Interaction of the scorpion toxin discrepin with Kv4.3 channels and A-type K + channels in cerebellum granular cells Cristiana Picco a, ⁎, Gerardo Corzo b, ⁎⁎, Lourival D. Possani b , Gianfranco Prestipino a a Istituto di Biofisica, CNR, Via De Marini 6, 16149 Genova, Italy b Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología/UNAM, Apartado Postal 510-3, Cuernavaca 62210, Mexico abstract article info Article history: Received 25 November 2013 Received in revised form 1 May 2014 Accepted 12 May 2014 Available online 17 May 2014 Keywords: A-type currents Discrepin Heterologous expression K + channel Scorpion toxin Background: The peptide discrepin from the α-KTx15 subfamily of scorpion toxins preferentially affects transient A-type potassium currents, which regulate many aspects of neuronal function in the central nervous system. However, the specific Kv channel targeted by discrepin and the molecular mechanism of interaction are still unknown. Methods: Different variant peptides of discrepin were chemically synthesized and their effects were studied using patch clamp technique on rat cerebellum granular cells (CGC) and HEK cells transiently expressing Kv4.3 channels. Results: Functional analysis indicated that nanomolar concentrations of native discrepin blocked Kv4.3 expressed channels, as previously observed in CGC. Similarly, the apparent affinities of all mutated peptides for Kv4.3 expressed channels were analogous to those found in CGC. In particular, in the double variant [V6K, D20K] the apparent affinity increased about 10-fold, whereas in variants carrying a deletion (ΔK13) or substitution (K13A) at position K13, the blockage was removed and the apparent affinity decreased more than 20-fold. Conclusion: These results indicate that Kv4.3 is likely the target of discrepin and highlight the importance of the basic residue K13, located in the α-helix of the toxin, for current blockage. General significance: We report the first example of a Kv4 subfamily potassium channel blocked by discrepin and identify the amino acid residues responsible for the blockage. The availability of discrepin variant peptides stimulates further research on the functions and pharmacology of neuronal Kv4 channels and on their possible roles in neurodegenerative disorders. © 2014 Elsevier B.V. All rights reserved. 1. Introduction K + channels responsible for A-type currents are of particular interest because they regulate firing frequency, spike initiation and waveform in excitable cells. They are thought to contribute to specialized functions like learning, memory and behavior. Physiological studies have revealed that A-type currents exhibit a wide range of biophysical and pharmaco- logical properties, according to their cellular function, as well as their molecular heterogeneities. In the heart, activation of the channels responsible for the A-type currents modulates the action potential duration and its shape. In the CNS, they play a critical role by regulating many aspects of neuronal function, and therefore, their pharmacological modulation potentially represents a powerful means of controlling CNS disorders, such as epilepsy, dementia, anxiety, pain, depression and stroke (for review, see references [1,2]). The characterization of new toxins, which could target A-type channels, is expected to reveal more about the role of K + channels in neuronal tissues. The K + channel specific peptides were divided in several families based on structural and functional properties; the most widely studied are the α-KTxs now subdivided into 26 sub-families ([3], see also UniProt databank), among which the subfamily α-KTx15 is the one reported to affect the transient I A currents [4–6]. Our group contributed by investigating the structure and pharmacology of K + channels expressing transient currents in cerebellum granular cell (I A -CGC) by means of synthetic peptides corresponding to various segments or variants of scorpion toxins [7–10]. Discrepin isolated from the venom of the Venezuelan scorpion Tityus discrepans belongs to the sub-family α-KTx15 [11] and its 3D-structure was determined by NMR [12]. The members of the α-KTx15 subfamily share very high sequence similarities (up to 98%). Discrepin (α-KTx15.6) displays only around 50% of sequence identity with the remaining members of the subfamily (Fig. 1). It has been shown that discrepin and different variants of this toxin can block A-type currents of rat CGC [13]. Cerebellum granular cells are located at the input stage of the cerebellum and play a critical role for regulating inputs coming from mossy fibers. The primary cell culture of cerebellum-neurons is widely used as a model of CNS, due to their abundance and the fact that they represent a highly homogeneous neuronal population [14]. They exhibit Biochimica et Biophysica Acta 1840 (2014) 2744–2751 ⁎ Corresponding author. Tel.: +39 010 6475569. ⁎⁎ Corresponding author. Tel.: +52 777 329 1669. E-mail addresses: picco@ge.ibf.cnr.it (C. Picco), corzo@ibt.unam.mx (G. Corzo). http://dx.doi.org/10.1016/j.bbagen.2014.05.009 0304-4165/© 2014 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagen