Unexpected synergism between Tetrodotoxin and μ-conotoxin in blocking voltage-gated sodium channels

Zhang, Min Min; McArthur, Jeffrey R.; Azam, Layla; Bułaj, Grzegorz; Olivera, Baldomero M.; French, Robert J.; Yoshikami, Doju

doi:10.4161/chan.3.1.7500

Cited by 48 publications

(75 citation statements)

References 24 publications

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“…Due to the network of inter-repeat H-bonds, the outer-pore lumen at the level of p52 is rather narrow, but in our model it still accommodates a TTX molecule. It will be interesting to further explore whether our narrow outer-pore model is consistent with intriguing data on synergistic and antagonistic interactions between tetrodotoxin and mu-conotoxin in blocking voltagegated sodium channels (37).…”

Section: Discussionsupporting

confidence: 58%

Possible Roles of Exceptionally Conserved Residues around the Selectivity Filters of Sodium and Calcium Channels

Tikhonov

Zhorov

2011

Journal of Biological Chemistry

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In the absence of x-ray structures of sodium and calcium channels their homology models are used to rationalize experimental data and design new experiments. A challenge is to model the outer-pore region that folds differently from potassium channels. Here we report a new model of the outer-pore region of the NaV1.4 channel, which suggests roles of highly conserved residues around the selectivity filter. The model takes from our previous study (Tikhonov, D. B., and Zhorov, B. S. (2005) Biophys. J. 88, 184 -197) the general disposition of the P-helices, selectivity filter residues, and the outer carboxylates, but proposes new intra-and inter-domain contacts that support structural stability of the outer pore. Glycine residues downstream from the selectivity filter are proposed to participate in knob-into-hole contacts with the P-helices and S6s. These contacts explain the adapted tetrodotoxin resistance of snakes that feed on toxic prey through valine substitution of isoleucine in the P-helix of repeat IV. Polar residues five positions upstream from the selectivity filter residues form H-bonds with the ascending-limb backbones. Exceptionally conserved tryptophans are engaged in inter-repeat H-bonds to form a ring whose -electrons would facilitate passage of ions from the outer carboxylates to the selectivity filter. The outerpore model of CaV1.2 derived from the NaV1.4 model is also stabilized by the ring of exceptionally conservative tryptophans and H-bonds between the P-helices and ascending limbs. In this model, the exceptionally conserved aspartate downstream from the selectivity-filter glutamate in repeat II facilitates passage of calcium ions to the selectivity-filter ring through the tryptophan ring. Available experimental data are discussed in view of the models.Voltage-gated ion channels are involved in the control of many physiological functions. Upon membrane depolarization, channels rapidly transit from the resting to the open state, then close to inactivated state(s), and return to the resting state upon membrane repolarization. The human genome encodes nine voltage-gated sodium (NaV1.1-NaV1.9) and ten voltage-gated calcium channels categorized as L (CaV1.1-CaV1.4), P/Q (CaV2.1), N (CaV2.2), R (CaV2.3), and T (CaV3.1-CaV3.3) types. The pore-forming ␣ 1 -subunit of calcium and sodium channels folds from a single polypeptide chain of four homologous repeats (Fig. 1A). Repeats I to IV are arranged clockwise when viewed extracellularly (1). Each repeat contains a voltage-sensing domain S1-S4, an outer helix S5, an inner helix S6, and a membrane-diving P-loop between S5 and S6. The P-loop includes a P-helix, a P-turn, and an ascending limb. Four ascending limbs, which line the outer pore, contain selectivity-filter residues in positions p50 (see Footnote 2 for residue labels). 2 Repeats I, II, III, and IV contain selectivity-filter glutamates in calcium channels (the EEEE ring) and Asp, Glu, Lys, and Ala residues in sodium channels (the DEKA ring). The ascending limbs also contain the outer carboxylates t...

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Section: Discussionsupporting

confidence: 58%

Possible Roles of Exceptionally Conserved Residues around the Selectivity Filters of Sodium and Calcium Channels

Tikhonov

Zhorov

2011

Journal of Biological Chemistry

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“…KIIIA blocks Na V 1.2 with 95% efficacy, as previously described in some detail (16,17; also see ref. 18).…”

Section: Resultsmentioning

confidence: 75%

“…The block of Na V 1.7 by KIIIA appears to have approximately a 90% efficacy. We presume KIIIA's "leaky" block of Na V 1.7 occurs by a mechanism similar to that hypothesized for KIIIA's incomplete block of Na V 1.2 (16,17), and studies are under way to investigate this further.…”

Section: Resultsmentioning

confidence: 87%

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μ-Conotoxins that differentially block sodium channels Na_V1.1 through 1.8 identify those responsible for action potentials in sciatic nerve

Wilson¹,

Yoshikami²,

Azam³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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128

181

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Voltage-gated sodium channels (VGSCs) are important for action potentials. There are seven major isoforms of the pore-forming and gate-bearing α-subunit (Na V 1) of VGSCs in mammalian neurons, and a given neuron can express more than one isoform. Five of the neuronal isoforms, Na V 1.1, 1.2, 1.3, 1.6, and 1.7, are exquisitely sensitive to tetrodotoxin (TTX), and a functional differentiation of these presents a serious challenge. Here, we examined a panel of 11 μ-conopeptides for their ability to block rodent Na V 1.1 through 1.8 expressed in Xenopus oocytes. Although none blocked Na V 1.8, a TTX-resistant isoform, the resulting "activity matrix" revealed that the panel could readily discriminate between the members of all pair-wise combinations of the tested isoforms. To examine the identities of endogenous VGSCs, a subset of the panel was tested on A-and C-compound action potentials recorded from isolated preparations of rat sciatic nerve. The results show that the major subtypes in the corresponding A-and C-fibers were Na V 1.6 and 1.7, respectively. Ruled out as major players in both fiber types were Na V 1.1, 1.2, and 1.3. These results are consistent with immunohistochemical findings of others. To our awareness this is the first report describing a qualitative pharmacological survey of TTX-sensitive Na V 1 isoforms responsible for propagating action potentials in peripheral nerve. The panel of μ-conopeptides should be useful in identifying the functional contributions of Na V 1 isoforms in other preparations. Hodgkin and Huxley developed a quantitative theory for the ionic basis of the action potential (1)-the molecular correlates of their pioneering efforts are the voltage-gated sodium channel (VGSC) and the voltage-gated potassium channel. Critical for the appreciation of the discrete biochemical nature of VGSCs were the investigations of the mechanism of action of the alkaloids tetrodotoxin (TTX) and saxitoxin (2-4). In the half century since these classic experiments, our understanding of the molecular structure of VGSCs has advanced dramatically (5). We now recognize that mammals have nine isoforms of the α-subunit of VGSCs, or Na V 1, the subunit containing both the Na + -conducting pore and voltage-sensing gate (5-7). Meanwhile, progress in the characterization of ligands that target VGSCs has also progressed (8, 9), although TTX remains a major pharmacological investigative tool for VGSCs.The nine mammalian Na V 1 isoforms can be categorized into those that are TTX-sensitive versus those that are resistant, with K d or IC 50 values in the nM versus μM ranges, respectively (7). Two, Na V 1.8 and Na V 1.9, are found in primary sensory neurons and are highly resistant to TTX (10-12). One, Na V 1.5, found in cardiac muscle, is moderately TTX resistant (13), and the remaining six Na V 1 isoforms are exquisitely sensitive to TTX. One of these, Na V 1.4 is found exclusively in skeletal muscle. Presently, the five neuronal subtypes that are TTX-sensitive (i.e., Na V 1.1, 1.2, 1.3, 1.6, and 1.7) cannot be re...

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“…We had shown previously that -KIIIA and its analogs block rNa V 1.2 and compete for, and can even co-occupy, site 1 with the guanidinium alkaloids tetrodotoxin, saxitoxin, and saxitoxin congeners (9,10,33). In the present experiments, we employed -PIIIA[R14Q] and rNa V 1.4.…”

Section: Discussionmentioning

confidence: 99%

Structural Basis for the Inhibition of Voltage-gated Sodium Channels by Conotoxin μO§-GVIIJ

Green

Gajewiak

Chhabra

et al. 2016

Journal of Biological Chemistry

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Cone snail toxins are well known blockers of voltage-gated sodium channels, a property that is of broad interest in biology and therapeutically in treating neuropathic pain and neurological disorders. Although most conotoxin channel blockers function by direct binding to a channel and disrupting its normal ion movement, conotoxin O §-GVIIJ channel blocking is unique, using both favorable binding interactions with the channel and a direct tether via an intermolecular disulfide bond. Disulfide exchange is possible because conotoxin O §-GVIIJ contains an S-cysteinylated Cys-24 residue that is capable of exchanging with a free cysteine thiol on the channel surface. Here, we present the solution structure of an analog of O §-GVIIJ (GVIIJ[C24S]) and the results of structure-activity studies with synthetic O §-GVIIJ variants. GVIIJ[C24S] adopts an inhibitor cystine knot structure, with two antiparallel ␤-strands stabilized by three disulfide bridges. The loop region linking the ␤-strands (loop 4) presents residue 24 in a configuration where it could bind to the proposed free cysteine of the channel (Cys-910, rat Na V 1.2 numbering; at site 8). The structure-activity study shows that three residues (Lys-12, Arg-14, and Tyr-16) located in loop 2 and spatially close to residue 24 were also important for functional activity. We propose that the interaction of O §-GVIIJ with the channel depends on not only disulfide tethering via Cys-24 to a free cysteine at site 8 on the channel but also the participation of key residues of O §-GVIIJ on a distinct surface of the peptide.Marine snails of the genus Conus employ a complex venom mixture to subdue prey and as an effective means of defending against predation. The active venoms contain an array of peptides that bind to and modulate the properties of ion channels, G-protein-coupled receptors, and neurotransmitter receptors (1). Several peptides modulate the activities of voltage-gated sodium channels (VGSCs), 4 which are implicated in numerous neurological disorders, as well as neuropathic pain. Four classes of conopeptides have been shown to affect VGSC activity. Peptides belonging to the -and ␦-families promote activation and inhibit inactivation, respectively, whereas the -and O-conotoxins inhibit VGSCs by either blocking the Na ϩ conductance pore or preventing channel activation, respectively (2, 3). Recently, the founding member of a fifth class of VGSC inhibitors was identified that blocks the channel through interaction with a previously unidentified neurotoxin binding site, site 8 (Fig. 1A) (4).O §-GVIIJ is a 35-residue peptide isolated from the venom of the piscivorous snail Conus geographus (Fig. 1B). In vitro folding of linear O §-GVIIJ with thiol-reactive oxidants (i.e. glutathione, L-cystine, or cystamine) resulted in adducts where Cys-24 was disulfide-bonded with glutathione, cysteine, or cysteamine, respectively (abbreviated as GVIIJ SSG , GVIIJ SSC , and GVIIJ SSEA , respectively; see Fig. 1C for structures). Of these, the GVIIJ SSC variant most closely resembled ...

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Unexpected synergism between Tetrodotoxin and μ-conotoxin in blocking voltage-gated sodium channels

Abstract: (2009) Unexpected synergism between Tetrodotoxin and µ-conotoxin in blocking voltage-gated sodium channels, Channels, 3:1, 32-38,

Cited by 48 publications

References 24 publications

Possible Roles of Exceptionally Conserved Residues around the Selectivity Filters of Sodium and Calcium Channels

Possible Roles of Exceptionally Conserved Residues around the Selectivity Filters of Sodium and Calcium Channels

μ-Conotoxins that differentially block sodium channels Na_V1.1 through 1.8 identify those responsible for action potentials in sciatic nerve

Structural Basis for the Inhibition of Voltage-gated Sodium Channels by Conotoxin μO§-GVIIJ

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Unexpected synergism between Tetrodotoxin and μ-conotoxin in blocking voltage-gated sodium channels

Abstract: (2009) Unexpected synergism between Tetrodotoxin and µ-conotoxin in blocking voltage-gated sodium channels, Channels, 3:1, 32-38,

Cited by 48 publications

References 24 publications

Possible Roles of Exceptionally Conserved Residues around the Selectivity Filters of Sodium and Calcium Channels

Possible Roles of Exceptionally Conserved Residues around the Selectivity Filters of Sodium and Calcium Channels

μ-Conotoxins that differentially block sodium channels NaV1.1 through 1.8 identify those responsible for action potentials in sciatic nerve

Structural Basis for the Inhibition of Voltage-gated Sodium Channels by Conotoxin μO§-GVIIJ

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μ-Conotoxins that differentially block sodium channels Na_V1.1 through 1.8 identify those responsible for action potentials in sciatic nerve