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

Abstract

Background 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. Methods 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. Results [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 ± 4.0 nM. 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 ± 2.5 nM, and depletes ~50% of Ca2+ load from skeletal sarcoplasmic reticulum vesicles. Conclusions We conclude that active recombinant Intrepicalcin was successfully obtained without the need of manual oxidation, enabling it to target RyR1s with high affinity. General Significance 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.

A Deeper Examination of Thorellius atrox Scorpion Venom Components with Omic Technologies

“…Intrepicalcin is a proven active calcin. It was heterologously expressed and shown to be active on RyRs [ 41 ].…”

Cryo-EM analysis of scorpion toxin binding to Ryanodine Receptors reveals subconductance that is abolished by PKA phosphorylation

“…Thus, on the basis of minimum radius and electrostatic surface potential, exit 1 may form the most likely exit pathway for Ca 2+ ions. In vejocalcin and intrepicalcin, two calcins with the highest degree of subconductance (2,14), Lys 30 is replaced with a neutral Gln and Lys 22 is replaced by Ser (fig. S5), which likely lowers the energetic barriers through multiple exits, explaining the higher subconductance.…”

“…Unlike other ion channels targeted by peptide toxins, RyRs are not exposed on the extracellular surface, yet calcins can efficiently reach them by permeating the plasma membrane (8,9). Upon engaging the RyR, calcins cause long-lived subconductance states, which are readily detected in single-channel recordings as decreased current levels at 20 to 60% of the full conductance (2,(10)(11)(12)(13)(14). The mechanism by which calcin binding results in subconductance, and how the subconductance level depends on the calcin's specific amino acid composition, had remain unsolved.…”

BmK NSPK, a Potent Potassium Channel Inhibitor from Scorpion Buthus martensii Karsch, Promotes Neurite Outgrowth via NGF/TrkA Signaling Pathway

“…Ion channels are the main targets of scorpion toxins. Studies have demonstrated that scorpion toxins can modulate the gating kinetics of many ion channels, including voltage-gated potassium channels (K v channels) [ 8 ], voltage-gated sodium channels (VGSCs) [ 7 , 9 ], voltage-gated calcium channels (Ca v channels) [ 10 ], chloride channels [ 11 ], and ryanodine receptors [ 12 ]. Due to their high affinity and selectivity, scorpion toxins are widely used as tools to explore the gating mechanism of ion channels [ 13 ] and their downstream cellular signaling pathways [ 14 ].…”