Crotamine Pharmacology Revisited: Novel Insights Based on the Inhibition of K_V Channels

Abstract

Crotamine, a 5-kDa peptide, possesses a unique biological versatility. Not only has its cell-penetrating activity become of clinical interest but, moreover, its potential selective antitumor activity is of great pharmacological importance. In the past, several studies have attempted to elucidate the exact molecular target responsible for the crotamine-induced skeletal muscle spasm. The aim of this study was to investigate whether crotamine affects voltagegated potassium (K V ) channels in an effort to explain its in vivo effects. Crotamine was studied on ion channel function using the two-electrode voltage clamp technique on 16 cloned ion channels (12 K V channels and 4 Na V channels), expressed in Xenopus laevis oocytes. Crotamine selectively inhibits K V 1.1, K V 1.2, and K V 1.3 channels with an IC 50 of ϳ300 nM, and the key amino acids responsible for this molecular interaction are suggested. Our results demonstrate for the first time that the symptoms, which are observed in the typical crotamine syndrome, may result from the inhibition of K V channels. The ability of crotamine to inhibit the potassium current through K V channels unravels it as the first snake peptide with the unique multifunctionality of cell-penetrating and antitumoral activity combined with K V channel-inhibiting properties. This new property of crotamine might explain some experimental observations and opens new perspectives on pharmacological uses.

Soluble prokaryotic expression and purification of crotamine using an N-terminal maltose-binding protein tag

“…Crotamine inhibited the hKv1.3 channel (but not the hKv1.5 channel), which is consistent with a previous study that reported that crotamine selectively inhibits hKv1.1, hKv1.2, and hKv1.3 channels, but not hKv1.4 or hKv1.5 channels (Peigneur et al, 2012). This functionality suggests that the purified recombinant protein is correctly folded.…”

“…This functionality suggests that the purified recombinant protein is correctly folded. Crotamine purified from crude venom exhibits an IC 50 value for hKv channel inhibition of approximately 300 nM (Peigneur et al, 2012). The crotamine purified here from E. coli inhibited hKv1.3 characterized by an IC 50 value of 67 nM, which is approximately 4 times more active in comparison with crotamine purified from snake venom.…”

“…However, cytotoxicity might also be related to the inhibition of several hKv channels, as Kv channels are well expressed and play a role in the cell proliferation of many types of cancer (e.g., breast cancer, prostate cancer, melanoma) (Peigneur et al, 2012;Wang et al, 2004). In addition, some autoimmune diseases are ameliorated by the inhibition of hKv1.3 in T-lymphocytes (Beeton et al, 2001), thereby widening the application potential of crotamine.…”

“…Subsequently, voltage-gated K (K v ) channels were suggested as potential targets of crotamine according to computational docking analyses (Yount et al, 2009). Recently, it was shown that crotamine selectively inhibits hKv1.1, hKv1.2, and hKv1.3 channels at an IC 50 value of approximately 300 nM, but other K channels are not affected (Peigneur et al, 2012). The structure of crotamine was determined using NMR (Fadel et al, 2005) and X-ray crystallography (Coronado et al, 2013), which revealed three intramolecular disulfide bonds.…”

Vipericidins, Snake Venom Cathelicidin-Related Peptides, in the Milieu of Reptilian Antimicrobial Polypeptides

“…One example of a multifunctional and versatile snake venom peptide that is structured by the central g-core and the canonical b-defensin fold is crotamine (Kerkis et al 2010; Rádis-Baptista and Kerkis 2011). Initially characterized from the venom of South American rattlesnake Crotalus durissus terrificus and described as a myotoxin with the ability to interfere with Na + -channel function, crotamine was demonstrated to possess multiple molecular functionalities, such as selective in vitro and in vivo anticancer cytotoxicity (Hayashi et al 2008;Pereira et al 2011;Kerkis et al 2014), biocide activity against bacteria and fungi (Oguiura et al 2011;Yamane et al 2013), selective interaction with the eukaryotic voltage K + channel (Yount et al 2009;Peigneur et al 2012), translocation across lipid bilayers and penetration into eukaryotic cells (Kerkis et al 2004), and interaction with nucleic acids and delivery of genes and probes (Nascimento et al 2007;Chen et al 2012). These properties equip crotamine with a unique versatility to be explored for the development of diagnostic probes and therapeutic peptides.…”