 1 mM CaCl₂, 11 mM EGTA and 5 mM HEPES (pH adjusted to 7.2 with 1 M CsOH) for whole-cell recording. This saline was not suitable for recording responses to ACh from outside-out patches because at negative membrane potentials inward potassium currents "contaminated" the recordings. Instead, the following saline was used: 140 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 11 mM EGTA and 5 mM HEPES (pH adjusted to 7.2 with 1 M KOH) for recording from outside-out patches. In this saline, the reversal potential for the potassium channels was about 60 mV, i.e., the potential at which the single nAChR studies were undertaken. The pipette resistances were ~5 M. Cells and membrane patches were constantly perfused at 8 to 13 ml min¹ with saline containing 135 mM NaCl, 5.4 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂ and 5 mM HEPES (pH adjusted to 7.4 with 1 M NaOH). For whole-cell and single channel recordings, ligands were applied as ~1-sec pulses using a rapid application technique. In brief, a pipette containing test solution was mounted on a steel plate that was electromagnetically moved laterally by ~100 µm through application of a step voltage of variable duration. Initially, the stream of test solution leaving the pipette bypassed the whole-cell or membrane patch under study, but during application of the step voltage the stream engulfed the cell or patch. The lateral movement of the pipette took ~60 msec, but only a fraction of this time includes the solution exchange time and the rise time to the peak of a response to ACh was typically ~10 msec. Most whole-cell and patch experiments were performed at a holding potential (V_H) of 60 mV and all studies were undertaken at 18 to 22°C. Whole-cell and patch currents were monitored using either an Axopatch 200 (Axon Instruments, Foster City, CA) or a List Electromedical L/M-EPC7 patch clamp amplifier. The output from the amplifier was low-pass filtered at 10 kHz, digitized using a Sony PCM and recorded on video tape. The single channel data were low-pass filtered at 1 kHz for analysis.

Chemicals. Liquid media (DMEM) and penicillin/streptomycin were purchased from Gibco BRL. Arcaine was purchased from RBI (Semat, UK). All other chemicals were purchased from Sigma Chemical Co., St. Louis, MO.

Analyses. All data analyses were undertaken on an IBM-compatible 486 computer using Axotape and pClamp 5.7.2 software (Axon Instruments). Curve fitting was performed using Grafit (Erithacus Software) and statistical analyses were undertaken using Sigmaplot (Jandel Scientific). Dose-response relationships were fit to a four parameter logistic equation: where M = maximum response, m = minimum response, E = EC₅₀, C = agonist concentration and S = Hill slope. The experimental results are mainly presented as means ± S.E. of data obtained from n cells or n patches. P values were determined using the unpaired Student's t test; difference between means was considered to be significant when P < .05.

	Results

Top Abstract Introduction Methods Results Discussion References

Characterization of responses to acetylcholine. The sensitivity of TE671 cells to ACh and the pharmacological properties of their nAChR were similar for different cell passages; thus, it was legitimate to pool data obtained from different passages. TE671 cells had resting membrane potentials of 30 to 60 mV. In a few cases, it was possible to clamp the cells at V_H between 150 and +100 mV, although in most experiments the V_H range was limited to between 125 and +50 mV. Inward currents of up to 4 nA at V_H = 60 mV were evoked by application of a 1-sec pulse of 10 µM ACh. The currents were characterized by an early peak followed by a decay to a low plateau (fig. 1A) (see also Siara et al., 1990). The characteristic responses to 0.1 to 1000 µM ACh at V_H = 60 mV presented in figure 1A show that the rate and extent of decay of a current from its peak was more pronounced at high ACh concentrations (see also fig. 5D and E). The dose-response relationship in figure 1B was fitted with the four parameter logistic equation given above where the EC₅₀ value for ACh (E) was 7.75 ± 0.14 µM, M was 101.1 ± 0.7%, m was 0.42 ± 0.45% and S was 1.09 ± 0.02 (n = 43 cells). Maximum responses were obtained with 100 to 1000 µM ACh, but at these concentrations desensitization of nAChR was pronounced. It is likely that desensitization caused S to deviate from its theoretical value of 2 for skeletal muscle nAChR. Similar observations were made by Franke et al. (1992). The current-voltage (I-V) relationship illustrated in figure 1D shows a slight inward rectification, but reverses close to 0 mV. Presumably, as the potential gradient was increased Cs⁺ became less permeant than Na⁺. Inward rectification of AMPA receptors is due to block by intracellular spermine (Kamboj et al., 1995). This raises the possibility that, in vivo, spermine causes an inward rectification of nAChR of TE671 cells. Because any spermine normally present in these cells would probably have been greatly diluted by the pipette solution during whole-cell recording, 1 mM spermine was added to the pipette solution to test this possibility. However, the presence of the polyamine intracellularly neither increased the inward rectification nor changed the reversal potential of the ACh-induced whole-cell current (fig. 3A).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Characterization of responses to acetylcholine. A, Whole-cell current responses from a TE671 cell exposed to brief applications of ACh at concentrations ranging from 0.1 to 1000 µM. The cell was clamped at 60 mV. B, Dose-response relationship for ACh. Peak amplitude data are plotted against ACh concentration as the percentage of the current elicited by 1000 µM ACh (in a few cells, 100 µM ACh produced the maximum response). Data points are means ± S.E. for n = 43 cells. The data were fitted by a four parameter logistic equation giving an EC50 value of 7.75 ± 0.14 µM. C, Recovery of nAChR from desensitization. Peak amplitudes of currents elicited by ACh for test pulses (I2) are expressed as a fraction (I2/I1) of peak amplitudes of currents to control responses (I1) and plotted against the time interval between control and test pulses. Data points are means ± S.E. for 10 µM ACh (, n = 7 cells) and 100 µM ACh (, n = 6 cells). For both concentrations of ACh, full recovery from desensitization was obtained after 20 sec (indicated by dashed line). D, Current-voltage relationship for peak responses to pulses (duration of 1 sec) of 10 µM ACh. The ligand was applied to 12 cells clamped at holding potentials (VH) between 125 mV and +75 mV. The data are normalized (In) expressed as a percentage of the peak currents obtained at 125 mV. Points are means ± S.E. In some cases the error bars are smaller than the symbols.

Desensitization onset. As described above, the whole-cell current evoked at V_H = 60 mV during application of a pulse of ACh rose rapidly (~10 msec) to a peak before declining during the ACh pulse to a low steady state, the relative amplitude (compared to the peak) of which was inversely proportional to the ACh concentration. The rate of decay of such a current from its peak gives an estimate of the rate of onset of nAChR desensitization (Katz and Thesleff, 1957). Not unexpectedly, the latter increased as the concentration of ACh was raised, e.g., 3.98 and 5.17 sec¹ with 10 µM and 1 mM ACh, respectively (fig. 5C and D. For brief single applications of ACh it was possible to fit this decay with a single exponential). The rate of onset of desensitization was also estimated from the decline in peak amplitude of the inward currents observed during application of 1 Hz trains (100-150 sec duration) of ACh pulses (0.5 sec duration) (Gration et al., 1980). With this approach, the decline in peak amplitude was best estimated by fitting the sum of two exponentials (fig. 5A; see below). The two rates obtained with 10 µM ACh were 0.114 ± 0.008 and 0.0071 ± 0.0003 sec¹. In both the single pulse and pulse train experiments the estimated rate constants were independent of V_H. It is appreciated that these estimates of desensitization onset rates are influenced by the ACh application protocols. For example, with very fast application of 1 mM ACh to outside-out patches of mouse muscle, desensitization rates of 20 to 50 sec¹ have been determined for nAChR (Franke et al., 1992). However, the protocols used in our studies were adequate for investigating qualitatively the effects of spermine on desensitization.

Recovery from desensitization. The rate of recovery from desensitization was determined by applying pairs of pulses (each of 1 sec duration) of ACh (either 10 or 100 µM), the pulses in a pair being separated by intervals of 0.5 to 60 sec (Gration et al., 1980). A plot of the ratio "peak amplitude of second response (I₂)/peak amplitude of first response (I₁)" against the interval between the two ACh pulses shows that recovery from desensitization was complete when the interval between the pulses was 10 to 20 sec (fig. 1C). The time course of recovery from desensitization was not significantly different for 10 and 100 µM ACh, a result that is consistent with that reported by Siara et al. (1990). In other experiments described herein, an interval of at least 30 sec was allowed between consecutive applications of ACh to ensure full recovery from desensitization.

Potentiation by spermine. Application (at V_H = 60 mV) of 10 mM spermine alone to TE671 cells did not elicit currents. At concentrations lower than 1 µM, spermine had no effect on the ACh-induced currents at V_H = 60 mV, whereas 1 to 100 µM spermine reversibly potentiated the currents (fig. 4A). Potentiation was characterized by a significant increase in the maximum response of the ACh dose-response relationship, but the EC₅₀ for ACh was not significantly changed (fig. 2A and 2B). Currents elicited by 10 µM ACh were potentiated similarly by 10 µM spermine and 100 µM spermine (fig. 2A and B) (i.e., 58.2 ± 12.6% (n = 4 cells) by 10 µM spermine and 41.5 ± 10.3% (n = 3 cells) by 100 µM spermine), whereas currents elicited by 100 µM ACh were potentiated more by 100 µM spermine than by 10 µM spermine (fig. 2A and B) (i.e., 12.2 ± 7.72% (n = 4 cells) by 10 µM spermine and 60 ± 13.12% (n = 3 cells) by 100 µM spermine, P = .02). Responses to 10 µM ACh obtained at V_H of +75 mV to 125 mV were compared with those to 10 µM ACh plus spermine obtained over the same V_H range (fig. 3B; table 1). Although 10 µM spermine potentiated the ACh-induced whole-cell current at all V_H (i.e., +75 mV to 125 mV), potentiation was slightly more marked at positive V_H. Qualitatively similar data were obtained with 100 µM spermine.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Dose-response relationships for ACh in the absence () and presence () of spermine at a concentration of A, 10 µM (n = 4 cells), B, 100 µM (n = 3 cells), C, 1 mM (n = 5 cells) and D, 10 mM (n = 6 cells). Percentages of maximum response in the absence of spermine are plotted against ACh concentration. Data are means ± S.E. All experiments were at a holding potential of 60 mV.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   A, Current-voltage relationships derived from whole-cell recordings with externally-applied 10 µM ACh without (, n = 7 cells) and with 1 mM spermine (, n = 3 cells) in the patch-pipette. Peak currents to ACh are normalized (In) as percentages of the response at 100 mV. Data points are means ± S.E. B, Voltage-dependence of the inhibition of responses to 10 µM ACh by spermine. Whole-cell currents induced by 10 µM ACh were recorded in the presence of 10 mM (, n = 3 cells), 5 mM (, n = 2 cells), 1 mM (, n = 5 cells), 100 µM (, n = 8 cells) and 10 µM (, n = 8 cells) spermine at holding potentials (VH) between +75 and 125 mV and compared to responses in the absence of spermine. Percentage of the control responses to ACh are plotted against VH. Data points are means ± S.E. C, Current-voltage relationships for 10 µM ACh in the presence () and absence () of 10 mM spermine (n = 4 cells). ACh currents are normalized (In) expressed as percentages of the current elicited at 125 mV in the absence of spermine. Data are means ± S.E.

Inhibition by spermine. Spermine (1 mM) caused a small but significant reduction in the maximum response elicited by 1 mM ACh (fig. 2C), but the EC₅₀ value for ACh was not significantly affected. In contrast, 10 mM spermine not only reduced the maximum response to ACh but also increased the EC₅₀ value from 9.2 to 52.8 µM (fig. 2D). Inhibition of ACh-elicited current by 1 mM spermine was voltage-dependent, i.e., at V_H = 125 mV it was ~49% whereas at V_H = +75 mV it was only ~9%. Inhibition by 5 mM spermine was maximal (~82%) at V_H = 100 mV and ~55% at V_H = +75 mV. Finally, inhibition by 10 mM spermine was maximal (~96%) even at +75 mV, which suggests that at this concentration, at least, inhibition by spermine has a voltage-independent component. The reversal potential was not changed by even 10 mM spermine (fig. 3C).

Effect of arcaine on spermine-induced potentiation of nAChR. If the potentiation by spermine of ACh-induced currents is due to interaction of this polyamine with a site on nAChR analogous to that present on NMDAR, then arcaine might be expected to compete with spermine for that site. Studies by Hsu (1994) on nAChR of frog muscle provide experimental evidence in favor of this conclusion. In our studies, neither 10 nor 100 µM arcaine elicited currents (V_H = +25 mV to 125 mV) when applied alone to TE671 cells (n = 6 cells) and when tested on a given cell at these concentrations it had no significant effect on the dose-response relationship for ACh. Figure 4A shows the effect of spermine (0.1 µM-10 mM) on the whole-cell current induced by 10 µM ACh (V_H = 60 mV) in the absence and presence of 10 µM arcaine. In the absence of arcaine, 1 to 100 µM spermine potentiated the ACh-induced current (by ~45% with 10 µM spermine), but when the polyamine was applied at these concentrations in the presence of 10 µM arcaine it was inhibitory (i.e., with 10 µM spermine there was an ~19% reduction in the amplitude of the response to ACh). This inhibition increased when the spermine concentration was raised above 100 µM. For example, 1 mM spermine alone did not significantly affect the amplitude of the ACh-induced current in the absence of arcaine (fig. 4A and fig. 2C), but it was markedly inhibitory in the presence of arcaine (i.e., ~40% reduction in amplitude of response to ACh). Figure 4B shows a dose-response relationship for ACh plus 100 µM arcaine obtained in the absence and presence of 1 mM spermine (V_H = 60 mV). On its own, spermine had little effect on the dose-response relationship for ACh, but in the presence of arcaine, the maximum response to ACh was significantly reduced by 1 mM spermine, although the EC₅₀ value for ACh (plus 100 µM arcaine) remained unaffected.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Effect of arcaine on spermine modulation of nAChR. A, Dose-inhibition/potentiation relationships for spermine effect on responses to 10 µM ACh in the absence () of and presence () of 10 µM arcaine. The cell was clamped at 60 mV. Percentage of the control responses to ACh are plotted against spermine concentration. Data are means ± S.E. (n = 3 cells). The dashed line indicates the control level for 10 µM ACh, i.e., 100%. B, Dose-response relationship for ACh plus 100 µM arcaine in the absence () and presence () of 1 mM spermine. Percentages of the maximum response are plotted against ACh concentration. Data are means ± S.E. (n = 3 cells) and were fitted by a four parameter logistic equation. The EC50s are 19.76 ± 2.32 µM (S.E.) and 17.64 ± 0.31 µM (S.E.) respectively. The holding potential was 60 mV. C, Voltage-dependence of inhibition of whole-cell currents evoked by 10 µM ACh plus 100 µM arcaine in the presence of 5 mM (, n = 4 cells), 1 mM (, n = 8 cells), 100 µM (, n = 8 cells) and 10 µM (, n = 3 cells) spermine. Percentage of the control ACh-elicited responses is plotted against holding potential (VH). Data are means ± S.E.

Effect of spermine on onset of desensitization. It was shown above that, within the constraints of the experimental protocols, the onset of ACh-induced desensitization (estimated by applying trains of ACh pulses) of nAChR of TE671 cells appears to be biphasic, comprising fast and slow components. In order to investigate the effects of spermine on the estimated desensitization onset rates, 1 Hz trains (75-150 sec duration) of pulses (0.5 sec duration) of ACh (10 µM  1 mM) were applied to TE671 cells during whole-cell recording in the absence and presence of 10 µM spermine (n = 7; fig. 5A-C). In the absence of spermine, the rate constants of the fast and slow phases of desensitization onset increased with ACh concentration. At 10 µM, spermine reduced the rate constants of both components at all ACh concentrations. The rate of desensitization onset estimated at V_H = 60 mV from the rate of decay of single currents induced during a pulse of ACh was also decreased by 10 µM spermine (fig. 5C and D), e.g., from 3.98 ± 0.21 to 1.81 ± 0.22 sec¹ (2.20-fold) for 10 µM ACh and from 5.17 ± 0.09 to 2.20 ± 0.12 sec¹ (2.35-fold) for 1 mM ACh.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   A-C, Effect of 10 µM spermine on the desensitization of nAChR measured as reduction in amplitude of ACh-induced whole-cell currents observed during application of trains of 75 pulses (0.5 sec duration) of ACh or 75 pulses (0.5 sec duration) of ACh plus spermine (10 µM). The amplitude of the ACh-induced inward current reached its peak value before the end of a 0.5 sec pulse of ACh. The pulses were applied at a frequency of 1 sec1 at VH = 60 mV. The ACh concentration was 10 µM (A), 100 µM (B) and 1 mM (C). The amplitudes of the ACh-induced currents (I) for each pulse train are plotted against time. The upper traces in A to C are data for ACh plus spermine. The plots are each best-fit by the sum of two exponentially decaying functions (solid curves). The rate constants are: (A) 0.114 ± 0.008 sec1, 0.007 ± 0.0003 sec1 for ACh and 0.069 ± 0.010 sec1, 0.0033 ± 0.0003 sec1 for ACh plus spermine; (B) 0.179 ± 0.005 sec1, 0.0072 ± 0.0006 sec1 for ACh and 0.089 ± 0.007 sec1, 0.008 ± 0.0004 sec1 for ACh plus spermine; and (C) 0.438 ± 0.018 sec1, 0.0239 ± 0.0009 sec1 for ACh and 0.0198 ± 0.007 sec1, 0.0011 ± 0.0002 sec1 for ACh plus spermine. D and E, Whole-cell currents elicited at VH = 60 mV in response to single pulses (1 sec duration) of 10 µM ACh (D) and 1 mM ACh (E) in the absence and presence of 10 µM spermine. Spermine was applied with ACh. The pulses of ACh (or ACh plus spermine) were applied at >30 sec intervals. Although spermine potentiated the responses to ACh in (D) and (E), the ACh-induced inward currents have been scaled to the same amplitude to allow better comparison of their decays.

Single channel studies. Single channel studies were only performed at 60 mV because many of the patches also contained potassium channels that reversed around this V_H with the pipette and bath solutions described (see "Methods"). With Cs⁺-filled pipettes, the reversal potentials were similar for the potassium channel and the nAChR channel. Application of 1.1-sec pulses of 10 µM ACh at V_H = 60 mV to outside-out patches excised from TE671 cells immediately elicited channel openings lasting for several hundred milliseconds (fig. 6). These were probably clusters of bursts of channel openings and in some cases groups of clusters of bursts, as burst durations associated with single agonist binding events are reported to last 10 to 20 msec (Oswald et al., 1989). In most patches, simultaneous openings of more than one nAChR were observed as superimposed inward currents of identical amplitude (fig. 6A), in which case the nAChR channel open probability (p_o) was calculated from the equation (Sokabe et al., 1991): Where p_c is the single channel closed probability (estimated from the relative area of the closed state peak of the all-points histogram) and N is the number of channels in the patch estimated from the number of peaks in the all-points histogram (this estimate is however a lower limit of N as all channels present may not be superimposed immediately following the application of ACh). In this series of experiments, p_o is defined as the open probability over the entire duration of the 1.1-sec ACh application.

View larger version (31K): [in this window] [in a new window]   Fig. 6.   Effect of spermine on single nAChR channel activity. A, Application of 10 µM ACh (indicated by arrow) to an outside-out patch excised from a TE671 cell in the absence (a) and in the presence of 10 µM spermine (b), 100 µM spermine (c) and 1 mM spermine (d). All of the data were collected from the same patch which was held at 60 mV and contained at least 2 nAChRs. B, Comparison of channel open probability (po) for different spermine concentrations. po during ACh application was determined by amplitude histogram analysis from seven outside-out patches, normalized for each patch such that it was equal to 1.0 in the absence of spermine and plotted against spermine concentration: viz, 0 µM (46 applications; seven patches), 10 µM (26 applications; seven patches, 100 µM (29 applications; seven patches) and 1 mM (12 applications; seven patches). C, Channel currents elicited by 10 µM ACh from an outside-out patch held at 60 mV, containing a single nAChR. Data were obtained in the absence (a) and presence of 10 µM (b), 100 µM (c) and 1 mM (d) spermine. Spermine promoted the subconductance state of ACh induced current, e.g., 10 µM ACh plus 100 µM spermine (c). D, po vs. spermine concentration for the patch illustrated in C. All data were low pass filtered at 1 kHz and sampled at 5 kHz.

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View larger version (34K): [in this window] [in a new window]   Fig. 7.   Averaged channel currents recorded from seven outside-out patches exposed to 10 µM ACh in the absence of spermine (A), and in the presence of 10 µM spermine (B), 100 µM spermine (C) and 1 mM spermine (D). Averaged currents (+) are plotted against time after the beginning of the ACh (or ACh plus spermine) pulse and fitted by a single exponential decay (solid line). Data were low pass filtered at 1 kHz and sampled at 1 kHz.

	Discussion

Top Abstract Introduction Methods Results Discussion References

The results of the electrophysiological determination of the sensitivity of nAChR of TE671 cells to ACh described in the present study are comparable to those obtained in binding studies (Lukas, 1986) and in ⁸⁶Rb⁺ efflux studies (Lukas, 1989). The electrophysiological properties of nAChR of TE671 cells reported herein are fully consistent with those obtained by Oswald et al. (1989), Siara et al. (1990) and Grassi et al. (1993).

This study confirms that spermine potentiates the responses of muscle-type nAChR to ACh. It is now shown that the major effect of the polyamine appears to be a reduction in the rate of onset of desensitization via an allosteric mechanism and/or an increase in the rate of recovery from desensitization of nAChR. Similarly, Lerma (1992) showed that spermine reduces the onset of NMDAR desensitization. Potentiation of whole-cell responses to 10 µM ACh and an increase in p_o in the single channel studies were most obvious with 10 µM spermine, less so with 100 µM spermine and were absent when the concentration of the polyamine was raised to 10 mM. However, with the higher concentrations (i.e., 100 µM or more) of spermine, the potentiating action was probably masked either partly or completely by its inhibitory action on nAChR. The apparent absence of either potentiation or inhibition at 1 mM spermine probably arose because the two actions of the polyamine were equal and opposite at that concentration. Interestingly, a study by Szczawinska et al. (1992), who investigated the effects of polyamines on nAChR using ⁸⁶Rb⁺-influx in receptor rich vesicles and -bungarotoxin binding, showed similar results to those reported herein, i.e., inhibition of ion flux and -bungarotoxin binding with spermine concentrations of more than 1 mM and potentiation of ion flux at lower spermine concentrations.

Further evidence for a polyamine potentiating site on nAChR of TE671 cells similar to that found on some NMDAR was obtained in the studies with arcaine. This compound competitively inhibited the observed potentiation of responses to ACh by <1 mM spermine. It is important to note that at these concentrations spermine is also an antagonist, but this antagonism is masked by the potentiation caused by the polyamine. When the potentiation was abolished by arcaine a voltage-independent antagonistic effect of spermine was uncovered, suggesting closed channel block probably due to an allosteric effect of spermine on the closed channel form of nAChR. With 1 to 5 mM spermine, the balance between potentiation and antagonism of ACh-induced currents shifts to the latter, the net effect being antagonism rather than potentiation. This antagonism appears as a voltage-dependent inhibition of nAChR. Interestingly, arcaine reduces the magnitude of this inhibition at highly negative V_H but unmasks inhibition at low negative and positive V_H such that the I-V characteristic for ACh plus spermine no longer shows voltage-dependence (compare fig. 3B and C). Therefore, we suggest that spermine acts at potentiating and voltage-dependent inhibitory sites on nAChR and that arcaine is a competitor at both of these sites. There is no evidence to suggest that arcaine influences the voltage-independent antagonism of nAChR by spermine.

Given its size relative to the nAChR channel, its linear conformation and its polycationic property at physiological pH, it would be surprising if spermine were not an open channel blocker of nAChR (Usherwood, 1987). The studies presently reported show that inhibition of nAChR by 1 mM spermine is noncompetitive and voltage-dependent, and may possibly involve open channel block of this receptor. However, in the presence of arcaine a noncompetitive, voltage-independent antagonism of nAChR by spermine was disclosed, suggesting an interaction of the polyamine with a closed channel form of nAChR. When the concentration of spermine was raised to 10 mM, a further form of antagonism was observed. This was a competitive and voltage-independent component and involved an increase in the EC₅₀ value for ACh.

Endogenous extracellular levels of spermine in mammals are reported to be in the low micromolar range (Schoemaker et al., 1994); thus, a small increase in the concentration of this polyamine could result in a positive modulation of nAChR receptor function, whereas a small decrease could result in a negative modulation. It may be possible therapeutically to use the polyamine potentiating site on nAChR, e.g., potentiation at this site could increase the effectiveness of nAChR in debilitating conditions such as myasthenia gravis. Studies are currently in progress to determine whether spermine potentiates neuronal nAChR.

In conclusion, we have shown that spermine potentiates and inhibits nAChR of TE671 cells. Thus, we have confirmed the results obtained by Hsu (1994) on nAChR of frog muscle. We propose that potentiation arises following interaction of spermine with a polyamine potentiating site, analogous to that present on NMDAR, causing a reduction in nAChR desensitization. It is proposed that inhibition of nAChR by spermine involves voltage-dependent and voltage-independent noncompetitive inhibition and, at high concentrations, competitive inhibition inferring that there are at least four sites for interaction of spermine with nAChR of TE671 cells.