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Research Communications |
a Department of Preclinical Veterinary Sciences, R.(D.)S.V.S., Summerhall, University of Edinburgh, Edinburgh EH9 1QH, U.K.; and
b Department of Pharmacology and Toxicology, Danish Centre For Experimental Parasitology, Royal Veterinary and Agricultural University, Frederiksberg C, Denmark
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: Oesophagostomum dentatum • nAChR • patch • clamp • heterogeneity • subtypes
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Not all acquired resistance, then, is due to changes in the target receptor of the therapeutic agent; however, there are only a limited number of general mechanisms by which resistance to a therapeutic agent may be acquired. Four types have been described by Albert (5) : type 1, site exclusion, the first type of acquired resistance to be recognized (4) , is due to the therapeutic drug failing to reach its site of action because the organism modifies the transport of the drug; type 2, DNA amplification, probably the most common mechanism of resistance, involves increases in production of the drug receptor in the pathogen or an increase in drug-destroying enzymes. An increase in the amount of the target dihydrofolate reductase produced by strains of pyrimethamine-resistant malaria, Plasmodium falciparum, is an example of this form of resistance (6) ; type 3, receptor modification, a form of `withdrawal of receptor' resistance (3) , includes receptor loss and reduction of the receptor affinity for the drug; and type 4, post receptor modification, is a form of resistance associated with modifications of pathways that follow receptor activation and can accommodate the effects of receptor stimulation.
Acquired resistance arises mainly by natural selection in infectious organisms produced by the selection pressure of drugs that kill sensitive individuals and thereby encourage the growth of resistant isolates. Resistance is not likely to arise from drug-provoked mutation because mutagenic drugs are normally avoided as therapeutic agents. In some medical texts, the misuse of chemotherapeutic agents is suggested as the cause of resistance. However, it seems that resistance is inevitable and follows regular use of drugs for treating infectious conditions; it is just that resistance occurs more rapidly when frequent subtherapeutic doses are used.
Annual WHO reports relate the enormous scale of suffering nematode parasites produce in humans. An example of a nematode parasite infecting humans is Ascaris lumbricoides, which has a prevalence of 30-60% in endemic areas (7) . Many species of nematode parasite live in the small intestine, producing symptoms of malnutrition, retardation of growth in children, diarrhea, abdominal pain, and, in a smaller proportion of cases, death. Similar nematode parasite infections occur in domestic animals, producing problems concerning animal welfare and economic loss.
A number of important drugs are used for treating nematode parasites that act rapidly by selective gating of membrane ion channels (8) . One such drug used to treat human and animal nematode infections is levamisole. This drug acts as a selective nicotinic agonist to produce spastic paralysis of the parasite, but has little effect on host nicotinic receptors (9) . The target site of levamisole is a pharmacologically distinctive ion channel that forms a nicotinic acetylcholine receptor (nAChR)1 on body muscle of nematodes (10) . The regular use of levamisole has given rise to the development of significant resistance, making the drug useless for particular nematode isolates (11) . Observations on the model soil nematode Caenorhabditis elegans have provided an understanding of the genetic basis of levamisole resistance, showing that up to 11 genes could be involved (12) ; three of these genes—unc-38, unc-29, and lev-1—encode protein subunits for nAChRs.
The information on levamisole resistance provided by the C. elegans studies is limited or difficult to interpret for several reasons. The resistance is produced in the laboratory where `natural selection' is not natural, but enhanced by mutagens and the derived offspring do not have to compete in the `real world' in the same way that a parasitic nematode has to. Uncoordinated levamisole resistant C. elegans (UNC mutants) may survive in the laboratory, but uncoordinated parasites would fail to complete their life cycle. The levels of levamisole resistance selected in vitro in the laboratory are much greater than those required for a parasitic nematode to overcome, because host toxicity prevents very high therapeutic levels of levamisole being used. Another problem is that physiological observations on intact levamisole receptors have not yet been achieved in C. elegans, so we have no direct information relating to their functional properties. In fact, the functions of five genes involved in levamisole resistance have not been identified and it is possible that resistance in parasitic nematodes is produced by a mechanism that does not involve the receptors.
The approach we have taken in studying levamisole resistance is
to choose a `real' nematode parasite that is suitable for patch-clamp
studies and may be passaged easily to produce resistance without
producing animal welfare problems. The parasite selected was
Oesophagostomum dentatum (Fig. 1
A). It is ~1 cm in length, with separate males and females
that inhabit the large intestine of pigs; sometimes the worm produces
small `nodules' in the intestine, and so the parasite is referred to
as the nodular worm, but infection is usually without clinical effects
(13)
. To begin the study, levamisole resistance (~x10) was produced
over 10 generations of the parasite by treating pigs with ascending
doses of levamisole and infecting the pigs with surviving offspring of
the O. dentatum isolate. Selection began with subtherapeutic
doses of levamisole to treat the sensitive isolate (14)
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In this study we compare the channel properties of the levamisole receptors in females of the sensitive isolate (SENS) with the channel properties of receptors of the resistant isolate (LEVR). We show that the receptors of SENS and LEVR are heterogeneous, even within a single patch, and that the resistance is type 3, which is associated with levamisole receptor changes. We also show that several channel properties conspire together to reduce the average current carried by the levamisole receptor-operated channels and that one of the subtypes of receptor is absent in the resistant isolate. Despite changes in the average properties of the receptor channels, we observed that properties of some individual channels from sensitive and resistant isolates could not be distinguished.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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At Edinburgh the adult O. dentatum (Fig. 1A
) were
removed from the thermos flask and placed in petri dishes containing
fresh maintenance solution. Parasites were incubated at 20°C in
maintenance solution, which was changed daily. The parasites survived
between 7 and 14 days under these conditions.
Preparation of muscle vesicles
The adult female parasite is larger than the male and therefore
easier to dissect; we also observed that the female parasites survived
considerably longer in vitro than the male. Consequently,
only female parasites were used for our experiments.
By using fine insect mount pins, we pinned a single female parasite through the head and tail regions in a 2.5 cm petri dish with Sylgard resin lining the base. The preparation was bathed in solution 1, a low Ca2+ maintenance solution containing (mM): NaCl, 35; Na acetate, 105; KCl, 2; MgCl2, 2; HEPES, 10; glucose, 3; EGTA, 1; ascorbic acid, 0.5, adjusted to pH 7.2 with NaOH. Solution 1 is adapted from solutions devised for electrophysiological experiments on Ascaris suum muscle cells (16) .
Under the dissecting microscope, a microscalpel, made by using a broken
razor blade, was used to cut longitudinally through the cuticle and
body wall along the length of the parasite. The digestive tract and
reproductive organs were then removed with fine forceps and discarded.
The resulting `muscle flap' (Fig. 1B
) was pinned out,
washed, and placed in solution 2 (identical to solution 1 except that
it contained no EGTA, but included collagenase at 1 mg/ml) at 37°C
for 10 min. The flap preparation was then washed (10x) in solution 1
and maintained at 37°C. Membrane vesicles (10–50 µm in size)
formed over the next hour from the muscle cells. Vesicles were
collected and used within 5 h of collagenase treatment.
Patch-clamp recordings
Individual vesicles were harvested with a Pasteur pipette
and transferred to the experimental chamber. Patch-clamp recordings
were made at room temperature (Fig. 1C
). The experimental
chamber was mounted on a Nikon TMS-PH3 inverted microscope and viewed
at x300 under phase contrast. The vesicles were bathed in high-Cs
(solution 3) to reduce K currents. Solution 3 contained (mM): CsCl, 35;
Cs acetate, 105; MgCl2, 2; dithiothreitol, 0.1;
HEPES, 10; and EGTA, 1, pH adjusted to 7.2 with CsOH. Our initial
recordings were made without dithiothreitol in the bathing solution. We
found the resulting membrane patches to be fragile and they frequently
broke down at potentials exceeding ± 50 mV. Although high
concentrations of dithiothreitol have been reported to inactivate
nicotinic channels (17)
, we found that the presence of a low
concentration (100 µM) substantially increased the stability of the
membrane patches and permitted recordings to be made in excess of ± 100 mV.
Most recordings were made using cell-attached patches at potentials
of ± 75 mV and ± 50 mV; -50 mV was included because this
is in the region of the resting membrane potential of nematode muscle,
-25 to -45 mV. Isolated inside-out patches were also used
occasionally to check for effects on reversal potentials, conductance,
and kinetics; isolation of the patch did not produce a significant
effect on these parameters. Patch electrodes were pulled from Garner
(7052) capillary glass to a resistance of 2 M
. The pipettes were
coated to near the tip with Sylgard to improve frequency responses.
Patch pipettes were filled with solution 4, which comprised (mM): CsCl,
140; MgCl2, 2; HEPES, 10; EGTA, 1. In addition,
solution 4 contained 10, 30, or 100 µM levamisole as a nicotinic
agonist. Both solutions 3 and 4 were Ca2+ free to
reduce contamination of the recordings with
Ca2+-activated channels.
Data processing and analysis
Single-channel currents were recorded with an Axopatch
200B amplifier onto DAT tape by using a Biological Inc. recorder.
Channel records were analyzed with an Axon Instruments Digidata 1200A
interface, an RM pentium computer and pClamp V6.0 software. The records
were digitally filtered at 1.5 kHz by the software; the sampling time
was 25 µs and the minimum detectable channel opening was 0.3 ms. The
threshold for channel opening was set at 50% of the single-channel
amplitude and the duration and amplitudes of the channel states were
measured. Exponential curves were fitted to the histograms of the
distributions of the open and closed durations using a simplex maximum
likelihood procedure. Gaussian curves were fitted to the distributions
of channel amplitudes using nonlinear simplex least squares to
determine the amplitude of single-channel openings at particular
potentials. The Gaussian fitting of the individual conductances of SENS
or LEVR to a probability density function (p.d.f.) of the form:
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ai=1)
Gi is the fitted mean conductance,
i is the fitted standard deviation,
x is the observed conductances of the single-channels, and
k equals the number of Gaussian terms fitted. For SENS,
k was 4; for LEVR, k was 3. The function was
fitted by a maximum likelihood procedure with the aid of the NAG
subroutine E04CCF, which uses a simplex procedure.
Minitab was used for statistical analysis; t tests were used
to assess differences in means. The Anderson-Darling test was used to
test for normality. ANOVA was used to compare effects of SENS and LEVR
populations on log10Po and 
values. The
nonparametric Kruskal-Wallis test was used to compare
Po values of SENS and LEVR because
Po values were not distributed
normally. 
2 was used to compare the number of
active and inactive patches of SENS and LEVR isolates. Results are
displayed as mean ± SE.
Chemicals and reagents
Levamisole hydrochloride, dithiothreitol, and collagenase type
1A were purchased from Sigma (St. Louis, Mo.).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Figure 2
A is a histogram illustrating the percentages of active
patches seen at the various concentrations of levamisole used. At 10
µM levamisole, both isolates exhibited a similar percentage of active
patches (SENS, 26.4%; LEVR, 30.2%). However, at 30 µM levamisole,
66.7% of patches in the SENS isolate contained active channels
compared with 28.6% in the LEVR isolate; at 100 µM levamisole, the
percentage of active patches was less for both isolates (SENS, 46.8%;
LEVR 9.0%). A 2 test across the concentration
range on the numbers of patches that contained active channels and
those that did not showed us that the difference between the two
isolates was highly significant (P<0.001).
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SENS and LEVR channel current–voltage relationships are linear and
both isolates have a similar mean conductance
Representative single-channel currents recorded from a SENS
cell-attached patch at +75 mV and -75 mV using 30 µM levamisole are
shown in Fig. 2B
along with the current–voltage plot. The
reversal potentials of all plots, including isolated inside-out
patches, were close to 0 mV, showing that the channel was nonselective
and permeable to Cs+ (reversal potential: 0 mV)
rather than to Cl- (reversal potential: -33
mV). Since the slopes of the plots were linear and without
rectification, linear least squares regression provided good estimates
of the conductances of the channels. The conductance of the channel
shown in Fig. 2B
was 46.2 ± 0.5 pS
(mean ±SE). In a proportion (9 of 67) of
patch recordings where channel openings were observed only at a single
membrane potential, the cord conductance rather than the slope
conductance was calculated using a reversal potential of 0 mV.
In the sensitive isolate, the conductance of the levamisole-activated channels ranged from 18.1 pS to 48.0 pS, with a mean of 37.7 pS ± 1.1 pS, n = 45. In the resistant isolate the conductance values ranged from 15.2 pS to 47.8 pS, with a mean of 36.6 pS ± 2.0 pS, n = 22. The mean conductance values of SENS and LEVR were similar and not significantly different (t test, P>0.05). Despite the similarity of the average conductance values, the distributions of the conductances for each isolate appeared different; this difference is considered later.
Patch Po is greater in SENS than LEVR
One way of achieving resistance to levamisole may be for the
receptor channels in the LEVR isolate to open for a shorter proportion
of time, giving rise to less depolarization and contraction. To test
for this effect, we compared the probability of channel opening,
Po, of levamisole receptors in patches
from SENS and LEVR parasites. In the protocol,
Po for each patch was determined
at ± 50 mV and ± 75 mV by measuring the total open time of
all the levamisole channels in a patch that had the same
(indistinguishable) conductances (amplitudes) divided by the total
duration of the recording. Because of uncertainties in the number of
channels present in each patch, we were not able to determine
Po for each channel, so we refer to
the patch Po values for individual
subtypes.
Figure 3
A shows mean ± SE patch
Po values at +50 mV and Fig. 3B
shows values at -50 mV for the SENS and LEVR isolates at
10, 30, and 100 µM levamisole. At each concentration and potential,
the SENS parasites have higher Po
values when compared with the LEVR parasites recorded under the same
conditions. Table 1
gives the mean patch Po values for
experiments at ± 50 mV as well as at ± 75 mV at each of the
different drug concentrations; like Fig. 3
, Table 1
shows that SENS
parasites have a higher patch Po (with
two exceptions) than the LEVR isolates under the same conditions. For
example, at +75 mV with 30 µM levamisole, the mean
Po was 17.87 ± 5.86 x
10-3 in SENS but 5.51 ± 2.47 x
10-3 in LEVR. To test the statistical
significance of the effect of isolate on patch
Po, we used ANOVA but carried out the
analysis on log-transformed Po data to
correct for normality. We found that the effect of the isolate type
SENS vs. LEVR was highly significant (P<0.001). In
addition, we carried out a nonparametric Kruskal-Wallis test on the
untransformed data and also found a significant (P<0.001)
difference between SENS and LEVR. These observations are consistent
with our hypothesis that LEVR patches have lower mean patch
Po values than SENS.
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Membrane potential also had significant (F test, P<0.001) effects on patch Po values, with smaller mean values being observed at hyperpolarized potentials, an effect that has been associated with voltage-sensitive channel block (18) . In addition, levamisole produced significant (F test, P<0.001) dose-dependent effects on patch Po values consistent with it acting as an agonist gating the receptor channel open as well as producing desensitization as concentration increases. These effects have been considered elsewhere (18) and are not relevant to this paper.
Mean open times are greater in SENS than LEVR
Reduced mean Po values may be
associated with the receptor-operated channel opening for a shorter
duration, so we compared mean open times, , from SENS and
LEVR isolates. Figure 4
A shows representative open time distributions and single
exponential fits from SENS and LEVR patches produced with 30 µM
levamisole at -75 mV. The mean open time of the SENS patch was 1.0 ms,
but the LEVR patch had a shorter mean open time: 0.6 ms. Figure 4B
is a histogram showing mean values at -75
mV for SENS and LEVR at 10, 30, and 100 µM levamisole: it can be seen
that the mean values for SENS at each concentration are
bigger than for LEVR, despite the dose-dependent reduction in
produced by open channel block. Table 1
summarizes the
mean ± SE values obtained at
±75 mV ± 50 mV at 10, 30, and 100 µM levamisole for the two
isolates and shows that, at both positive and negative membrane
potentials, mean values were shorter for LEVR than SENS,
except at 100 µM at positive membrane potentials. ANOVA confirmed the
statistical significance of the effect of the SENS and LEVR isolates on
values (F test, P<0.001).
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The effect of lower Po and associated
shorter values in the resistant isolate is that, for any
given levamisole concentration, the channels in the LEVR isolate will
pass less current across the membrane to produce less depolarization
and contraction than for the same concentration of levamisole in the
SENS isolate.
Open channel block occurs in SENS and LEVR
Levamisole acts as a nicotinic agonist in other nematode parasites
like Ascaris suum; because it is a cation, it can produce a
flickering open channel block at negative membrane potentials at higher
concentrations (18)
. We observed a similar open channel block in our
preparations of O. dentatum. Figure 2B
shows a
characteristic flickering block at -75 mV with 30 µM levamisole,
where two bursts of openings lasting longer than 10 min are seen; at
positive potentials (+75 mV), the flickering bursts were not present
and single long openings occur like the 10 ms opening illustrated.
A simple channel block model (closed-open-blocked) (19)
may be used to
describe the open channel block of levamisole (18)
: the forward
blocking rate, k+b, for
levamisole is determined from the slope of the plot 1/
vs. levamisole concentration; the unblocking rate,
k-b, is equal to 1/mean
block time; and the dissociation constant
(KD) is
k-b/k+b. If
the channel pore of the two isolates were different, we might expect
that the blocking rate constants and dissociation constant for the
channel would also be different.
We compared the forward blocking and unblocking constants,
k-b and
k+b, and the dissociation
constant KD for channels from the SENS
and LEVR isolates for channels in the conductance range 38–48 pS.
Table 2
summarizes the k+b,
k-b, and KD
values obtained at -50 mV and -75 mV for each isolate at -50 mV—for
example, KD = 73.6 µM for SENS and
84.9 µM for LEVR. The values for KD
and the other rate constants are similar for each isolate, suggesting
that the block site in the pore of the channel is conserved between
isolates. We were unable to detect a difference between the SENS and
LEVR isolates.
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The distribution of single-channel conductances differs between
isolates
Figure 5
A shows examples of current-amplitude vs. open time scatter
plots and selected openings from a SENS patch and a LEVR patch at +75
mV (30 µM levamisole). Figure 5B
shows the
current-amplitude histograms for openings obtained from these patch
recordings. The SENS patch had three open current levels, marked I, II,
and III and corresponding to cord conductances of 26, 41, and 47 pS;
the LEVR patch had two open current levels, marked IV and V,
corresponding to cord conductances of 25 and 48 pS. Channels with these
same cord conductances at different patch potentials were observed for
both patches, suggesting the presence of at least three conductance
subtypes of levamisole receptor even in a single SENS patch and the
presence of at least two subtypes of receptor even in a single LEVR
patch. We did not observe more than three open current levels for SENS
or more than two open current levels for LEVR patches in single
patches.
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The presence of the different conductance levels in each patch
might be explained by the presence of a single levamisole
receptor that has multiple open conductance states or each patch may
contain multiple subtypes of levamisole receptor, each with a
distinctive conductance. In the majority of our other patches (e.g.,
Fig. 2
), only a single conductance level was observed. We interpret
these observations to indicate that the different conductance levels
observed in Fig. 5A, B
were due to the presence of multiple
subtypes of levamisole receptor in the patches. We also point out that
transitions from one conductance level to another in the channel
records were rare and not frequent enough to be consistent with a
channel with multiple subconductance states.
Figure 6
A shows a histogram of the conductances of all levamisole
receptors observed in SENS patches obtained with the concentrations
10–100 µM levamisole. The distribution has four peaks, was skewed
toward the higher conductances, and was significantly different from a
normal distribution (Anderson-Darling test for normality,
P<0.01) so that the conductance histogram is not consistent
with a single population of levamisole receptors. We described the data
in Fig. 6A
by fitting the conductance distribution to the
sum of four Gaussian distributions with peaks at 21.4 ± 2.3 pS,
labeled G25; 33.0 ± 4.8 pS, labeled G35;
38.1 ± 1.2 pS, labeled G40, and 44.3 ± 2.2 pS,
labeled G45. We have used G labeling before for
convenience in order to refer to peaks in the conductance distribution
(20)
.
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Figure 6B
shows the histogram of conductances obtained from
levamisole receptors recorded from all LEVR patches using 10-100 µM
levamisole. The LEVR distribution has similar peaks to the SENS
conductance distribution but lacks the G35 peak. Again, the
LEVR distribution was significantly different from a normal
distribution (Anderson-Darling test for normality, P<0.01)
and could not be described by a single normal distribution. When the
conductance distribution was fitted by the sum of 3 Gaussian
distributions, the peaks were 25.2 pS ± 4.5 pS, labeled
G25; 41.2 ± 1.7 pS, labeled G40; and 46.7
pS ± 1.1 pS, labeled G45. The difference between the
conductance distributions of the SENS and LEVR populations is that the
LEVR population lacks channels with conductances in the range 33-37 pS
(G35). There were considerable overlaps between the
conductance and the opening properties of levamisole receptors observed
in the SENS and LEVR isolates to the extent that often it was not
possible to determine from the channel properties alone from which
isolate the recording had been made. Nucleotide variation in parasitic
nematodes is marked (21)
and may have contributed to the distribution
in the characteristics of the channels.
Loss of a G35 subtype in LEVR does not account for
all differences between isolates
We have already commented on the differences in average
Po and values of SENS
and LEVR levamisole receptor populations. It was of interest to
determine whether we could account for the difference in population
Po and values as a loss
of the G35 subtype by the LEVR parasites. To test for this,
we removed the data sets for channels with conductances in the range
33-37 pS from the SENS data set. We compared properties of channels
with conductances < 33 pS (G25) from the SENS isolates
with that of the LEVR isolates and found no significant differences in
either log10 Po or
values when using ANOVA. When we compared channel
properties of channels with conductances > 37 pS (G40
and G45), however, we did find significant differences
between SENS and LEVR isolates for both log10Po and (F tests,
both P<0.001). The differences between the two isolates was
statistically significant, arguing that loss of the G35
subtype alone cannot explain the differences in the properties of the
receptor populations.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Resistance ratio SENS:LEVR
By using the patch-clamp technique, we sought to gain better
insight into the mechanisms of anthelmintic resistance by looking at
properties of individual levamisole receptors in sensitive and
resistant isolates of O. dentatum. We postulated that
acquired resistance to levamisole might be explained by changes in
receptor channel properties: type 3 resistance.
Levamisole is well distributed throughout body water because it has a
pKa of 8.0 and easily crosses membrane barriers
(24)
; therefore, the usual therapeutic dose of levamisole at 7.5 mg
Kg-1 is likely to be equivalent to a
concentration of ~30 µM levamisole at receptor sites in the
parasites. We calculated the levamisole resistance ratios,
RRlev, at 30 µM from:
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Varady et al. (14) determined a resistance ratio of 7.3 for these same isolates of O. dentatum using an assay based on movement of larvae hatching from eggs. The similarity between their observations based on muscle movement in intact larvae and our value of 10 suggests that levamisole resistance in the LEVR isolate is attributable to levamisole ion channel receptor changes (type 3 resistance) rather than a change in drug transport, muscle contractility, or other types of resistance. The change in channel properties seen in LEVR isolates in O. dentatum also suggests that it is the genes that determine the structure of the nAChR receptor subunits (Unc-38, Unc-29, and Lev-1) (12) , which give rise to the development of acquired resistance through reduced opening and enhanced desensitization.
In another pathogenic nematode parasite, Haemonchus contortus, acquired resistance to levamisole has been studied by using whole adult worm contraction assays (11) . This study also revealed the presence of enhanced desensitization and an increase in levamisole concentration required to produce contraction in resistant isolates. Together with our observations on O. dentatum, enhanced desensitization with reduced Po appears to be an important mechanism of anthelmintic resistance.
Heterogeneous subtypes of levamisole receptor
The distribution of levamisole receptor conductances from
the SENS isolate in this study was skewed toward G45 and
fitted by the sum of four Gaussian distributions, suggesting the
presence of four main subtypes of levamisole receptor, which we refer
to as G25, G35, G40, and G45. In our previous
study (20)
using 10 µM levamisole, we distinguished two main
subtypes—G35 and G45—and suggested the presence
of other subtypes. In this series of experiments we have found that, on
a defined SENS isolate, it is possible even within a single SENS patch
of membrane to observe the presence of up to three of the subtypes, so
we are able to conclude that individual parasites have at least three
heterogeneous receptor subtypes. However, it is known that all species
of parasitic nematode have a high nucleotide diversity, with averages
of 0.019–0.027 substitutions per site occurring between individuals
from the same population (21)
. The presence of the different peaks in
our distribution of conductances of the levamisole receptors may be
explained by the presence of different subtypes in muscle patches of
individuals, with perhaps additional variance being produced by
variation between the nucleotide structure of individual nematode
parasites.
We have previously suggested an explanation for the presence of
multiple receptor subtypes within an individual parasite (20)
based on
variations in the stoichiometry of the pentameric subunit composition
of the nAChR ion channel in the parasite. In brief, we suggest there
are three genes in O. dentatum analogous to the C.
elegans genes (unc-38, coding an
-subunit; lev-1 and unc-29, coding
for different ß-subunits) (12)
and that each may
contribute one or more subunits of the pentameric levamisole nAChR ion
channel 25, 26)
. The distribution of channel conductances would then
consist of a number of peaks (rather than a single peak) and could be
skewed, depending on the frequency of the subunit combinations.
Although C. elegans and O. dentatum have widely
different lifestyles (free-living in contrast to parasitic), recent
molecular studies have emphasized that they are closely related
phylogenetically (27)
. Therefore, it is likely that our extrapolation
of genetic information from C. elegans to O.
dentatum is appropriate and informative. This extrapolation is
further supported by the 91% similarity of the -subunit Tar
1 gene in the sheep nematode parasite Trichostrongylus
colubriformis with the unc-38 gene of C.
elegans 28, 29)
.
Differences in receptor subtypes between isolates
The sensitivities of different nAChR subtypes to agonists
(in our case, levamisole) and biophysical properties, including
desensitization, are expected to depend on the subunit composition of
the nAChR channel 25, 30)
. One possible mechanism for the development
of resistance could be a shift in the relative proportions of subtypes
due to a change in the probabilities for either UNC-29 or LEV-1
subunits being present to produce a change in the averaged properties
of the receptor population.
When we compared log10
Po and of the G25
values of SENS and LEVR isolates, we found no significant differences.
In contrast, we found that a comparison of SENS: G40 and
G45 vs. LEVR: G40 and G45 revealed
significant differences (F test,
P<0.001 for log10
Po and ). Our data showed no
changes in the properties (and, by implication, structure) of the
G25 LEVR subtype, but changes in other subtypes. The
simplest structural explanation for our observations is that the
acquired resistance is associated with changes in one of the
ß-subunits (UNC-29 or LEV-1) because changes in the UNC-38, the
-subunit, would be expected to change the properties of all
subtypes, including the G25 subtype. The failure of
experiments that have cloned -subunits of levamisole
receptors from other resistant and sensitive nematode parasites
[Tar-1, Trichostrongylus colubriformis 28, 29)
;
Hea-1, Haemonchus contortus (31)
] identify the
molecular basis of resistance as consistent with this explanation. We
caution, however, that mutation of other genes (12)
that do not encode
levamisole receptor subunits may also be involved in levamisole
resistance in parasitic nematodes.
A variety of molecular changes in nAChR subunit structure may enhance
desensitization, including changes to agonist binding sites on
-subunits (32)
, modification of the M3 transmembrane regions (33)
,
or changes to the M2 transmembrane regions including leucine 247, the
block site of QX222 in the ion pore (34)
. We did not detect a
change in the KD for levamisole
in our study, suggesting that this site remains unchanged in the
resistant parasitic nematodes. Changes in the molecular structure
of the channel associated with an increased desensitization may also be
associated with reduced opening rates and shorter open times, as we
have observed here (33)
.
To conclude, we have identified receptor changes associated with
levamisole resistance. The simplest explanation for our observations
appears to be that one of the ß-subunits (equivalent to C.
elegans UNC-29 or LEV-1) in SENS is altered in the LEVR isolate,
so that levamisole receptor subtypes other than G25 have
reduced Po and values
and desensitize more rapidly; consequently, they carry less current
when exposed continuously to levamisole. Future molecular experiments
might examine changes in the ß-subunits of the levamisole receptor.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Abbreviations: ANOVA, analysis of variance;
nAChR, nicotinic acetylcholine receptor; LEVR, levamisole resistant
isolate; SENS, levamisole sensitive isolate.
Received for publication September 15, 1998.
Revision received October 30, 1998.
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REFERENCES |
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  H. Qian, A. P. Robertson, J. A. Powell-Coffman, and R. J. Martin Levamisole resistance resolved at the single-channel level in Caenorhabditis elegans FASEB J, September 1, 2008; 22(9): 3247 - 3254. [Abstract] [Full Text] [PDF]
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 D. Rayes, M. Flamini, G. Hernando, and C. Bouzat Activation of Single Nicotinic Receptor Channels from Caenorhabditis elegans Muscle Mol. Pharmacol., May 1, 2007; 71(5): 1407 - 1415. [Abstract] [Full Text] [PDF]
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H. Qian, R. J. Martin, and A. P. Robertson Pharmacology of N-, L-, and B-subtypes of nematode nAChR resolved at the single-channel level in Ascaris suum FASEB J, December 1, 2006; 20(14): 2606 - 2608. [Abstract] [Full Text] [PDF]
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 S. M. Trailovic, A. P. Robertson, C. L. Clark, and R. J. Martin Levamisole receptor phosphorylation: effects of kinase antagonists on membrane potential responses in Ascaris suum suggest that CaM kinase and tyrosine kinase regulate sensitivity to levamisole J. Exp. Biol., December 15, 2002; 205(24): 3979 - 3988. [Abstract] [Full Text] [PDF]
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 A. P. Robertson, C. L. Clark, T. A. Burns, D. P. Thompson, T. G. Geary, S. M. Trailovic, and R. J. Martin Paraherquamide and 2-Deoxy-paraherquamide Distinguish Cholinergic Receptor Subtypes in Ascaris Muscle J. Pharmacol. Exp. Ther., September 1, 2002; 302(3): 853 - 860. [Abstract] [Full Text] [PDF]
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) and LEVR (
) parasites.
B) Representative channel opening and current-voltage
relationship from a cell-attached patch with 30 µM levamisole. Note
the presence of the `flickering' block shown by the channel currents
at -75 mV.
