Recombinant expression and functional characterization of martentoxin: a selective inhibitor for BK channel (α + β4).

Jie Tao,Xiao Chen,Yong Ji,Bin Wu,Jian Shi,Zhi Zhou

doi:10.3390/toxins6041419

Abstract

Martentoxin (MarTX), a 37-residue peptide purified from the venom of East-Asian scorpion (Buthus martensi Karsch), was capable of blocking large-conductance Ca2+-activated K+ (BK) channels. Here, we report an effective expression and purification approach for this toxin. The cDNA encoding martentoxin was expressed by the prokaryotic expression system pGEX-4T-3 which was added an enterokinase cleavage site by PCR. The fusion protein (GST-rMarTX) was digested by enterokinase to release hetero-expressed toxin and further purified via reverse-phase HPLC. The molecular weight of the hetero-expressed rMarTX was 4059.06 Da, which is identical to that of the natural peptide isolated from scorpion venom. Functional characterization through whole-cell patch clamp showed that rMarTX selectively and potently inhibited the currents of neuronal BK channels (α + β4) (IC50 = 186 nM), partly inhibited mKv1.3, but hardly having any significant effect on hKv4.2 and hKv3.1a even at 10 μM. Successful expression of martentoxin lays basis for further studies of structure-function relationship underlying martentoxin or other potassium-channel specific blockers.

Highlights

The large-conductance, calcium-activated potassium channels (BK, termed MaxiK) distributed in both excitable and non-excitable cells are involved in many cellular functions such as smooth muscle tone [1], neuronal firing [2], endocrine cell secretion [3], cell proliferation [4] and migration [5]
Martentoxin, a 37-residue short-chain peptide belonging to α-KTx16 subfamily, is found to be specific to blocking large-conductance Ca2+-activated K+ (BK) channels and intensively studied
These results suggested that the bioactivity of recombinant martentoxin was not affected by this system

Summary

Introduction

The large-conductance, calcium-activated potassium channels (BK, termed MaxiK) distributed in both excitable and non-excitable cells are involved in many cellular functions such as smooth muscle tone [1], neuronal firing [2], endocrine cell secretion [3], cell proliferation [4] and migration [5].Functional BK channels are a tetramer of four pore-forming α subunits encoded by a single geneSlowpoke (Slo) [6]. Functional BK channels are a tetramer of four pore-forming α subunits encoded by a single gene. Owing to the tissue-specific regulatory β-subunits and alternative splicing of Slo gene, BK channels possess a rather complex diversity of subtype family, which endow various physiological and pharmacological properties in different organisms [7,8,9]. Martentoxin, a 37-residue short-chain peptide belonging to α-KTx16 subfamily, is found to be specific to BK channels and intensively studied. Martentoxin showed a poor sequential similarity (35%–50%) with those toxins in ChTX and KTX groups including BmTX1, BmTX2, BmTX3 and BmKTX from the same venom, but a high sequential identity

Results

Discussion

Conclusion