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Induction of cytosolic Ca2+ elevation mediated by Mas-7 occurs through membrane pore formation
1Department of Life Science and Basic Science Research Institute, Pohang University of Science and Technology, Pohang, 790-784, Republic of Korea.
Abstract:
Mas-7, a mastoparan derivative, induces elevation of intracellular free Ca2+ concentration ([Ca2+]i) along two independent pathways. The minor contribution occurs via phospholipase C activation and is negatively regulated by treatment with phorbol 12-myristate 13-acetate, a protein kinase C activator. The major contribution involves plasma membrane pores allowing not only Ca2+, Mn2+, and Na+ to enter but also the uptake of ethidium bromide (314 Da) and lucifer yellow (457 Da), but not fura-2 (831 Da), Evans blue (961 Da), and fluorescein-conjugate phalloidin (1,175 Da). Mas-7-induced current, as measured in planar lipid bilayers, reveals that Mas-7-induced pores have two slope conductances, 290 and 94 pS, and that the pores are nonselective for cations. The results also indicate that Mas-7 can produce pores by direct interaction with the plasma membrane without the involvement of membrane proteins and cytosolic factors. Besides in human neuroblastoma cells, similar Mas-7 effects were also observed in other cell lines such as HL-60, 1321N1 human astrocytoma, and bovine chromaffin cells. The data suggest that the Mas-7-induced [Ca2+]i elevation is the combined result of Ca2+ release from stores via phosphoinositide turnover and prolonged Ca2+ influx through membrane pores.
Insights
Mas-7 peptide opens plasma membrane pores, increasing intracellular calcium ([Ca2+]i) via direct membrane interaction. This pore formation allows ion and small molecule influx, contributing significantly to calcium elevation alongside minor phospholipase C activation.
Area of Science:
- Cell Biology
- Biophysics
- Pharmacology
Background:
- Mastoparan derivatives, like Mas-7, are known to affect cell membranes.
- Intracellular calcium concentration ([Ca2+]i) plays a critical role in various cellular processes.
- Understanding calcium signaling pathways is crucial for drug development.
Purpose of the Study:
- To elucidate the mechanisms by which Mas-7 elevates intracellular calcium ([Ca2+]i).
- To characterize the nature and properties of Mas-7-induced membrane pores.
- To investigate the cell-type specificity of Mas-7 effects.
Main Methods:
- Measurement of intracellular calcium ([Ca2+]i) levels.
- Electrophysiological recordings in planar lipid bilayers to study Mas-7-induced currents.
- Assessment of molecule permeability through Mas-7-induced pores using various dyes.
- Experiments involving protein kinase C activators.
Main Results:
- Mas-7 elevates [Ca2+]i through two pathways: minor phospholipase C activation and major plasma membrane pore formation.
- Mas-7-induced pores allow passage of Ca2+, Mn2+, Na+, ethidium bromide, and lucifer yellow, but not larger molecules.
- Pores exhibit two slope conductances (290 and 94 pS) and are nonselective for cations.
- Mas-7 directly interacts with the plasma membrane to form pores, independent of proteins and cytosolic factors.
- Similar effects were observed across multiple cell lines, including neuroblastoma, HL-60, astrocytoma, and chromaffin cells.
Conclusions:
- Mas-7 induces significant intracellular calcium ([Ca2+]i) elevation primarily through direct plasma membrane pore formation.
- These pores facilitate the influx of various ions and small molecules.
- The findings reveal a novel mechanism of calcium regulation by Mas-7, with potential implications for cellular signaling research.