Recombinant expression of Intrepicalcin from the scorpion Vaejovis intrepidus and its effect on skeletal ryanodine receptors

• Published in: Biochimica et biophysica acta 2017-04, Vol.1861 (4), p.936-946
• Main Authors: Vargas-Jaimes, Leonel, Xiao, Liang, Zhang, Jing, Possani, Lourival D., Valdivia, Héctor H., Quintero-Hernández, Verónica
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Calcin; Calcium - metabolism; Escherichia coli - genetics; Escherichia coli - metabolism; Intrepicalcin; Models, Molecular; Muscle, Skeletal - metabolism; Peptides - genetics; Peptides - metabolism; Rabbits; Rats; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Ryanodine - metabolism; Ryanodine receptor; Ryanodine Receptor Calcium Release Channel - metabolism; Sarcoplasmic reticulum; Sarcoplasmic Reticulum - metabolism; Scorpion; Scorpion Venoms - genetics; Scorpion Venoms - metabolism; Scorpions - genetics; Scorpions - metabolism; Thioredoxins - metabolism; Transcriptome - genetics; Vaejovis intrepidus
• ISSN: 0304-4165; 0006-3002; 1872-8006; 1878-2434
• DOI: 10.1016/j.bbagen.2017.01.032

Scorpion venoms contain toxins that modulate ionic channels, among which are the calcins, a small group of short, basic peptides with an Inhibitor Cystine Knot (ICK) motif that target calcium release channels/ryanodine receptors (RyRs) with high affinity and selectivity. Here we describe the heterologous expression of Intrepicalcin, identified by transcriptomic analysis of venomous glands from Vaejovis intrepidus. Recombinant Intrepicalcin was obtained in Escherichia coli BL21-DE3 (periplasm) by fusing the Intrepicalcin gene to sequences coding for signal-peptide, thioredoxin, His-tag and enterokinase cleavage site. [3H]Ryanodine binding, used as a functional index of RyR activity, revealed that recombinant Intrepicalcin activates skeletal RyR (RyR1) dose-dependently with Kd=17.4±4.0nM. Intrepicalcin significantly augments the bell-shaped [Ca2+]-[3H]ryanodine binding curve at all [Ca2+] ranges, as is characteristic of the calcins. In single channel recordings, Intrepicalcin induces the appearance of a subconductance state in RyR1 with a fractional value ∼55% of the full conductance state, very close to that of Vejocalcin. Furthermore, Intrepicalcin stimulates Ca2+ release at an initial dose=45.3±2.5nM, and depletes ~50% of Ca2+ load from skeletal sarcoplasmic reticulum vesicles. We conclude that active recombinant Intrepicalcin was successfully obtained without the need of manual oxidation, enabling it to target RyR1s with high affinity. This is the first calcin heterologously expressed in the periplasma of Escherichia coli BL21-DE3, shown to be pharmacologically effective, thus paving the way for the generation of Intrepicalcin variants that are required for structure-function relationship studies of calcins and RyRs. •Active recombinant Intrepicalcin was obtained in the periplasma of E. coli BL21 DE3•Recombinant Intrepicalcin activates skeletal RyR (RyR1) with Kd=17.4±4.0nM.•Intrepicalcin induces the appearance of a subconductance state in RyR1.•Intrepicalcin stimulates Ca2+ release at an initial dose=45.3±2.5nM.