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Characterization of a novel Bm?TX47 toxin modulating sodium channels: the crucial role of expression vectors in toxin pharmacological activity.

ABSTRACT: Long-chain scorpion toxins with four disulfide bridges exhibit various pharmacological features towards the different voltage-gated sodium channel subtypes. However, the toxin production still remains a huge challenge. Here, we reported the effects of different expression vectors on the pharmacological properties of a novel toxin Bm?TX47 from the scorpion Buthus martensii Karsch. The recombinant Bm?TX47 was obtained using the expression vector pET-14b and pET-28a, respectively. Pharmacological experiments showed that the recombinant Bm?TX47 was a new ?-scorpion toxin which could inhibit the fast inactivation of rNa(v)1.2, mNa(v)1.4 and hNa(v)1.5 channels. Importantly, the different expression vectors were found to strongly affect Bm?TX47 pharmacological activities while toxins were obtained by the same expression and purification procedures. When 10 µM recombinant Bm?TX47 from the pET-28a vector was applied, the values of I(5ms)/I(peak) for rNa(v)1.2, mNa(v)1.4 and hNa(v)1.5 channels were 44.12% ± 3.17%, 25.40% ± 4.89% and 65.34% ± 3.86%, respectively, which were better than those values of 11.33% ± 1.46%, 15.96% ± 1.87% and 5.24% ± 2.38% for rNa(v)1.2, mNa(v)1.4 and hNa(v)1.5 channels delayed by 10 µM recombinant Bm?TX47 from the pET-14b vector. The dose-response experiments further indicated the EC50 values of recombinant Bm?TX47 from the pET-28a vector were 7262.9 ± 755.9 nM for rNa(v)1.2 channel and 1005.8 ± 118.6 nM for hNa(v)1.5 channel, respectively. Together, these findings highlighted the important role of expression vectors in scorpion toxin pharmacological properties, which would accelerate the understanding of the structure-function relationships of scorpion toxins and promote the potential application of toxins in the near future.

SUBMITTER: Li T

PROVIDER: S-EPMC3968363 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Characterization of a novel BmαTX47 toxin modulating sodium channels: the crucial role of expression vectors in toxin pharmacological activity.

Li Tian T Xu Lingna L Liu Honglian H He Yawen Y Liang Songping S Li Wenxin W Wu Yingliang Y

Toxins 20140226 3

Long-chain scorpion toxins with four disulfide bridges exhibit various pharmacological features towards the different voltage-gated sodium channel subtypes. However, the toxin production still remains a huge challenge. Here, we reported the effects of different expression vectors on the pharmacological properties of a novel toxin BmαTX47 from the scorpion Buthus martensii Karsch. The recombinant BmαTX47 was obtained using the expression vector pET-14b and pET-28a, respectively. Pharmacological e ...[more]

PMID: 24577584

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Project description:Scorpion ?-toxin 4 from Centruroides suffusus suffusus (Css4) enhances the activation of voltage-gated sodium channels through a voltage sensor trapping mechanism by binding the activated state of the voltage sensor in domain II and stabilizing it in its activated conformation. Here we describe the antagonist and partial agonist properties of a mutant derivative of this toxin. Substitution of seven different amino acid residues for Glu(15) in Css4 yielded toxin derivatives with both increased and decreased affinities for binding to neurotoxin receptor site 4 on sodium channels. Css4(E15R) is unique among this set of mutants in that it retained nearly normal binding affinity but lost its functional activity for modification of sodium channel gating in our standard electrophysiological assay for voltage sensor trapping. More detailed analysis of the functional effects of Css4(E15R) revealed weak voltage sensor trapping activity, which was very rapidly reversed upon repolarization and therefore was not observed in our standard assay of toxin effects. This partial agonist activity of Css4(E15R) is observed clearly in voltage sensor trapping assays with brief (5 ms) repolarization between the conditioning prepulse and the test pulse. The effects of Css4(E15R) are fit well by a three-step model of toxin action involving concentration-dependent toxin binding to its receptor site followed by depolarization-dependent activation of the voltage sensor and subsequent voltage sensor trapping. Because it is a partial agonist with much reduced efficacy for voltage sensor trapping, Css4(E15R) can antagonize the effects of wild-type Css4 on sodium channel activation and can prevent paralysis by Css4 when injected into mice. Our results define the first partial agonist and antagonist activities for scorpion toxins and open new avenues of research toward better understanding of the structure-function relationships for toxin action on sodium channel voltage sensors and toward potential toxin-based therapeutics to prevent lethality from scorpion envenomation.

Dataset Information

Characterization of a novel Bm?TX47 toxin modulating sodium channels: the crucial role of expression vectors in toxin pharmacological activity.

Publications

Characterization of a novel BmαTX47 toxin modulating sodium channels: the crucial role of expression vectors in toxin pharmacological activity.

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