Recombinant expression of Intrepicalcin from the scorpion Vaejovis intrepidus and its effect on skeletal ryanodine receptors.
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.
Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62271, México.
Leonel
L
Vargas-Jaimes
Department of Marine Biotechnology, Faculty of Naval Medicine, Second Military Medical University, Shanghai, 200433, China.|Center for Arrhythmia Research, Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA.
Liang
L
Xiao
Department of Marine Biotechnology, Faculty of Naval Medicine, Second Military Medical University, Shanghai, 200433, China.
Jing
J
Zhang
Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62271, México.
Lourival D
LD
Possani
Center for Arrhythmia Research, Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA.
Héctor H
HH
Valdivia
Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62271, México.|CONACYT- Laboratorio de Ecología Molecular Microbiana, Centro de Investigaciones en Ciencias Microbiológicas-Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, Ciudad Universitaria, C.P. 72570, Puebla, México.
Verónica
V
Quintero-Hernández
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000378:metabolism*; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism*; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D012604:Scorpion Venoms / Q000235:genetics* / Q000378:metabolism*; D012605:Scorpions / Q000235:genetics / Q000378:metabolism*; D013879:Thioredoxins / Q000378:metabolism; D059467:Transcriptome / Q000235:genetics
D010455:Peptides; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D012604:Scorpion Venoms; D012433:Ryanodine; D013879:Thioredoxins; D002118:Calcium
Calcin; Intrepicalcin; Ryanodine receptor; Sarcoplasmic reticulum; Scorpion; Vaejovis intrepidus