Inhibition of calcium release-activated calcium current by Rac/Cdc42-inactivating clostridial cytotoxins in RBL...

Area of Science:

• Cell Biology
• Immunology
• Molecular Biology

Background:

• RBL 2H3 hm1 cells are a model for studying mast cell activation.
• Rho GTPases play critical roles in various cellular processes, including exocytosis.
• The high-affinity IgE receptor (FcεRI) signaling pathway is crucial for mast cell activation.

Purpose of the Study:

• To investigate the role of Rho GTPases in FcεRI-mediated activation of RBL 2H3 hm1 cells.
• To determine which specific Rho GTPases are involved in calcium mobilization and exocytosis.

Main Methods:

• Utilized large clostridial cytotoxins (Clostridium difficile toxin B, Clostridium sordellii lethal toxin, C2IN-C3, Clostridium botulinum C2 toxin) to selectively inhibit Rho GTPases.
• Measured hexosaminidase release as an indicator of exocytosis.
• Assessed intracellular calcium (Ca2+) mobilization using Fura-2 or similar dyes.
• Performed patch-clamp recordings to analyze calcium release-activated calcium currents (ICRAC).

Main Results:

• Clostridium difficile toxin B and Clostridium sordellii lethal toxin inhibited hexosaminidase release and Ca2+ mobilization.
• These toxins reduced Ca2+ influx, primarily by inhibiting the calcium release-activated calcium current (ICRAC).
• Neither Rho-specific C3-fusion toxin nor actin-specific C2 toxin affected these early activation events.

Conclusions:

• Rac and Cdc42 GTPases, but not Rho, are involved in early FcεRI-mediated Ca2+ mobilization.
• Rac and Cdc42 likely regulate Ca2+ influx through calcium release-activated calcium channels.
• These GTPases are implicated in both early Ca2+ signaling and later exocytosis events.