A disulfide tether stabilizes the block of sodium channels by the conotoxin μO§-GVIIJ.
A cone snail venom peptide, μO§-conotoxin GVIIJ from Conus geographus, has a unique posttranslational modification, S-cysteinylated cysteine, which makes possible formation of a covalent tether of peptide to its target Na channels at a distinct ligand-binding site. μO§-conotoxin GVIIJ is a 35-aa peptide, with 7 cysteine residues; six of the cysteines form 3 disulfide cross-links, and one (Cys24) is S-cysteinylated. Due to limited availability of native GVIIJ, we primarily used a synthetic analog whose Cys24 was S-glutathionylated (abbreviated GVIIJSSG). The peptide-channel complex is stabilized by a disulfide tether between Cys24 of the peptide and Cys910 of rat (r) NaV1.2. A mutant channel of rNaV1.2 lacking a cysteine near the pore loop of domain II (C910L), was >10(3)-fold less sensitive to GVIIJSSG than was wild-type rNaV1.2. In contrast, although rNaV1.5 was >10(4)-fold less sensitive to GVIIJSSG than NaV1.2, an rNaV1.5 mutant with a cysteine in the homologous location, rNaV1.5[L869C], was >10(3)-fold more sensitive than wild-type rNaV1.5. The susceptibility of rNaV1.2 to GVIIJSSG was significantly altered by treating the channels with thiol-oxidizing or disulfide-reducing agents. Furthermore, coexpression of rNaVβ2 or rNaVβ4, but not that of rNaVβ1 or rNaVβ3, protected rNaV1.1 to -1.7 (excluding NaV1.5) against block by GVIIJSSG. Thus, GVIIJ-related peptides may serve as probes for both the redox state of extracellular cysteines and for assessing which NaVβ- and NaVα-subunits are present in native neurons.
Department of Biology and Department of Medicinal Chemistry, L. S. Skaggs Pharmacy Institute, University of Utah, Salt Lake City, UT 84112.
Joanna
J
Gajewiak
Layla
L
Azam
Julita
J
Imperial
Aleksandra
A
Walewska
Brad R
BR
Green
Pradip K
PK
Bandyopadhyay
Shrinivasan
S
Raghuraman
Beatrix
B
Ueberheide
Marshall
M
Bern
H Mimi
HM
Zhou
Natali A
NA
Minassian
Rebecca H
RH
Hagan
Mack
M
Flinspach
Yi
Y
Liu
Grzegorz
G
Bulaj
Alan D
AD
Wickenden
Baldomero M
BM
Olivera
Doju
D
Yoshikami
Min-Min
MM
Zhang
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D020916:Conotoxins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D003545:Cysteine / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004220:Disulfides / Q000378:metabolism*; D008969:Molecular Sequence Data; D062551:NAV1.2 Voltage-Gated Sodium Channel / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D051381:Rats; D017422:Sequence Analysis, DNA; D053719:Tandem Mass Spectrometry; D061567:Voltage-Gated Sodium Channel Blockers / Q000378:metabolism / Q000633:toxicity*
D020916:Conotoxins; D017931:DNA Primers; D018076:DNA, Complementary; D004220:Disulfides; D062551:NAV1.2 Voltage-Gated Sodium Channel; D061567:Voltage-Gated Sodium Channel Blockers; D003545:Cysteine
NaVβ-subunit; S-cysteinylation; disulfide exchange; tethered toxin; voltage-gated sodium channel