Masayoshi Okada1, Gerardo Corzo2, Gustavo A Romero-Perez3, Fredy Coronas2, Hiroko Matsuda3, Lourival D Possani2. 1. Department of Pysiology, Kansai Medical University, Hirakata, Osaka 573-1010, Japan. Electronic address: mokada-tky@umin.ac.jp. 2. Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62210, Mexico. 3. Department of Pysiology, Kansai Medical University, Hirakata, Osaka 573-1010, Japan.
Abstract
BACKGROUND: Arachnoid venoms contain numerous peptides with ion channel modifying and cytolytic activities. METHODS: We developed a green fluorescent protein (GFP)-based assay that can monitor the changes in currents through overexpressed inwardly rectifying K(+) channels (Kir2.1), in which GFP expression was increased by blockade of Kir2.1 current. Using this assay, we screened venom of many spider species. A peptide causing GFP decreasing effect was purified and sequenced. Electrophysiological and pain-inducing effects of the peptide were analyzed with whole-cell patch-clamp recordings and hot-plate test, respectively. RESULTS: Among venoms we screened, soluble venom from Lachesana sp. decreased the GFP expression. Purification and sequencing of the peptide showed that the peptide is identical to a pore-forming peptide purified from Lachesana tarabaevi venom. Whole cell patch-clamp recordings revealed that the peptide had no effect on Kir2.1 current. Instead, it induced a current that was attributable to the pore-formation of the peptide. The peptide was selectively incorporated into hyperpolarized, i.e., Kir2.1 expressing, cells and for this reason the peptide decreased GFP expression in our Kir2.1 assay. The pore-formation positively shifted the reversal potential and induced burst firings in the hippocampal neurons in a synaptic current-independent way. The application of the Lachesana sp. peptide induced pain-related behavior in mice. CONCLUSIONS: The peptide, which was found in Lachesana sp. venom, formed pores and thereby depolarized neurons and induced pain. GENERAL SIGNIFICANCE: Our data suggested an additional physiological role of the pore-forming peptides.
BACKGROUND:Arachnoid venoms contain numerous peptides with ion channel modifying and cytolytic activities. METHODS: We developed a green fluorescent protein (GFP)-based assay that can monitor the changes in currents through overexpressed inwardly rectifying K(+) channels (Kir2.1), in which GFP expression was increased by blockade of Kir2.1 current. Using this assay, we screened venom of many spider species. A peptide causing GFP decreasing effect was purified and sequenced. Electrophysiological and pain-inducing effects of the peptide were analyzed with whole-cell patch-clamp recordings and hot-plate test, respectively. RESULTS: Among venoms we screened, soluble venom from Lachesana sp. decreased the GFP expression. Purification and sequencing of the peptide showed that the peptide is identical to a pore-forming peptide purified from Lachesana tarabaevi venom. Whole cell patch-clamp recordings revealed that the peptide had no effect on Kir2.1 current. Instead, it induced a current that was attributable to the pore-formation of the peptide. The peptide was selectively incorporated into hyperpolarized, i.e., Kir2.1 expressing, cells and for this reason the peptide decreased GFP expression in our Kir2.1 assay. The pore-formation positively shifted the reversal potential and induced burst firings in the hippocampal neurons in a synaptic current-independent way. The application of the Lachesana sp. peptide induced pain-related behavior in mice. CONCLUSIONS: The peptide, which was found in Lachesana sp. venom, formed pores and thereby depolarized neurons and induced pain. GENERAL SIGNIFICANCE: Our data suggested an additional physiological role of the pore-forming peptides.
Authors: Anna Beatriz R Mayor; Leonardo A Guevarra; Myla R Santiago-Bautista; Librado A Santiago Journal: J Venom Anim Toxins Incl Trop Dis Date: 2020-08-03