Glutamate-gated chloride channels and the mode of action of the avermectin/milbemycin anthelmintics.
ABSTRACT The macrocyclic lactones are the biggest selling and arguably most effective anthelmintics currently available. They are good substrates for the P-glycoproteins, which might explain their selective toxicity for parasites over their vertebrate hosts. Changes in the expression of these pumps have been implicated in resistance to the macrocyclic lactones, but it is clear that they exert their anthelmintic effects by binding to glutamate-gated chloride channels expressed on nematode neurones and pharyngeal muscle cells. This effect is quite distinct from the channel opening induced by glutamate, the endogenous transmitter acting at these receptors, which produces rapidly opening and desensitising channels. Ivermectin-activated channels open very slowly but essentially irreversibly, leading to a very long-lasting hyperpolarisation or depolarisation of the neurone or muscle cell and therefore blocking further function. Molecular and genetic studies have shown that there are multiple GluCl isoforms in both free-living and parasitic nematodes: the exact genetic make-up and functions of the GluCl may vary between species. The known expression patterns of the GluCl explain most of the observed biological effects of treatment with the macrocyclic lactones, though the reason for the long-lasting inhibition of larval production in filarial species is still poorly understood.
- SourceAvailable from: PubMed Central[Show abstract] [Hide abstract]
ABSTRACT: Neglected tropical diseases (NTDs), malaria, and tuberculosis have a devastating effect on an estimated 1.6 billion people worldwide. The World Intellectual Property Organization (WIPO) Re:Search consortium accelerates the development of new drugs, vaccines, and diagnostics for these diseases by connecting the assets and resources of pharmaceutical companies, such as compound libraries and expertise, to academic or nonprofit researchers with novel product discovery or development ideas. As the WIPO Re:Search Partnership Hub Administrator, BIO Ventures for Global Health (BVGH) fields requests from researchers, identifies Member organizations able to fulfill these requests, and helps forge mutually beneficial collaborations. Since its inception in October 2011, WIPO Re:Search membership has expanded to more than 90 institutions, including leading pharmaceutical companies, universities, nonprofit research institutions, and product development partnerships from around the world. To date, WIPO Re:Search has facilitated over 70 research agreements between Consortium Members, including 11 collaborations focused on anthelmintic drug discovery.International Journal for Parasitology: Drugs and Drug Resistance. 12/2014;
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ABSTRACT: Evaluate use of spherical nanocarbon (Grafex) injected into known Human Prostatic carcinoma to increase absorption of microwave energy, specifically into tumor cells. This study was selected to evaluate if nanocarbon and microwave can be used as primary treatment and selected to study heat sink associated with microwave treatment. This specific nanocarbon has demonstrated a range of energy absorption capabilities superior to other non-metallic materials.Materials and Methods10 Nude nu/nu mice were injected with DU145 (ATC#HTB-81) 1x 10^7 Human Prostate carcinoma cells introducted into the dermis and allowed to grow to >1cm. One control mouse received no treatment. One control mouse received only an injection of nanocarbon. Remaining 8 mice received treatment with the microwave and the nanocarbon. Medwave and BSD magnetron generators with temperature probes were used. Nanocarbon and a viscous carrier were injected into the tumors. Short cycle power using 10 watts at 15 seconds used as baseline settings. Target temperature within the tumor was 60 degrees C.ResultsThe control non treated mouse was euthanized due to metastatic prostate cancer 3 weeks after initial injection. Seven of the original treated mice have resolved their prostate tumors with no apparent ill effects from the nanocarbon. One mouse had a non treated skin ulcer from the non cooled microwave probe during the treatment process and was also euthanized ,however, was responding to treatment. The one mouse that received just the nanocarbon treatment, is alive, however the tumor growth has not receded or advanced. All mice were kept in isolation and were observed to have no side effects from the nanocarbon.Conclusion Nanocarbon assisted microwave therapy maximizes energy transfer. Conversion of microwave energy causes thermal ablation to the cancer cells. This reduces heat sink effect and surrounding tissue damage by using much shorter treatment times and lower power output wattage. This suggests Grafex nanocarbon causes enhancement of the dielectric properties of the tumor causing cytotoxic heating. To date, there is no observed toxic side effects in mice from this small study.Journal of Vascular and Interventional Radiology 04/2013; 24(4):S116-S117. · 2.15 Impact Factor
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ABSTRACT: The cattle tick Rhipicephalus (Boophilus) microplus is the most economically important ectoparasite affecting the cattle industry in tropical and subtropical areas around the world. The principal method of tick control has relied mainly on the use of chemical acaricides, including ivermectin; however, cattle tick populations resistant to ivermectin have recently been reported in Brazil, Mexico, and Uruguay. Currently, the molecular basis for ivermectin susceptibility and resistance are not well understood in R. microplus. This prompted us to search for potential molecular targets for ivermectin. Here, we report the cloning and molecular characterization of a R. microplus glycine-like receptor (RmGlyR) gene. The characterized mRNA encodes for a 464-amino acid polypeptide, which contains features common to ligand-gated ion channels, such as a large N-terminal extracellular domain, four transmembrane domains, a large intracellular loop and a short C-terminal extracellular domain. The deduced amino acid sequence showed around 30% identity to GlyRs from some invertebrate and vertebrate organisms. The polypeptide also contains the PAR motif, which is important for forming anion channels, and a conserved glycine residue at the third transmembrane domain, which is essential for high ivermectin sensitivity. PCR analyses showed that RmGlyR is expressed at egg, larval and adult developmental stages. Our findings suggest that the deduced receptor is an additional molecular target to ivermectin and it might be involved in ivermectin resistance in R. microplus.Parasite 01/2014; 21:43. · 0.82 Impact Factor
Glutamate-gated chloride channels and the mode of action
of the avermectin/milbemycin anthelmintics
A. J. WOLSTENHOLME* and A. T. ROGERS
Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK.
The macrocyclic lactones are the biggest selling and arguably most effective anthelmintics currently available. They are
good substrates for the P-glycoproteins, which might explain their selective toxicity for parasites over their vertebrate
hosts. Changes in the expression of these pumps have been implicated in resistance to the macrocyclic lactones, but it is
clear that they exert their anthelmintic effects by binding to glutamate-gated chloride channels expressed on nematode
neurones and pharyngeal muscle cells. This effect is quite distinct from the channel opening induced by glutamate, the
endogenous transmitter acting at these receptors, which produces rapidly opening and desensitising channels. Ivermectin-
activated channels open very slowly but essentially irreversibly, leading to a very long-lasting hyperpolarisation or
depolarisation of the neurone or muscle cell and therefore blocking further function. Molecular and genetic studies have
shown that there are multiple GluCl isoforms in both free-living and parasitic nematodes: the exact genetic make-up and
biological effects of treatment with the macrocyclic lactones, though the reason for the long-lasting inhibition of larval
production in filarial species is still poorly understood.
Key words: Ivermectin, Caenorhabditis elegans, Haemonchus contortus, Filaria, ionotropic receptor, chemotherapy.
The avermectins and milbemycins (A/M), often also
referredtoas the macrocyclic lactones,arethe largest
selling anthelmintics in the world. As well as being
widely used in veterinary medicine for the treatment
of gastro-intestinal nematode infections and ecto-
parasite infestations (Vercruysse & Rew, 2002), they
are used in agriculture for the control of insect pests
and in human medicine for the treatment of filarial
nematode infections, most notably in the treatment
and control of onchocerciasis (Omura & Crump,
2004). This enormous success, which has only very
recently been clouded by reports of resistance in
veterinary parasites in some parts of the world
(Jackson & Coop, 2000; Anziani et al. 2001;
Familton, Mason & Coles, 2001; Loveridge et al.
2003), is due to their rapid effects on, and specificity
for, the target organisms. Though extremely effec-
tive at controlling arthropods as well as nematodes,
the A/M are ineffective against flatworms and tape-
worms. This article will concentrate on the nemato-
cidal effects of these drugs.
When the A/M anthelmintics are applied to
nematodes two main effects, rapid paralysis of
either move or to feed and, for most infections, the
from the host. The major group of nematodes
refractory to A/M treatment, adult macrofilariae,
may survive drug treatment because neither move-
ment nor pharyngeal pumping are required for their
continued survival in tissues. However, in this case a
further effect of the drugs is revealed, a long-lasting
reduction in the production of new larvae (Campbell
et al. 1983). It is this third effect, coupled with their
extreme toxicity to the motile microfilariae, which is
the control of onchocerciasis. These three effects
predict that the target of these drugs must have roles
in the locomotion, pharyngeal pumping and repro-
duction of parasitic nematodes.
GLUTAMATE-GATED CHLORIDE CHANNELS
Soon after the introduction of ivermectin, the first
A/M to be commercialised, in 1980, electro-
physiological experiments showed that the drug
caused an increase in the chloride conductance of
mammalian neuronal membranes, and that this
effect was blocked by picrotoxin (Kass et al. 1980;
Supavilai & Karobath, 1981; Graham, Pfeiffer &
Betz, 1982; Pong & Wang, 1982). Ivermectin has
continued to be the drug used in most published
studies on the A/M family, since it is both the largest
selling of these compounds and also the least
hydrophobic of those readily available. Given the
known role of GABA receptors in nematode loco-
motion (McIntire et al. 1993) and that picrotoxin is a
* Author for correspondence. Tel: 01225 386553. Fax:
01225 386779. E-mail: A.J.Wolstenholme@bath.ac.uk
Parasitology (2005), 131, S85–S95.
f 2005 Cambridge University Press
Printed in the United Kingdom
potent GABA-gated chloride channel blocker (Olsen
& Tobin, 1990), it was a natural hypothesis that the
A/M were acting at GABA receptors, and early data
on nematode preparations offered some support for
this (Holden-Dye et al. 1988; Martin & Pennington
1989; Holden-Dye & Walker, 1990). However, the
concentrations required were rather too high for this
effect to be clinically relevant and in some prepara-
tionsivermectininhibited rather than opened GABA
receptor channels (Martin & Pennington, 1988;
Holden-Dye & Walker, 1990) and, though it is clear
that the A/M do have effects on nematode GABA
receptors (Feng et al. 2002), attention turned to
alternative chloride channels as the likely target.
Experiments in which total mRNA isolated from
Caenorhabditis elegans was injected into Xenopus
oocytes revealed a glutamate-gated chloride channel
(GluCl) that was sensitive to ivermectin (Arena et al.
1991, 1992). Fractionation of this total mRNA into
smaller and smaller pools eventually resulted in
the identification of two GluCl subunits, designated
a and b, from C. elegans (Cully et al. 1994).
Expression of the a subunit in the Xenopus oocyte
resulted in the appearance of an ivermectin-gated
channel, the b subunit produced a glutamate-
co-expression of the two subunits resulted in an
ivermectin-sensitive, glutamate-gated channel. The
properties of this channel were very similar to
that produced by the total mRNA preparation, and
it was swiftly established that its pharmacology was
very similar to that expected for the avermectin
target (Arena et al. 1995). The genes encoding these
proteins were later designated glc-1 for the GluCla
subunit and glc-2 for the GluClb subunit (glc=
glutamate-gated chloride channel).
This rather simple picture rapidly became more
complex as complementary molecular biological and
genetic approaches led to the identification of two
further a subunits, GluCla2A and GluCla2B, from
C. elegans (Dent, Davis & Avery, 1997; Vassilatis
et al. 1997a). These subunits produced channels
gated by both glutamate and ivermectin and were
products of the alternatively spliced avr-15 (avr=
evidence that the GluCl were indeed the in vivo
avermectin targets. Reporter gene constructs, in
which the putative promoter regions of the genes
under study are attached to lacZ or GFP (Fire,
Harrison & Dixon, 1990), demonstrated that both
the GluCla2 and b subunits were expressed in
pharyngeal muscle cells and, in the case of the
GluCla2 subunits, more widely in the nematode
motor nervous system, (Dent et al. 1997; Laughton,
Lunt & Wolstenholme, 1997a) consistent with the
paralysis of the pharynx and body-wall muscle
observed when ivermectin is applied. Thegenetics of
avermectin resistance suggested that this was not the
end of the story, since mutations in avr-15 alone did
not cause the worms to be drug-resistant and a
mutation in a second gene, avr-14 was also necessary
for moderate resistance and in a third, glc-1, for high
level resistance (Dent et al. 2000). The second gene,
avr-14, is identical to the gene also referred to, by us,
as gbr-2 (GABA receptor related) (Laughton, Lunt
& Wolstenholme, 1997b). It too is alternatively
spliced and encodes two subunits, GluCla3A and
GluCla3B, which share a common N-terminal half
but differ in the C-terminal, channel forming, half of
the protein. GluCla3B produces glutamate- and
ivermectin-sensitive channels when expressed in
Xenopus oocytes, but the GluCla3A subunit has yet
to be shown to produce any functional channels
(Dent et al. 2000; Rogers & Wolstenholme, unpub-
lished). The complete sequencing of the C. elegans
genome allowed bioinformatics to be added to the
cDNA cloning and genetic methods previously used.
This led to the identification of a fourth a-subunit
encoding gene, glc-3: its product, GluCla4, also
produced glutamate- and ivermectin-gated chloride
channels when expressed
(Horoszok et al. 2001). An additional putative
GluCl-encoding gene, glc-4, has also been identified
(Cully, Wilkinson & Vassilatis, 1996) that may also
be alternatively spliced. The sequence of the pre-
dicted glc-4 encoded subunits is rather distant from
either the a or b subunits previously described,
suggesting that it may belong to a different, c class.
The current list of C. elegans GluCl genes and the
subunits they encode is given in Table 1.
Though the paralytic effects of the A/M on para-
sitic nematodes had been observed at an early stage,
and parasite preparations, especially from Ascaris
suum, have been widely used to study the effects of
the drugs on non-recombinant targets (Kass et al.
1980; Kass, Stretton & Wang, 1984; Holden-Dye
et al. 1988; Martin & Pennington 1989; Holden-Dye
& Walker, 1990; Martin, 1996; Adelsberger, Scheur
& Dudel, 1997; Brownlee, Holden-Dye & Walker,
1997), progress in the molecular characterization of
the GluCl has been much slower in parasites. The
sequences of the C.elegans GluClsubunitswereused
as the basis of a reverse transcriptase-PCR approach
using degenerate primers based on highly conserved
regions of amino acids. The parasite species most
widely studied in this way has been Haemonchus
contortus, from which several GluCl cDNAs have
been cloned (Delany, Laughton & Wolstenholme,
1998; Forrester et al. 1999; Jagannathan et al. 1999).
From this work it is clear that some of these subunits
are very similar to those found in C. elegans,
including the HcGluCla3A, 3B and HcGluClb
(Hc=Haemonchus contortus) subunits. Others, such
as the HcGluCla or a subunit, are less closely related
to any particular C. elegans gene product (Yates,
Portillo & Wolstenholme, 2003), raising the in-
triguing possibility that the GluCl gene family varies
between nematode species. Of the various subunits
A. J. Wolstenholme and A. T. Rogers
described to date, GluCla3 has been found in the
largest number of species, with sequences reported
from H. placei (Mes, 2004), A. suum (Jagannathan
et al. 1999), Cooperia oncophora (Njue et al. 2004;
Njue & Prichard, 2004), Dirofilaria immitis (Cully
et al. 1996; Yates & Wolstenholme, 2004), and
Onchocerca volvulus (Cully et al. 1996). Mutations
and polymorphisms in GluCla3 subunits have been
associated with avermectin resistance in both C.
2004; Njue & Prichard, 2004) which could be inter-
preted to suggest that they are important in vivo drug
targets. The GluCl subunits currently known in
parasitic nematodes are summarised in Table 2.
The sequence of the GluCl subunits clearly
revealed them to be members of the ‘Cys-loop’
family of ligand-gated ion channels, closely relatedto
vertebrate GABAAand glycine receptors, and more
distantly related to the nicotinic acetylcholine
receptors. In fact their closest homologues in verte-
brates are probably the glycine-gated chloride
has the characteristic topology of 4 membrane-
C-termini. As with other members of this family, the
GluCl are predicted to be pentameric structures,
with the native channels consisting of more than one
subunit type. Table 1 clearly shows that in C. elegans
there are more than five predicted subunits and this
immediately implies that there must be multiple
types of native GluCl, and hence avermectin targets,
in this organism, and therefore presumably in other
nematodes, each made up of different combinations
of subunits and presumably expressed on different
tissues. This molecular complexity presents a major
challenge to a complete understanding of the A/M’s
actions,sinceitis likely that eachofthese GluClswill
have different sensitivities and accessibilities to the
drug, and that activation of them will have different
effects on the organism. If the potential for variations
in the number and expression of the individual
subunits are added to the mix, then it is obvious that
there is scope for considerable differences in the
effects of the A/M between nematode species. The
determination of the subunit composition of native
GluCl is therefore of major importance, but is not
trivial. One advantage may be that nematodes are
anatomically rather simple, with relatively few
Recombinant nematode GluCl are activated by
L-glutamate, but not aspartate, GABA, glycine,
histamine or any other amino acid or candidate
neurotransmitter. Ibotenate, a conformationally con-
strained analogue of glutamate, is at least a partial
agonist at most GluCl (Cully et al. 1994; Dent et al.
1997; Vassilatis et al. 1997a; Dent et al. 2000;
Horoszok et al. 2001; Forrester et al. 2003; Yates &
Wolstenholme, 2004). Glutamate activation of most
a-subunit- containing receptors produces a rapid
opening of the channel that rapidly desensitises
(Fig. 1A). If the receptor is composed solely of
b subunits, then the receptors do not desensitise, but
do close rapidly once agonist is removed (Cully et al.
1994; Njue et al. 2004). The EC50for activation of
recombinant C. elegans GluCl by L-glutamate is
usually about 1–2 mM if an a-subunit is present,
although the GluCla2B is more sensitive (EC50=
0.14 mM) (Cully et al. 1994; Dent et al. 1997;
Vassilatis et al. 1997a; Dent et al. 2000; Horoszok
et al. 2001), and those composed of b subunits are
also more sensitive to the agonist (EC50=0.38 mM)
(Cully et al. 1994). There have been fewer quanti-
tative data published for recombinant parasite
GluCl: the EC50 for glutamate at the D. immitis
GluCla3B receptor is also approximately 1 mM
(Yates & Wolstenholme, 2004) but, intriguingly the
Table 1. The GluCl genes of Caenorhabditis elegans, and the subunits they encode
avr-14 mutations cause moderate avermectin resistance
in combination with avr-15, and very high level
resistance as a triple mutant with avr-15 and
glc-1. GluCla3B forms glutamate- and ivermectin-gated
channels: no channels have been reported for GluCla3A.
Laughton, Lunt & Wolstenholme,
1997b; Dent et al. 2000
In avr-15 mutants pharyngeal pumping is insensitive to
ivermectin. GluCla2 subunits form glutamate- and
ivermectin-gated channels and can co-assemble with
GluCla1 forms ivermectin-gated channels: glutamate binds
but does not open the channel. When expressed with
GluClb, ivermectin-potentiated, glutamate-gated channels
GluClb forms glutamate-gated channels.
GluCla4 forms glutamate- and ivermectin-gated channels.
Dent, Davis & Avery, 1997;
Vassilatis et al. 1997a;
Pemberton et al. 2001
Cully et al. 1994; Etter et al. 1996
Cully et al. 1994
Horoszok et al. 2001
Cully, Wilkinson & Vassilatis, 1996
GluCl and the A/M anthelmintics
C. oncophora GluCl subunits are much more sensi-
tive, with the a3 subunit forming a receptor with an
EC50of y0.03 mM and the b subunit having a EC50
for glutamate of y0.18 mM (Njue et al. 2004). This
increased agonist sensitivity of the GluCl subunits
may be conserved in H. contortus, since the
HcGluCla subunit forms receptors with an EC50for
glutamate of only 8.4 mM (Forrester et al. 2003) and
the HcGluCla3B receptor has a very similar quan-
titative pharmacology to that from C. oncophora
(Rogers & Wolstenholme, unpublished). By analogy
with the other members of the ‘cys-loop’ family of
ligand-gated ion channels, glutamate would be
expected to bind to the extracellular domains of
the receptor, possibly at the subunit interface
(Cascio, 2004). The agonist-binding site would be
made up of six loops, three from each of the adjacent
subunits. Consistent with this prediction, a mutation
in the extracellular domain of the C. oncophora a3
subunit (L256F) reduced the efficacy of glutamate
approximately three-fold and a combination of two
polymorphisms in the N-terminal domain (V60A
and R100H) abolished the glutamate sensitivity of
the b subunit (Njue et al. 2004). The combination of
mutations in both subunits reduced the EC50 for
glutamate at the a3b heteromeric receptor by
13-fold. It is possible to correlate the amino acid
sequence of many GluCla3 subunits around this
position with their sensitivity to glutamate (Fig. 2),
which may indicate that this part of the subunit
either forms part of the binding site or is involved in
the efficient coupling of ligand-binding to channel
Application of ivermectin to recombinant GluCl
produces a markedly different effect from glutamate
(Fig. 1B). The channels open very slowly, and the
rate of channel opening may be dose-dependent
channel opening is essentially irreversible, and the
(Cully et al. 1994; Vassilatis et al. 1997a; Horoszok
et al. 2001; Forrester et al. 2003; Yates &
Wolstenholme, 2004). Ivermectin has no effect on
GluClb subunits (Cully et al. 1994; Njue et al. 2004).
Because of the extremely unusual properties of
ivermectin and theirreversible channelsit opens, itis
very difficult to obtain good quantitative data with
this drug, but EC50values in the range of 0.1 to
10 mM have been reported for recombinant GluCl
500 nA |__
Fig. 1. Examples of chloride currents produced by
activation of recombinant GluCl in Xenopus oocytes.
HcGluCla3B cRNA was transcribed in vitro and
microinjected into oocytes as described (Yates &
Wolstenholme, 2004). Recordings were made 3–5 days
later. A) shows the response to 1 mM L-glutamate and
B) to 1 mM ivermectin: the horizontal bars above the
recordings indicate when agonist was applied. Note the
rapid onset and desensitisation of the L-glutamate-
induced currents, compared to the slow onset and
essentially irreversible current induced by ivermectin.
Table 2. The glutamate-gated chloride channel (GluCl) subunits of parasitic nematodes
Subunit Species in which foundPropertiesReferences
Forms glutamate- and
Forms a high affinity
ivermectin binding site.
GluCla3A does not express in
oocytes or mammalian cell
lines. GluCla3B forms
glutamate- and ivermectin-
gated channels and a high
affinity ivermectin binding
Forrester et al. 1999, 2003;
Cheeseman et al. 2001;
Forrester, Prichard &
Ascaris suum*, Cooperia
contortus, H. placei,
Cully, Wilkinson & Vassiliatis,
1996; Jagannathan et al.
1999; Cheeseman et al. 2001;
Njue & Prichard, 2004; Njue
et al. 2004; Yates &
channels. Can be co-
expressed with GluCla3B.
Does not form a high-affinity
ivermectin binding site.
Delany et al. 1998; Cheeseman
et al. 2001; Njue et al. 2004
* Only a single GluCla3 subunit has been identified in these species.
A. J. Wolstenholme and A. T. Rogers
(Arena et al. 1991; Cully et al. 1994; Vassilatis et al.
1997a; Dent et al. 2000; Horoszok et al. 2001;
Forrester et al. 2003; Njue et al. 2004). These are
rather higher than the concentrations at which the
drug shows anthelminticeffects, and also higher than
the Kdvalues (26–100 pM) obtained in radioligand
binding experiments to membranes from mam-
malian cells expressing recombinant H. contortus
GluCl(Cheesemanet al.2001;Forrester,Prichard &
Beech, 2002). Very recent experiments have found
that the H. contortus GluCla3B subunit can be acti-
vated by ivermectin concentrations as low as 0.1 to
1.0 nM (Rogers and Wolstenholme, unpublished),
much closer to the concentrations suggested by the
One very interesting phenomenon is the inter-
action between glutamate and ivermectin. The very
first paper on the cloning of the GluCl subunits from
C. elegans (Cully et al. 1994) showed that con-
activate the channels would nonetheless potentiate
the effects of simultaneously applied sub-maximal
concentrations of glutamate. Similar results were
obtained (Martin, 1996) using two-electrode patch
clamp recordings from the pharyngeal muscle of A.
suum, where milbemycin D caused a dose-dependent
potentiation of the observed response to glutamate.
human glycine receptors: ivermectin acted as an
(>0.3 mM) but at lower concentrations (30 nM) it
(Shan, Haddrill & Lynch, 2001). Ivermectin has
similar allosteric effects at the vertebrate a7 nicotinic
receptor (Krause et al. 1998) and here mutations in
the TM2 channel domain, believed to change the
conformational equilibrium that exist between the
active and desensitised states of the receptor, alter
ivermectin potentiation. This result was interpreted
to suggest that ivermectin binding also changes this
equilibrium, leading to an increased probability of
channel opening. More recent experiments have also
shown the reverse phenomenon, that glutamate
potentiates the activity and binding of ivermectin, at
Forrester, Beech & Prichard, 2004). Glutamate and
ivermectin do not compete for the same binding site
(Hejmadi et al. 2000) so these data suggest that the
two sites exert complementary, and possibly addi-
tive, effects on the conformational changes needed
for the channels to open. It is possible that inter-
actions between exogenous anthelmintic and endo-
genous glutamate explain the extraordinary potency
of the A/M for killing worms and the much lower
concentrations needed to activate native GluCl
(Pemberton et al. 2001) than those reported for
recombinant channels expressed in Xenopus oocytes
(Arena et al. 1991; Cully et al. 1994; Vassilatis et al.
1997a; Dent et al. 2000; Horoszok et al. 2001;
Forrester et al. 2003; Njue et al. 2004). Binding of an
A/M to a GluCl molecule will both increase the
probability of that channel opening but, in addition,
greatly stabilise the open state once that opening has
taken place, resulting in the essentially irreversible
In summary, in vitro molecular cloning and
expression of recombinant subunits has provided
unequivocal evidence that the GluCl are the
nematode targets of the ML anthelmintics. An
examination of the in vivo roles of these receptors
should explain how the drugs exert their anthel-
mintic effects, and vice versa.
P-GLYCOPROTEINS AND THE
It is apparent from the previous section that, in vitro,
the A/M do not interact only with the invertebrate
GluCl, but also with other invertebrate and ver-
tebrate ligand-gated chloride channels. Is the in vivo
selectivity of these compounds for the invertebrate
parasites over their vertebrate hosts due solely to
a higher affinity at the GluCl over these other
channels, or are other factors important? An
important finding has been that certain collie dogs
are extremely sensitive to treatment with the A/M,
and that this sensitivity is associated with mutations
in the mdr-1 gene that encodes a P-glycoprotein
(Mealey et al. 2001; Roulet et al. 2003). The drugs
are extremely good substrates for these pumps in
vertebrates and it is likely that their reduced toxicity
for host versus parasite is due to their removal from
the CNS by non-specific pumps of the blood-brain
are also good substrates for nematode P-glyco-
proteins (Kerboeuf et al. 2003) and it has been sug-
gested that changes in the expression of these pumps
might contribute to drug resistance (Blackhall et al.
1998; Sangster et al. 1999; Drogemuller, Schnieder
Subunit Sequence from position 253EC50 Glutamate
C. oncophora IVMS
V K L L L R R
C. oncophora IVMR
V K L L R RF
V K L L L R R
V L L R R
V L L L R R
V K L L L R R
Fig. 2. Alignment of the partial amino-acid sequence of
the GluCla3B subunits from several nematode species,
correlated with their sensitivity to activation by
glutamate. IVMSand IVMRare ivermectin sensitive and
resistant, respectively. Amino-acids that are different
from the ivermectin sensitive version of the C. oncophora
subunit are shown in white on a black background.
GluCl and the A/M anthelmintics
& von Samson-Himmelstjerna, 2004). However,
there are no suggestions that the anthelmintic effects
of the A/M are due to their interactions with the P-
glycoproteins and it seems this is solely due to their
GLUCL IN THE NEMATODE PHARYNX
The nematode pharynx is a muscular organ and its
function is to take in and partially process food prior
to pumping in into the gut. The structure of this
organ varies widely between nematode species and is
one of the most characteristic features of their mor-
phology. It contains distinct muscle, nerve and gland
cells and is almost a self-contained system (Bird &
Bird, 1991). As such, it is amenable to electro-
physiological recordings and pharyngeal prepara-
tions have been widely used to study the effects of the
A/M. It is also easy to observe and measure the rate
a relaxation phase triggered by the glutamatergic
1993a,b; Raizen, & Avery, 1994; Lee et al. 1999),
which implies the presence of inhibitory glutamate
receptors on the post-synaptic muscle cells.
Pharyngeal pumping in nematodes is extremely
sensitive to the A/M, and in many species the phar-
ynx is the most sensitive organ to the effects of the
drugs, with reported EC50 values of 0.2 to 10 nM
(Bottjer & Bone, 1985; Avery & Horvitz, 1990;
Geary et al. 1993; Gill et al. 1995; Paiement et al.
1999; Sheriff et al. 2002). This may mean that the
primary anthelmintic effect of the A/M is on the
pharynx and that its paralysis leads to worm death,
pressure. The inhibition of pumping is due to the
(Martin, 1996; Adelsberger et al. 1997; Pemberton
et al. 2001). The irreversible activation of these
receptors by ivermectin leads to a depolarisation of
the muscle (Pemberton et al. 2001), presumably due
to high internal [Clx], and a cessation of pumping.
In C. elegans, reporter gene experiments have
shown that the avr-15 and glc-2 genes, encoding the
GluCla2 and b subunits, are indeed expressed in
pharyngeal muscle cells (Dent et al. 1997; Laughton
et al. 1997a). Pemberton et al. (2001) employed a
pharyngeal preparation from wild type and mutant
C. elegans to make direct recordings of the effect of
glutamate and ivermectin on muscle activity. Some
of the properties of these preparations were consist-
ent with the suggestion that the pharyngeal receptor
in C. elegans is composed of a2 and b subunits, in
particular the loss of ivermectin sensitivity of this
preparation in avr-15 mutant worms (Pemberton
et al. 2001), but some of the pharmacological details
are not. The channels found in avr-15 worms do not
possess the picrotoxin sensitivity predicted for a
GluClb receptor and the sensitivity to ivermectin of
the preparation is rather higher than that seen for the
GluCla2+b combination in the Xenopus oocyte. So
is there a third GluCl subunit present in pharyngeal
muscle and, if so, which is it? So far the only gene
that can be ruled out is avr-14, where neither
reporter gene patterns nor the physiological data
supports expression in these cells (Dent et al. 2000;
Pemberton et al. 2001).
For parasitic species, we have less information.
The physiological experiments have been, by and
large,carriedoutonlargeworms such asA.suum and
these clearly indicate the presence of GluCl on
pharyngeal muscle, but the only localisation studies
have been carried out on H. contortus using anti-
bodies raised against synthetic peptides corre-
sponding to poorly conserved regions of the
individual subunits. These studies have not yielded
any strong evidence for expression of any H. con-
tortus GluCl subunit in pharyngeal muscle cells, and
in particular the HcGluClb subunit was not found
there (Delany et al. 1998), but have suggested the
possibility that HcGluCla3B may be expressed
in pharyngeal neurones (Portillo, Jagannathan &
receptors, which are presumably presynaptic, could
explain the inhibition of pumping observed in this
species (Geary et al. 1993). However, it may also
be that so-far undiscovered GluCl subunits may be
present on H. contortus pharyngeal muscle: the
absence of the HcGluClb subunit could well mean
that the composition, and hence pharmacology, of
GLUCL AND THE CONTROL OF LOCOMOTION
The second major effect of the A/M on nematodes is
an apparent paralysis of body-wall muscle, rendering
them immobilised. However, all the evidence points
to this being an indirect effect, rather than a direct
inhibition of neuromuscular transmission and there
arenoindications that GluCl areexpressed in muscle
cells. Locomotion in nematodes is controlled by both
excitatory and inhibitory motor neurones, organised
into ventral and dorsal nerve cords, each of which
innervate body-wall muscle. Waves of reciprocal
excitation and inhibition pass down the body, so that
the dorsal muscles are relaxed as the ventral muscles
are contracted, and vice versa. This results in the
characteristic sinusoidal swimming motion. Studies
on C. elegans showed that the motor neurones are in
turn controlled by command interneurones in the
head of the worm that regulate the rate of locomotion
and also the frequency at which the worm reverses
and moves backwards(Whiteetal.1986;Zhengetal.
Early reports showed that, in A. suum, avermectin
blocked transmission between interneurones and
excitatory motor neurones in the ventral cord, and
A. J. Wolstenholme and A. T. Rogers
also ventral inhibitory transmission (Kass et al.1980,
1984). This would predict that GluCl subunits are
expressed on those motor neurones and this predic-
tion has been confirmed in C. elegans, using reporter
gene constructs, and H. contortus, using antibodies
(Fig. 3). Both avr-14 and avr-15 are expressed in
C. elegans motor neurones (Dent 1997, 2000) and, in
H. contortus, the HcGluCla, a3A, a3B and b sub-
units have all been detected on motor neurones
Jagannathan & Wolstenholme, 2003). All of the H.
contortus subunits were detected on motor neurone
commissures, structures connecting the dorsal and
ventral nerve cords and which form synapses with
interneurones on the lateral and sub-lateral nerve
cords. The use of anti-GABA antibodies suggested
that these were inhibitory motor neurons (Portillo
et al. 2003). Application of A/M to channels on these
inhibitory motor neurones would therefore pre-
sumablyresult in an irreversible hyperpolarisationof
the cells and their consequent inability to produce
action potentials. This would prevent inhibitory
transmission at the neuromuscular junction and
hence the abolition of the waves of muscular relax-
ation required for movement.
In addition, some GluCl subunits have been
detected on interneurones, which might represent
the command interneurones. The C. elegans avr-14
gene is expressed in extra pharyngeal head neurones
(Dent et al. 2000) and use of an antibody that
recognised both GluCla3 subunits produced im-
munofluorescence in the nerve ring of H. contortus
(Jagannathan et al. 1999). Direct confirmation that
GluCl are present in at least some command inter-
neurones was provided by the detection of a
glutamate-gated chloride current in the AVA inter-
neurone of C. elegans using in vivo recording
techniques (Mellem et al. 2002). Mutations in the
excitatory glutamate receptors also found in these
neurones reduce the frequency at which the worms
reverse direction (Brockie et al. 2001). We have
examined the effects of mutations in several GluCl
genes on this behaviour and have found the opposite
phenotype, that is the worms reverse more fre-
quently and the durations of the forward movements
are reduced (N. Aptel, A. Cook, L. Holden-Dye &
A. Wolstenholme, unpublished). The GluCl thus
have a major role in regulating nematode locomotion
by A/M anthelmintics causes an effective paralysis.
GLUCL IN OTHER ORGANS
The A/M anthelmintics do not efficiently kill some
tissue-dwelling adult worms, especially the macro-
filariae. Nonetheless, they are effective for the treat-
ment and prophylaxis of filarial infections such as
Onchocerca volvulus due to their microfilariacidal
effects and because they dramatically reduce the
production of new microfilariae for several months
(Klager et al. 1993; Lok et al. 1995). Similar effects
on fecundity have been observed in other species
(Petersen et al. 1996), implying that the GluCl have a
rolein reproduction. C.eleganscarrying mutationsin
some GluCl genes do show a reduction in egg pro-
duction (N. Aptel, V. Portillo & A. Wolstenholme,
unpublished), but the nature of that role is currently
unknown. No GluCl have been reported to be
present on any reproductive tissue.
An intriguing recent suggestion is that GluCl may
be present on sensory neurones, or involved in the
pathways linking sensory stimulation to behavioural
effects. Studies on Brugia pahangi have shown that
ivermectin can block physiological responses to
chemical stimuli in amphids (Perry, 2001; Rolfe,
Barrett & Perry, 2001) and, in H. contortus, the
HcGluCla3A subunit was detected in a structure
that closely resembled a sensory neurone (Portillo
et al. 2003). This is even more intriguing in light of
the finding that ivermectin-resistant H. contortus
have defects in amphid structure (Freeman et al.
2003): amphids are the sensory organs present in the
nematode head that contain the sensory neurones.
One of their functions would be expected to be to
detect the stimuli that allow adult filaria to locate
each other and mate and there are suggestions that
ivermectin interferes with this process (Duke et al.
1992). In C. elegans, mechanical stimulation inhibits
pharyngeal pumping in adult worms and it was
suggested that GluCl containing the avr-14 and
pathway linking mechanosensation to pharyngeal
pumping (Keane & Avery, 2003). There is therefore
considerable evidence that the GluCl play a role in
Fig. 3. Schematic representation of the distribution of GluCl in nematodes. The cuticle is outlined in grey and the
pharynx in black. Structures reported to express GluCl are indicated by arrows.
GluCl and the A/M anthelmintics
sensory signalling in nematodes, but as yet it is
not clear how important this role is as a target for
the A/M anthelmintics. One possibility is that the
amphids act as a route of entry for the drugs into the
It is generally accepted that the molecular targets of
the avermectin/milbemycin anthelmintics are the
glutamate-gated chloride channels. The number of
identified GluCl subunits indicates that nematodes
contain multiple forms of these channels, which may
differ in their sensitivity to the current drugs, and at
least some of these are expressed in the nematode
neuromuscular system. The elucidation of the
subunit structure of these different forms and its
relationship to drug sensitivity remains a major
challenge. The GluCl are widely expressed in the
most of the effects of the A/M, with the possible
exception of the reproductive effects, can be ex-
plained by this distribution. The implication is that
these drugs can kill, or damage, nematodes in a var-
iety of ways and, given that there are multiple forms
of GluCl, the most important mechanism may vary
between species. This has important implications for
the development of resistance, a topic outside the
scope of this review, which is becoming a major
concern in veterinary parasites (Kaplan, 2004). The
mechanisms of A/M resistance are still somewhat
controversial (Wolstenholme et al. 2004), but it may
be that the multiplicity of GluCl subtypes and
functions may be reflected in a similar multiplicity of
Work on the nematode GluCl in the authors’ laboratory
is and has been funded by the BBSRC (awards 86/
GAN13134 and BBS/B/07594) and the Wellcome Trust
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