Signal transduction by cholera toxin: processing in vesicular compartments does not require acidification

Area of Science:

• Cell Biology
• Molecular Biology
• Gastroenterology

Background:

• Cholera toxin (CT) triggers a cAMP-dependent chloride secretion in intestinal epithelial cells.
• CT enters cells via endosomes and its mechanism involves acidic compartments.
• The role of intravesicular pH in CT signal transduction remains unclear.

Purpose of the Study:

• To investigate the effect of intravesicular pH on CT-induced signal transduction in T84 cells.
• To determine if acidic vesicular compartments are necessary for CT's downstream effects.

Main Methods:

• T84 cell monolayers were treated with pH-altering reagents (chloroquine, methylamine, NH4Cl, nigericin, bafilomycin A1).
• Intravesicular pH gradients were confirmed using acridine orange and confocal microscopy.
• cAMP-dependent chloride secretion (Isc) was measured in response to CT and other agonists (VIP, forskolin, 8-bromo-cAMP).
• CT binding to ganglioside GM1 was assessed in vitro at different pH levels.

Main Results:

• Reagents collapsing intravesicular pH gradients inhibited CT-induced Isc, with nigericin showing the most significant inhibition.
• Adenylate cyclase activity and Gs protein coupling were unaffected by the pH-modulating treatments.
• CT binding to GM1 was pH-independent between pH 5.0 and 10.
• The maximal secretory response to CT relative to VIP was not altered by most treatments, except nigericin.

Conclusions:

• Low intravesicular pH has minimal to no impact on CT binding to its receptor GM1.
• Subsequent CT processing and signal transduction events are largely independent of intravesicular pH.
• The study suggests that acidic vesicular compartments are not essential for cholera toxin's signaling pathway in T84 cells.