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with either wild-type β or 
subunits and with carboxy-terminal truncated β (βT) or (T) subunits in Xenopus laevis oocytes. β and βT channels (9.6 and 8.7 pS, respectively, with 150 mM Li+) were found to be constitutively open. Only upon inclusion of 1 µM amiloride in the pipette solution could channel activity be resolved; both channel types had short open and closed times. Mean channel open probability (Po) for β was 0.54 and for βT was 0.50. In comparison, and T channels exhibited different kinetics: channels (6.7 pS in Li+) had either long open times with short closings, resulting in a high Po (0.78), or short openings with long closed times, resulting in a low Po (0.16). The mean Po for all channels was 0.48. T (6.6 pS in Li+) behaved as a single population of channels with distinct kinetics: mean open time of 1.2 s and closed time of 0.4 s, with a mean Po of 0.6, similar to that of . Inclusion of 0.1 µM amiloride in the pipette solution reduced the mean open time of T to 151 ms without significantly altering the closed time. We also examined the kinetics of amiloride block of β, βT (1 µM amiloride), and T (0.1 µM amiloride) channels. β and βT had similar blocking and unblocking rate constants, whereas the unblocking rate constant for T was 10-fold slower than βT. Our results indicate that subunit composition of ENaC is a main determinant of Po. In addition, channel kinetics and Po are not altered by carboxy-terminal deletion in the β subunit, whereas a similar deletion in the subunit affects channel kinetics but not Po.
Key Words: epithelial sodium channel • open probability • amiloride block • Xenopus oocyte
Abbreviations: CH, chimera; ENaC, epithelial sodium channel
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introduction |
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, β, and that share 
35% identity at the amino acid level (Canessa et al., 1993
, 1994). Each subunit has two transmembrane domains linked by a large extracellular loop, and the amino and carboxy termini (COOH termini) in the cytoplasm. Biophysical properties of ENaC at the single channel level consist of a small conductance (5 pS), ionic selectivity of Li+ > Na+ >> K+, and high affinity (Ki of 0.1 µM) for the open-channel blocker amiloride (reviewed extensively by Garty and Palmer, 1997).
Injection of the three subunits of ENaC in Xenopus laevis oocytes induces the expression of channels with properties similar to the ones exhibited by channels in native tissues (Canessa et al., 1994). subunits alone can induce amiloride-sensitive currents, but the very low level of expression (1% of β current) has precluded their characterization at the single channel level. Coexpression of and β or and , but not β and , subunits induces whole-cell amiloride-sensitive currents that reach 10–20% of the magnitude obtained with , β, and together.
Previous characterization of whole-cell currents induced by β and channels showed that these channels differ in many properties (McNicholas and Canessa, 1997). For instance, β channels have 10-fold higher Ki for amiloride than channels (1 and 0.1 µM, respectively); and the ion selectivity of β channels is Li+ 
Na+, whereas for channels it is Li+ > Na+. These findings, together with the demonstration of differential subunit expression within epithelial tissues (Farman et al., 1997), may explain the variability in single channel properties of amiloride-sensitive ENaCs in native tissues (e.g., Palmer and Frindt, 1986; MacGregor et al., 1994) and suggest that alteration of subunit composition may be physiologically important.
In this study, we sought to define the single channel properties of β and channels. We examined single channel conductances and amiloride kinetics of channels formed by and wild-type or truncated β (βT) and (T) subunits. Furthermore, we also examined the kinetics and open probability (Po) of β and channels and compared them with those of COOH-terminally truncated channels βT and T. The importance of the COOH termini of the β and subunits on the activity of ENaC was first shown by mutations that produce deletions of part or almost all the COOH termini of β or subunits in patients with Liddle's syndrome, a hereditary form of autosomal dominant hypertension (Shimkets et al., 1994; Hansson et al., 1995). Indeed, expression of COOH-terminally truncated β or subunits in Xenopus oocytes induces amiloride-sensitive whole-cell currents that are three- to fivefold larger than those observed with wild-type subunits (Schild et al., 1995). At least two mechanisms have been proposed to account for the increase in current observed with the truncated subunits. The first mechanism is an increase in channel number at the plasma membrane, and the second is an increase in channel Po. Using cell-attached single channel patches from oocytes expressing either wild-type (β or ) or truncated (βT or T) subunits, we have examined whether the COOH terminus alters channel kinetics and Po.
The findings of this study demonstrate that subunit composition confers distinct kinetics of amiloride block to β and channels and that amiloride block is not affected by deleting the COOH termini. We also show that subunit composition is a major determinant of channel Po. We found that β channels exhibited a very high Po that approximated 1, whereas channels exhibited a mean Po of 0.5. Expression of subunit with a β chimera (CH), which has only the M2 domain and short segment of amino acids preceding M2 from β, and the rest of the sequence from the subunit, induced channels with very high Po similar to β channels, indicating that this region of the β subunit confers the very high Po to the channels. The contribution of the COOH terminus of β and subunits to channel kinetics was assessed by examining β and βT channels and and T channels. Channel kinetics and Po of β and βT channels were not affected by the COOH terminus of the β subunit; the Po of both types of channels remained close to 1. In contrast, COOH-terminal deletion of the subunit changed the gating kinetics without an overall effect on the mean Po. The main effect of deleting the COOH termini of β or on channel activity was an increase in the density of channels at the cell surface that could account for the three- to fivefold increase in whole-cell currents.
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cRNAs were in vitro transcribed from plasmid psD5 with SP6 RNA polymerase using Message Machine (Ambion Inc., Austin, TX) according to the supplier's instructions. Oocytes were injected 24 h after isolation and defolliculation. Equal amounts of subunit cRNA were used at all times (1–3 ng) in a constant injectate volume of 50 nl. After injection, oocytes were kept in the supplemented ND96 medium with 1 µM amiloride was added to prevent sodium loading of oocytes upon expression of ENaC. Recordings were made from oocytes 24–48 h after injection, depending on the subunit composition of the channels.
Electrophysiology
Before patch clamping, the vitelline membrane was removed manually using fine forceps after allowing the cell to shrink for 5 min in a hypertonic solution containing (mM): 220 N-methyl- D-glucamine, 220 aspartic acid, 2 MgCl2, 10 EGTA, 10 HEPES, pH 7.4.
Two standard solutions were used throughout this study. They were a Li+ pipette solution containing (mM): 150 LiCl, 1 CaCl2, 1 MgCl2, 5 HEPES, pH 7.4, and a K+ bath solution containing (mM): 150 KCl, 5 EDTA, 5 HEPES, pH 7.4. Amiloride was included in the pipette solution where indicated. All experiments described herein were performed at ambient room temperature.
Patch pipettes were pulled from fine borosilicate capillary glass (LG16 glass; Dagan Corp., Minneapolis, MN) in two stages, using a microelectrode puller (PP-83; Narishige International USA Inc., East Meadow, NY). They were not fire polished and typically had tip resistances between 3 and 5 M
when partially filled with pipette solution.
Channel currents were recorded from oocytes using the cell- attached configuration of the patch clamp technique (Methfessel et al., 1986). Recordings were made, lasting anywhere between several minutes (for β) to up to 1 h (for ), using an Axopatch 200A amplifier (Axon Instruments, Foster City, CA) interfaced, using a DigiData 1200 interface (Axon Instruments), to an IBM-compatible 166 MHz Pentium PC (Gateway 2000 Inc., N. Sioux City, SD). Data was acquired at 5 kHz, filtered at 250 Hz during acquisition using an eight-pole low pass Bessel Filter (Frequency Devices Inc., Haverhill, MA), and stored directly as data files on the hard drive of a personal computer. The pClamp 6 (Axon Instruments) suite of software was used for data acquisition and its subsequent analysis.
Data was further filtered digitally at 50 kHz during analysis and display. In all channel current traces, a downward deflection represents channel opening. Open and closed time histograms were generated using Pstat within pClamp. Po, and in some cases nPo for patches with more than one channel, was calculated using a specialized nPo program that was written by Jinliang Sui (Mount Sinai School of Medicine, New York) and is available online (http://www.axonet.com/pub/userware/popen). nPo was converted to mean channel Po in patches where the number of active channels could accurately be determined; in most cases, only single channel patches were used or patches with at most two active channels, based upon the number of current transitions observed using channel recordings lasting from several minutes to 1 h. Statistical analysis, where appropriate, was performed using the Student's paired t test.
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results |
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β- or βT-expressed large amiloride-sensitive whole-cell currents measured with two-electrode voltage clamp (between 1–3 µA for β and 
10 µA for βT, not shown), but we could not discern channel activity when these oocytes were patch clamped. Upon inclusion of 1 µM amiloride in the pipette solution, which is the Ki for β channels (McNicholas and Canessa, 1997), we detected channel currents. Fig. 1 shows representative examples of the currents observed for β and βT at –40 mV; at this potential, both channel types displayed fast, flickery transitions between an open and a closed level. A common observation from all β and βT single channel records was the absence of long closed states.
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channels were found to exist in two modes. One gating mode had long closed states with shorter less frequent openings and had a low channel Po. Conversely, the other gating mode had long open states with infrequent transitions to shorter closed states and had a high channel Po. No channels with intermediate Po were found. All T channels, on the other hand, existed in only one state and had shorter and more frequent openings and closings when compared with . However, the frequency of the open states exceeded that of the closed states. When 0.1 µM amiloride was included in the pipette, fewer open states were observed and their length was decreased.
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βT or T, the incidence of channel activity was greater than in oocytes injected with β or . The observation percentage (number of patches with channel activity/number of patches tested) was 40.7% (24/59) for βT, 34.7% (108/309) for T, 12.8% for β (10/78) with 1 µM amiloride (0/24 without amiloride), and 17.3% (21/ 121) for the combined populations of channels. Multiple channel-containing patches (between four and eight channels per patch) were very frequent with βT - or T -expressing oocytes, whereas most patches from oocytes injected with β or contained usually one or at most two channels. Therefore, the analysis of Po and kinetics in the following sections primarily came from patches that contained only one (kinetics) or at most two electrically active channels (Po).
Single channel conductance (slope conductance) was estimated by measuring currents at a range of voltages with 150 mM Li+ in the pipette. Fig. 3 shows the conductance–voltage relationships for the various channel types. There was no difference between β and βT (9.6 vs. 8.7 pS, P > 0.4) or and T (6.7 vs. 6.6 pS, P > 0.4), although the channels containing β and βT subunits had slightly larger conductances than those with the corresponding subunits.
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β and βT channels. These measurements were made with 1.0 µM amiloride in the pipette. β channel Po was 0.65 at 0 mV and 0.5 at –80 mV. As can be seen, the plots of Po against membrane voltage for β and βT were almost identical and were not greatly affected by voltage. The high channel Po in the presence of an amiloride concentration equal to the Ki, and the absence of channel gating without amiloride, suggests that when amiloride is absent, channel Po would be close, if not equal, to 1.
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β (0.54 ± 0.03) and βT (0.50 ± 0.03) were almost identical, suggesting that truncation of the COOH terminus of the β subunit did not alter channel Po (P > 0.15). We were unable to obtain similar plots of Po vs. voltage for the and T channels due to the channel kinetics. For example, to obtain an accurate and meaningful plot for , we would have had to record from a cell-attached patch for at least 90 min (e.g., 0, –40, –80 mV for at least 30 min each), but found that oocytes became difficult to patch after 1 h in the bath. Therefore, we chose to measure channel Po (and single channel kinetics) for and T at –40 mV. This voltage was selected to minimize the activity of a variety of channels endogenous to oocytes (Stühmer and Parekh, 1995). A mean Po of 0.16 ± 0.04 was calculated for the low Po and 0.78 ± 0.05 for the high Po channels, with the mean Po for the entire channel types being 0.47. Preliminary experiments indicate that after excision, patches containing high Po channels adopt a low Po mode instantaneously (Fyfe, G.K., and C.M. Canessa, unpublished observations). The mean Po of T was found to be 0.60 ± 0.01, a value that is not significantly different to the mean value for the channels (P > 0.19). Inclusion of 0.1 µM amiloride in the pipette (the Ki value of amiloride for T channels determined by two-electrode voltage clamp; McNicholas and Canessa, 1997) reduced the mean Po of the T channels to 0.30 ± 0.02.
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subunit and a β chimera. This CH combined the amino terminus, the first transmembrane region (M1), and the extracellular domain of the subunit with the second transmembrane (M2) region and COOH terminus of the β subunit. With 1.0 µM amiloride in the pipette, the mean Po of these β CH channels was 0.62 ± 0.06 (n = 3).
Fig. 5 shows two plots of Po vs. time. Fig. 5 A shows a representative example of a single channel high Po patch that was recorded for 1 h. Note that although the mean Po for this example was 0.67, the Po of the channel (measured every 30 s) varied greatly from close to 1 to near 0 constantly throughout the entire recording. In Fig. 5 B, a representative example of an T patch in the absence and presence of 0.1 µM amiloride is shown. Although the length of these recordings is shorter, it appears truncation of the COOH terminus has given the T channel a more stable Po; i.e., a variation of 0.5–0.8, as opposed to that of 0–1 for .
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β, βT, and T in the presence of amiloride. The kinetics for β and βT were very similar. On the other hand, T had relatively longer open and closed times.
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, the high and low Po modes. High Po channels had a relatively long mean open time of 6.9 s and a shorter mean closed time (1.9 s). Conversely, low Po channels had a relatively short open time lasting <1 s and a longer closed time lasting almost 5 s. T channels exhibited different kinetics compared with channels. They had a mean open time of 1.2 s and a relatively short mean closed time of 400 ms. Channel Po calculated from the mean open and closed times using the equation
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Having obtained the open and closed times for β and βT at a range of voltages between 0 and –80 mV in the presence of 1 µM amiloride, we were able to calculate the blocking (KON) and unblocking (KOFF) rate constants for amiloride at each of the voltages. KON was calculated from the equation
 | (2) |
 | (3) |
Fig. 7 shows the plot of KON and KOFF vs. voltage for β and βT. There was very little difference between the two channel types. The KON and KOFF, dissociation constant at 0 mV (Kd(0)), and dissociation constant at –40 mV (Kd(–40)) values are shown in Table III. Amiloride block is very weakly voltage dependent because the Kd(0) and Kd(–40) for both β (2.7 and 1.5 µM) and βT (1.7 and 1 µM) are close to each other. The difference in the amiloride blocking kinetics when comparing βT (and β) to T is in the off rates: the off rate for βT was calculated to be 39 s–1 and for T was 3 s–1, 1/13 of the βT value. At –40 mV, the Kd of amiloride for T was calculated to be 0.05 µM, a value similar to the amiloride Ki (0.1 µM). We also estimated 
, the electrical distance sensed by amiloride, for β and βT. Both values were found to be 0.3.
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with β subunits favored a conformation in the open state, giving rise to β channels with a very high Po. In the presence of 1 µM amiloride in the pipette (concentration that corresponds to the amiloride Ki of β channels), the Po was 0.5, indicating that without amiloride the Po of β channels is close to one. These channels do not exhibit the characteristic long closed states as seen in oocytes injected with , β, and in cells from native tissues. β channels seem to be constitutively open, no discernible channel transitions were observed in seals obtained in the absence of amiloride, suggesting that β channels do not have a closed state.
Channels formed by the subunit and a β CH when patched with 1.0 µM amiloride in the pipette also exhibited a very high Po similar to β channels (amiloride Ki of CH channels is 1.0 µM). However, in recordings from CH channels, we occasionally detected very brief closed states that were never seen in β channels. The CH comprises the first amino-terminal 2/3 of the subunit and the last COOH-terminal 1/3 of the β subunit that includes the intracellular COOH terminus, the M2 domain, and a short segment of amino acids preceding M2 that has been implicated in amiloride binding (Waldmann et al., 1995; Schild et al., 1997). Similarity in channel kinetics observed in β and CH indicates that the 1/3 of the β subunit is responsible for conferring the high Po on these channels. As we discuss later, the intracellular COOH terminus of the β subunit does not affect the Po of β channels; therefore, the high Po is a property of M2 and/or the region preceding it.
In contrast to β channels, channels exhibited kinetics that were characteristic of wild-type β with closed states that varied from a few milliseconds to several seconds. Two populations of channels were clearly distinguishable: one consistently had a low Po (0.16 ± 0.04) with long closed states, and the other had a high Po (0.78 ± 0.05) with long open states. The mean Po for the whole population of channels was 0.47. Similar findings have been documented for ENaC expressed in cortical collecting tubules where channels exhibited either predominately low or high Po; but, when all channels were averaged, the mean Po was 0.5 (Palmer and Frindt, 1996).
Although the low and high Po channels may represent the extremes of continuous open probabilities, this does not seem to be the case because all the channels we observed fitted well into the low or high Po modes, no intermediate Po values were seen. In spite of the long recording times, we did not see conversion of channels from a low to a high Po mode or vice versa when cell attached. Furthermore, channels in the two gating modes can coexist; occasionally, we obtained patches with two channels: one with high and the other low Po. Although not shown here, our observation that high Po channels, when excised, adopt a low Po state instantaneously before running down several minutes later suggests that some intracellular factor or process may be required to maintain a high Po. It has been reported that hyperpolarization of the membrane potential and low concentration of extracellular Na+ can shift channels to a high Po state (Palmer et al., 1998). For reasons explained in the RESULTS, we did not attempt these maneuvers; all of our experiments were performed at –40 mV with 150 mM Li+ in the pipette.
The COOH terminus of the subunit may contain elements that allow channels to adopt a high or low Po state. Phosphorylation/dephosphorylation of residues in may induce conformational changes that stabilize either high or low Po channels. Indeed, we have recently demonstrated that ENaC is phosphorylated in the subunit (Shimkets et al., 1998). Alternatively, binding of accessory proteins could be another way of inducing conformational changes.
Effect of COOH Terminal Deletion on Po and on Channel Density at the Plasma Membrane
Previous experiments have shown that oocytes expressing ENaC containing β and subunits truncated at their COOH termini had larger whole-cell currents than oocytes expressing wild-type β or . There are at least three mechanisms by which an increase in current could arise in cells expressing truncated subunits: increased single channel conductance, increased channel Po, or increased expression of conducting channels at the plasma membrane. Single channel conductance was found to be unaltered (Schild et al., 1995; Snyder et al., 1995), and the number of channels expressed at the cell surface was found to be increased, but an effect on channel Po has remained controversial. For example, Firsov et al. (1997), using a flag epitope inserted into the extracellular domain of the β subunit, demonstrated that cells expressing truncated subunits had an increased number of channels in the plasma membrane when compared with oocytes expressing wild-type β subunits. The calculated number of channels at the cell surface did not account for the total increase in amiloride-sensitive current measured in the same cells, leading to the inference that channel Po had also been altered to further increase whole-cell currents. Awayda et al. (1997), using a similar approach (fluorescent antibodies to detect surface expression of channels), arrived at the same conclusion because they found only a small rise in the fluorescent signal when comparing oocytes expressing wild-type β and βT subunits.
We have addressed this problem by measuring directly the single channel conductance and Po of β, βT, , and T channels using the cell-attached configuration of the patch clamp technique. Oocytes injected with βT or T channels expressed three- to fivefold larger whole-cell currents than oocytes injected with β or . The single channel conductances of β and were unaltered by COOH-terminal truncations (Fig. 3). Therefore, with β or , changes in single channel conductance cannot explain the increase in whole-cell currents. A slight difference in the channel conductances of β and βT vs. and T may reflect differences in the channel pore structure.
Fig. 4 and Table I show that the Po of β and βT channels in the presence of 1 µM amiloride in the pipette were 0.54 and 0.50, respectively. These results indicate that both types of channels have a very high endogenous Po, 1, and that it is not affected by deletion of the COOH terminus of the β subunit.
channels existed in two gating modes corresponding to low and high Po (Fig. 2 and Table I); each of these modes appeared with equal frequency such that the mean Po of the entire population was 0.47. Truncation of the COOH terminus of the subunit produced a single population of T channels with more homogenous kinetics, distinct from channels. The mean open and closed times of T channels were shorter and less variable when compared with channels (Table II). Also, the Po of channels fluctuated less through time (Fig. 5, A and B). Although the mean Po of T channels (0.6) was slightly higher than that of channels (0.48), the difference does not account for the observed increase in whole-cell current.
We think that our estimate of channel Po is accurate for the following reasons. Due to the nature of the kinetics of β and βT channels (with amiloride in the pipette), multichannel patches were evident instantly and overestimation of channel Po was unlikely. Regarding and T channels, the kinetics were slower and, in most cases, multichannel patches were not evident until 1–2 min after seal formation. Therefore, we recorded patches with for longer periods to be confident of the number of electrically active channels present in the patch; only patches with one or two channels were used to calculate Po. Our results clearly demonstrate that truncations of the COOH termini of the β or subunits do not increase channel Po.
In contrast, the number of active channels at the plasma membrane was larger in oocytes injected with βT or T subunits. The number of successful seals that contained channels and the frequency of patches having more than one channel was greater in oocytes expressing βT or T, indicating that truncated channels are expressed at a higher density. These results are consistent with the presence of endocytosis signals in the COOH terminus of β and that, when deleted, increase the number of channels in the cell surface (Shimkets et al., 1997).
Staub et al. (1997) have proposed another mechanism to explain the increase in number of channels at the plasma membrane. According to these investigators, the protein Nedd4 binds to the COOH termini of the , β, and subunits and subsequently ubiquitinates several conserved lysines in the amino terminus of and , but not β. Only ubiquitinated channels could be removed from the plasma membrane; whereas ubiquitination of had a modest effect on the rate of endocytosis, ubiquitination of appeared to be a prerequisite. Therefore, the hypothesis predicts that oocytes expressing β and βT channels should express a similar number of channels at the cell surface. Instead, we found that the whole-cell currents and the density of channels in the plasma membrane were larger in cells expressing βT channels.
Amiloride Block
Amiloride is an open channel blocker with high affinity for ENaC (Kd of 0.1 µM). The binding site(s) for amiloride is located in the extracellular side of the channel protein. Previous work has shown that mutations of residues in M2 of the subunit (Waldmann et al., 1995) and the region preceding M2 of , β, and subunits (Schild et al., 1997) change the affinity for amiloride. The Kd's for amiloride of β and channels, measured by inhibition of whole-cell currents, are 1 and 0.1 µM, respectively (McNicholas and Canessa, 1997). Here, we have examined the kinetics of amiloride block of β and channels. Direct measurements of KON and KOFF for amiloride on β channels (Table II) showed that these channels have a high Kd for amiloride, and that the decrease in affinity is due to a 10-fold larger KOFF when compared with values reported for β channels (4 s–1; Palmer and Frindt, 1986) or βT (2.5 s–1; Schild et al., 1995). Both the KON and KOFF of amiloride for β channels showed a weak voltage dependence, with a calculated electrical distance sensed by amiloride of 30%. As expected, deletion of the COOH terminus, which is an intracellular domain of the β subunit, did not affect the kinetics of amiloride block as shown in Figs. 1 and 7. The KON and KOFF values for β and βT were almost identical (Table II). CH channels resemble β channels in their amiloride affinity with a Kd of 1 µM measured by inhibition of whole-cell currents. The kinetics of amiloride block of CH channels were also indistinguishable from β channels, a result that agrees with the notion that amiloride interacts with M2 and the amino acids preceding it.
The kinetics of amiloride block of channels were markedly different from β channels. Although the KONs were similar for βT and T when measured with 1 and 0.1 µM amiloride, respectively, in the patch pipette (Table III), the KOFF of amiloride for T channels was much slower, suggesting that amiloride may either occupy an inhibitory site on the channel (or reside in the channel pore) for longer times. The observation that the closed time for T in the presence of amiloride remained unaltered when compared with the absence of amiloride (365 and 400 ms, respectively, Table II), and that the corresponding histogram was described well by one exponential, suggests that the channel closed time and amiloride block time were similar or had the same order of magnitude. We are confident that the value attributed to the mean blocked time for amiloride is correct because the Kd (0.05 µM) calculated from the KON and KOFF is similar to the values obtained with two independent measurements: inhibition of whole-cell current (Ki = 0.1 µM) and mean Po (a 50% reduction in Po with 0.1 µM amiloride).
The kinetics of amiloride block of channels seemed very similar to those described for β. Overall, except for the lower magnitude of whole-cell current due to the lower number of channels expressed at the cell surface, channels exhibited similar properties to β channels.
This work was done under the tenure of an American Heart Association Postdoctoral Fellowship (G.K. Fyfe) and was supported by the Edward Mallinckrodt Jr. Foundation (C.M. Canessa).
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ACKNOWLEDGMENTS |
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Submitted: 7 May 1998
Accepted: 2 July 1998
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-subunits, Am J Physiol, 1997, 273, C1889–C1899.[Medline], β and subunit mRNAs of the epithelial sodium channel along the rat respiratory tract, Am J Physiol, 1997, 272, C131–C141.[Medline], β, and subunits of the epithelial sodium channel (ENaC) involved in amiloride block and ion permeation, J Gen Physiol, 1997, 109, 15–26.
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