Role of Alpha5-Containing Nicotinic Receptors in Neuropathic Pain And
Nicotinic Receptors and Anthelmintic Resistance
description
Transcript of Nicotinic Receptors and Anthelmintic Resistance
Nicotinic Receptors and Anthelmintic ResistanceRichard J. Martin, Adrian J. Wolstenholme, S. M. Williamson & Alan P. RobertsonDepartment of Biomedical Sciences, Iowa State University, Ames, Iowa USA & Dept of Infectious Diseases, University of Georgia, Athens, Georgia
Nicotinic Anthelmintics
1. Imidazothiazoles: levamisole, agonist2. Tetrahydropyrimidines: pyrantel , agonist3. Quaternary Amines: bephenium , agonist4. AADs: monepantel , agonist5. Macfortines: paraherquamide &
derquantel, antagonist
Complementary site on adjacent subunit
Primary site
on α subunit
A
B
C
DE
F
Y93 W86
Y190 C192
C193 Y198
W149 Y151
Y111 Y117
W55/57
D180
A B C
Na+, Ca2+
α
Agonist binding site
outside
inside
α
C. elegans nicotinic receptor subunitsJones et al., 2007: 29 subunits, 5 core structures
AADs
levamisole
Muscle nAChRs in C. elegans
C. elegans levamisole sensitive receptor
G= 30 pS
(Qian et al, 2008)
ACR 16
ACR 16
ACR 16
ACR 16
ACR 16
LEV 8
UNC
63
UNC
29
LEV 1
UNC
38
C. elegans nicotine sensitive receptor
Nicotinic anthelmintic
5 min
1g
10 nM
100 nM300 nM
1 M30 M
30 nM
Control dose-response trace
Pharmacological Diversity in Ascaris
3 receptor types in Ascaris suum:
N-type(low sensitivity to paraherquaminde)
L-type(sensitive to paraherquamide
and 2 desoxo-paraherquamide: derquantel)
B-type(most sensitive to
2-desoxo-paraherquamide:
derquantel)
N-type
L-typeB-type
C
O1
O2
O3 B-type
L-type
N-type
1.5 2.0 2.5 3.0 3.5 4.0 4.5
0
10
20
30
40
50
Nu
mbe
r o
f obs
erv
atio
ns
(N)
Amplitude (pA)
N-type
L-type
B-type
3 channel currents & 3 nAChR subtypes
N-subtype
24pS
nicotine
L-subtype
35pS
levamisole
paraherquamide
B-subtype
45pS
bephenium
2-desoxoparaherquamide
(derquantel)
& paraherquamide
A B
C
mfglleallwlvvcgtsLTVGNEDAKRLYDDLMVNYNKHRRPAMSPHEPVTIKLKLRLSQIIDVHEIDQIMTYSVW LKQVWIDKKLSWDPKNYGGVSVLYVPYEMIWVPDIVLYNNADSNYNITISTKATLHYSGEVTWEPPAIFKSMCQID VRWFPFDEQKCHLKFGSWTYSEDLLVLELLDGEPHYELETNEFGEVDNITIVDDGIDLSDYYPSVEWDIMSRVAIR RTKNYPSCCPQSDAYIDIMYYLELRRKPLFYTVNLVFPCVGISFLTILVFYLPSDSGEKVTLCISILVALTVFFLL LTEIIPATSISLPLIGKYLLFTMVMVTLSVVVTVISLNLHFRTPTTHRMPEWVKWLFLKFLPKVLFMRRPLADTDD TYRRVSQRRGDNCEKVAINYHEHRVSRDIGRALSTSPVDERIQKLYYSPAVVKAFENVCFIAELLKKKDRDDKVDE DWKYVAMVLDRLFLLLFSFACFIGTVTILLQAPTLYDSREAIDLQYRPANISTPVTQ mhlpspaliivyyvlvallragvvlcSDDEERLMVDVFRGYNSLIQPVRNLSDTPIIVKIALQLVLLINVDERDQV MHTNVWLTLKWHDFQMKWNPVNYGEIQNIRVAPDKVWLPDIVFFNNADGNYEVSFMCNVVISHKGDMLWVPPAIYK SSCIIDVEFFPFDEQTCHLIFGSWTYNENEIKLEFEQAEWVDLSEYAPSSIWDVMDAPASLVNKRSRIEFQVRIRR KTLFYTVVLIIPTVLMAFLSMAVFFLPTDSGEKMTLTISVLLSIVVFLLLVSKILPPTSSTIPLMAKYLLLTFVLN VITILVTVIIINVYFRGPTTHRMPKWVRTTFLQVMPKLLCMQRPKQLLRKMAAHAAAQQANGAAVATALPGIGEFT LNPSAHHPFCPSADDSRFHPPSIELTHVDSVRQFERDPMTSAYYPLSADALRAIDAIEYITDHLKQDEEYKMYRDD WKYVAMIIDRLLLYVFFGITVGGTCGILFSAPYVFQGVDQRAELQRLIHLYKNGGEI
Asu-UNC38
Asu-UNC29
L-subtype receptor
UNC29
UNC29
UNC29
UNC38
UNC38
UNC29
UNC29
UNC29
UNC38
UNC38
N-subtype receptor
UNC29
UNC38
UNC29
UNC38
UNC38
UNC29
UNC38
UNC29
UNC38
UNC38
1:5 Ascsu unc-38: unc-29
5:1 Ascsu unc-38: unc-29
levamisole
levamisole
nicotine
nicotine
Kopp et al. 2008 IJP: qtPCR A. caninum pyrantel-sensitive vs -resistant. Expression of aar-38, aar-63 and aar-29 reduced
Ref gene Isolate aar-38 aar-63 aar-29 aar-19 aar-15
aar-8 PR/NT
60S PR 0.11 ± 0.013 0.29
0.66 ± 0.0090
0.55 0.94 ± 0.046 0.62 0.54 ±
0.0210.99
0.31 ± 0.018
0.890.41 ± 0.037
0.92
NT0.37 ± 0.022a
1.2 ± 0.070a
1.5 ± 0.035a
0.55 ± 0.0082
0.35 ± 0.091
0.45 ± 0.018
Also no changes in E153 and Q57 – pyrantel-specific sites Rayes et al. (2006)
1.aar-29 -I50L, S206T,I309L (adults and larvae), I422L in both isolates and L466M in the NT isolate,
2.aar-63-L393I(adults and larvae)
3.No 38 amino-acid changes
4.15-46 nucleotide changes
N-subtype
nAChR subtypes and resistance
L-subtype
B-subtype
levamisole resistance
Monepantel resistance: Hco-mptl-1; Hco-des -2H; Hco-deg-3H; Rufener et al.,
Monepantel resistance: Hco-mptl-1, some lack splice acceptor (TTTCAG)
Molecular detection of resistance: levamisole & monepantel
• Kopp et al., Did not find specific SNPs associated with resistance
• E153 & Q57 not associated with pyrantel resistance• Expression levels of UNC-38, UNC-29 and UNC-63 are
reduced in pyrantel resistance • Rufener et al., Hco-mptl-1 & Hco-des-2H, 1. a panel of mutations (null mutants) including deletions
at intron-exon boundaries giving mis-spliced transients & premature stop codons2. Altered expression levels of Hco-mptl-1 & Hco-des-2H and & Hco-deg-3H
rNAInner nuclear
membrane
Putative L-type receptor subunits:
UNC-38, UNC-29, UNC-63, LEV-1, ACR-13LEV-10
NRA-1
SOC-1 TPA-1
TAX-6
UNC-22
LEV-11
Muscle contraction
UNC-50 RIC-3
Translation
UNC-74
Processing & assembly
Ca CaER
UNC-68
C. elegans proteins involved in levamisole resistance
Resistance may be due to a panel of functional null mutant genes with deletions and insertions and reduced expression of the target receptor genes + other sites!
Acknowledgments• Alan Robertson• Sally Williamson• Sreekanth Puttachary• Sam Buxton• Cheryl Clark• Adrian Wolstenholme• Steve Koppe• Andrew Kotze• Supported by NIH: R01 R 01 AI 047194
UNC-38 LEV-8
UNC-29
UNC-63 LEV-1
C. elegans levamisole-sensitive channel
G= 30 pS