Amino acid sequence determination and chemical synthesis of CllErg1 (gamma-KTx1.5), a K+ channel blocker peptide isolated from the scorpion Centruroides limpidus limpidus

Abstract

Uma nova toxina denominada CllErg1 (nomenclatura sistemática -KTx1.5) foi purificada do veneno do escorpião Centruroides limpidus limpidus e a sua seqüência de amino ácidos foi determinada. Ela tem 42 resíduos de amino ácidos entrecruzados por quatro pontes de disulfetos e bloqueia especificamente um canal de potássio da família eter-a-go-go (ERG). O peptídeo completo foi quimicamente sintetizado e adequadamente enrolado, mostrando que é capaz de bloquear o canal-ERG humano (HERG) com uma afinidade idêntica ao peptídeo nativo. A toxina CllErg1 sintética pode ser produzida em quantidades suficientes para compensar a sua baixa concentração encontrada no veneno natural. Isto prepara o caminho para conduzir estudos enfocando a identificação dos padrões estruturais do HERG que são críticos para o funcionamento adequado do canal iônico. Adicionalmente, outro peptídeo análogo CllErg2 (nome sistemático -KTx4.1) foi purificado e a sua seqüência completa de amino ácidos foi determinada. Este contém 43 resíduos de amino ácidos, mantidos de forma compacta por quatro pontes de disulfetos.A novel toxin named CllErg1 (systematic nomenclature -KTx1.5) was purified from the venom of the scorpion Centruroides limpidus limpidus and its amino acid sequence was determined. It has 42 amino-acid residues cross-linked by four disulfide bridges and blocks specifically a potassium channel of the family ether-a-go-go (ERG). The full peptide was chemically synthesized and properly folded, showing that it blocks the human ERG-channels (HERG) with identical affinity to that of the native peptide. Synthetic CllErg1 can be produced in quantities enough to compensate its low concentration in the natural venom. It paves the way to conduct studies aimed at the identification of the structural motifs of HERG critical for proper channel function. Additionally, another analogous peptide CllErg2 (systematic name -KTx4.1) was purified and had its full amino acid sequence determined. It contained 43 amino acid residues, maintained closely packed by four disulfide bridges. 1 The electric potential of these cells is maintained by active pumps and ion-channels.2 One of these channels is the inward rectifier potassium channel (IKr).3 The IKr voltage gated potassium ion current contributes to the repolarization phase of cardiac action potential and the non-specific blockade of cardiac IKr current is a significant contributor to fatal cardiac arrhythmias associated with prolongation of the QT interval. 4 The human ether-a-go-go related gene (h-erg) encodes a potassium channel (HERG) that is responsible for the current Ikr. 5,6 Mutations in HERG predispose patients to long QT syndrome and sudden cardiac arrest. 5,7,8 Blockage of the channel by commonly used medications, such as class III antiarrhythmics, antihistaminics and antipsychotics can also lead to long QT. In most circumstances arrhythmias associated with long QT and sudden death is a result of a combination of genetic, environmental and pharmacological factors. 9 The HERG channels, are also expressed...

Two Novel Ergtoxins, Blockers of K+-channels, Purified from the Mexican Scorpion Centruroides elegans elegans

“…CeErg4 is 97.6% identical to CeErg5. When compared to other known scorpion toxins, by application of ClustalW (Table 2), CeErg4 showed to be identical to toxin CllErg1 from the scorpion C. limpidus limpidus [42]. This toxin (CllErg1) was partially characterized electro-physiologically, using human erg1 gene expressed heterologously as cRNA into Xenopus laevis oocytes, from which a K D of 10 nM was obtained, similar to what was found and described here.…”

“…It suffices to recall that changing only amino acid residue in certain types of scorpion toxins can modify the affinity by three orders of magnitude, as shown for the case of charybdotoxin [43]. Also the fact that CeErg4, reported here, and CllErg1 [42] have the same amino acid sequence is not unusual. Butantoxin, an a-KTx toxin (systematic number aKTx 12.1) was found to be present in the venom of three species of scorpion from the genus Tityus [44].…”

“…CeErg1 is about 93% identical to CeErg4 reported here. From the large list of possible ergtoxins [37] until now only five of them were actually purified from the corresponding venoms and assayed: CnErg1 [30], ErgTx2 [24], BeKm-1 [31], CsEKerg1 [32] and CllErg1 [42]. Thus far the best studied are CnErg1 and BeKm-1 (reviewed in [36]).…”

“…The same panels show the theoretical curves resulting from the KD obtained during kinetics experiments shown in the right panels, for rERG2 (up-right) and hERG3 (downright). Time constants and K D values are reported in Table 1 This table summarizes the blocking properties of CeErg4; n.d., means not determined; n.e., means no effect found; n is the number of times the experiment was performed and the signal minus (-) means not applicable Toxin sequences were from: CnErg1 of C. noxius noxius [37]; CsErg1 of C. sculpturatus [37]; CllErg1 of C. limpidus limpidus [42]; CeErg1 [37]; CeErg4 and CeErg5 of C. elegans [this work]; CexErg1 of C. exilicauda [37]; CgErg1 of C. gracilis [37]. The complete sequence is shown for CnErg1; for the other toxins only the residues that are different from CnErg1 are shown (theoretical conservative and non-conservative changes are marked in dark grey and middle grey, respectively), light grey background indicate the cysteine positions channels showed at least 11 positions that are occupied by different amino acids in the vestibule of these three variants, and data published by Zhang et al [46] indicate that some of these amino acid positions are those that get in contact with ergtoxins (BemK-1).…”

Scorpion venom components that affect ion-channels function

“…These peptides were found in scorpions of the genus Centruroides, Mesobuthus and Buthus (Zeng et al, 2006; Jiménez-Vargas et al, 2012). The first peptide isolated was ErgTx1 ( C. noxius; Gurrola et al, 1999a), followed by the BeKm-1 ( B. eupeus; Korolkova et al, 2001); other 26 members of this subfamily were Ergtoxin-like peptides isolated from scorpions of the genus Centruroides (Lecchi et al, 2002; Nastainzky et al, 2002; Corona et al, 2002; Coronas et al, 2005; Restano-Cassulini et al, 2008; Jiménez-Vargas et al, 2012) and BmKKx2 (from M. martensii; Zeng et al, 2006). The γ-KTxs were described as mainly targeting hERG channels (Coronas et al, 2002).…”