title: Expression and characterization of recombinant kurtoxin, an inhibitor of T-type voltage-gated calcium channels.
abstract: Kurtoxin, a 63-amino acid peptide stabilized by four disulfide bonds, is the first reported peptide inhibitor of T-type voltage-gated calcium channels. Although T-type calcium channels have been implicated in a number of disease states, including epilepsy, chronic pain, hypertension and cancer, the lack of selective inhibitors has slowed progress in understanding their precise roles. Kurtoxin is a potentially valuable tool with which to study T-type calcium channels. However, because of the limited availability of the native protein, little is known about the structure and molecular mechanism of kurtoxin. Here we report the expression of kurtoxin in Escherichia coli and the structural and functional characterization of the recombinant protein. The disulfide bond pairings and secondary structure of recombinant kurtoxin were characterized through enzymatic cleavage, mass analysis and CD spectroscopy. Recombinant kurtoxin almost completely inhibited the T-type calcium channel in a manner identical to the native toxin. The availability of recombinant kurtoxin that is identical to the native toxin should help in the study of T-type calcium channels and enable development of new strategies for producing even more-selective T-type calcium channel inhibitors and for investigating the molecular basis of the toxin-channel interactions.
authors.affiliation: Department of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea.
authors.forename: Chul Won
authors.initials: CW
authors.lastname: Lee
authors.forename: Young-Jae
authors.initials: YJ
authors.lastname: Eu
authors.forename: Hye Jung
authors.initials: HJ
authors.lastname: Min
authors.forename: Eun-Mi
authors.initials: EM
authors.lastname: Cho
authors.forename: Jun-Ho
authors.initials: JH
authors.lastname: Lee
authors.forename: Ha Hyung
authors.initials: HH
authors.lastname: Kim
authors.forename: Seung-Yeol
authors.initials: SY
authors.lastname: Nah
authors.forename: Kenton J
authors.initials: KJ
authors.lastname: Swartz
authors.forename: Jae Il
authors.initials: JI
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D020747:Calcium Channels, T-Type / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D012604:Scorpion Venoms / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*
chemical_list: D020747:Calcium Channels, T-Type; D011994:Recombinant Proteins; D012604:Scorpion Venoms; C118444:kurtoxin; D003545:Cysteine