Biochemistry Article Figure 7. Comparison of the amino acid sequences of the indicated voltage-gated ion channels and the surface profiles of kurtoxin and scorpion α- toxins. (A) Comparison of the amino acid sequences of the domain IV S3−S4 linker in different voltage-gated ion channels (Cav, voltage-gated calcium channels; Nav, voltage-gated sodium channels; and Kv2.1, voltage-gated potassium channels). These sequences were aligned using ClustalX. Highly conserved residues are shaded in black or gray. The red rectangular boxes highlight the proposed kurtoxin binding site on Cav3 (T-type) Ca2+ channels. The blue rectangular boxes highlight the sequence involved in the binding sites of gating modifiers (Ile273, Phe274, and Glu277 in K+ channels; Glu1613 in Na+ channels; and Glu1658 in P/Q-type Ca2+ channels). (B) Surface profiles of kurtoxin, AaH II, LqqhαIT, and Bmk M1: yellow for hydrophobic residues (Ala, Cys, Gly, Leu, Ile, Phe, Pro, Trp, Tyr and Val), blue and red for basic (Arg and Lys) and acidic (Asp and Glu) residues, respectively, and white for other residues. The surface hydrophobic patch residues and charged residues are indicated. between the two channels and that corresponds to the HaTx/ GrTx binding site on voltage-gated K+ and Ca2+ channels.55 The region conserved in Cav3 (T-type) Ca2+ channels contains three glutamate residues [Glu1661, Glu1662, and Glu1664 for Cav3.1 (Figure 7A)]. Notably, the region in the Cav3 (T-type) Ca2+ channel makes a larger negative patch than in Na+, K+, or other types of Ca2+ channels, and this negative domain is conserved in all Cav3 (T-type) Ca2+ channel subtypes. As mentioned, kurtoxin is highly electropositive because of the presence of an electropositive patch formed by Lys13, Arg14, and five positively charged residues in the C-terminal segment (Arg54, Lys56, Arg57, Arg60, and Arg62). We therefore speculate that the positively charged surface of kurtoxin is an important determinant of its binding to the conserved negative domain in Cav3 (T-type) Ca2+ channels. Figure 7B shows a comparison of the surface profiles of kurtoxin and three scorpion α-toxins. Kurtoxin exhibits surface characteristics arising from a surface hydrophobic patch in combination with a large electropositive patch. By contrast, scorpion α-toxins show a rather small surface hydrophobic patch with a mixed charged surface. These distinct surface profiles may explain why only kurtoxin is able to interact with the binding domain on Cav3 (T-type) Ca2+ channels, which is composed of electronegative and hydro- phobic residues. We are currently in the process of preparing alanine mutants of kurtoxin and the Cav3.1 channel to examine the molecular basis of the interaction between the toxin and channel. ■ CONCLUSION We investigated the three-dimensional structure of the first peptide toxin known to inhibit Cav3 (T-type) voltage-gated Ca2+ channels and suggest that its unique surface properties are likely responsible for its binding selectivity. Interestingly, kurtoxin can interact with high affinity with native neuronal high-threshold L-type, N-type, and P-type Ca2+ channels in central and peripheral neurons, producing complex gating modifications specific to each channel type.56 When the channels are expressed in Xenopus oocytes, however, kurtoxin interacts only with the α-subunit of Cav3.1 (α1G T-type) voltage-gated Ca2+ channels; it does not interact with any other type of Ca2+ channel.55 At present, the mechanism by which kurtoxin interacts with Ca2+ channels remains unknown. The structural studies of kurtoxin reported here provide clues about the molecular mechanism by which Cav3 (T-type) Ca2+ channel activity is regulated by selective ligands and could contribute to the development of highly specific Cav3 (T-type) Ca2+ channel inhibitors. ■ ASSOCIATED CONTENT AUTHOR INFORMATION *S Supporting Information Figures S1−S3. This material is available free of charge via the Internet at http://pubs.acs.org. Accession Codes The coordinates for kurtoxin have been deposited in the Protein Data Bank as entry 1T1T. ■ Corresponding Author *Department of Life Science, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea. E- mail: jikim@gist.ac.kr. Telephone: +82-62-715-2494. Fax: +82- 62-715-2484. Funding This research was supported by grants from the Next- Generation BioGreen 21 Program (PJ008158); the Rural Development Administration, Republic of Korea; a National Research Foundation of Korea grant funded by the Korean Government (MEST) (NRF-C1ABA001-2011-0018559); the Brain Research Center of the 21st Century Frontier Research 1869 dx.doi.org/10.1021/bi201633j | Biochemistry 2012, 51, 1862−1873