Journal
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS
Volume 1861, Issue 4, Pages 936-946Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbagen.2017.01.032
Keywords
Scorpion; Vaejovis intrepidus; Intrepicalcin; Ryanodine receptor; Sarcoplasmic reticulum; Calcin
Categories
Funding
- DGAPA-UNAM [IN203416]
- National Institute of Health, USA [R01HL055438, R01-HL134344, R01-HL120108]
- National Council of Science and Technology (CONACyT) [287191]
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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: [H-3]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+]-[H-3]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 RyRls 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. (C) 2017 Elsevier B.V. All rights reserved.
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