The effect of PRR ligands on the membrane potential of intestinal epithelial cells

Disproportionate signaling through intestinal epithelial pattern recognition receptors (PRR) plays a role in IBD (inflammatory bowel disease) pathophysiology. Diarrhea is a clinical trademark of IBD and altered activity of K⁺ channels (KC) may contribute to the low sodium absorption state. Here we sought to study the impact of PRR activation on the membrane potential of human intestinal epithelial cells and the role of KC in it.

All assays were performed in cultured HT-29 cells. KC activity was assessed by spectrofluorometry, measuring changes in cell membrane potential (MP) with the anionic fluorophore DiBAC₄(3). PRRs were activated by specific ligands (MDP, LTA, MPLA, flagellin, loxoribine and ODN2216). KC modulators employed were BaCl₂, pinacidil, noxiustoxin and AMP-PNP.

Activation of NOD2, TLR5, TLR7 and TLR9 hyperpolarized the membrane (at 10³ng/ml, the normalized AUC of the fluorescence intensity variation from the control were respectively (mean±SEM): -725.3±111.5; -1517.4±95.0; -857.8±61.1 and -995.6±53.6), while TLR2 and TLR4 stimulation induced membrane depolarization (1110.4±73.1 and 3890.3±342.7 at 10³ng/ml, respectively). MPD effect on MP was abolished by BaCl₂, partially reversed by AMP-PNP (aK_ATP channel inhibitor) and insensitive to noxiustoxin (a voltage-gated KC inhibitor).

It was shown for the first time that PRR activation affects MP in human intestinal epithelial cells. KC appear to be important mediators in this phenomenon; in particular, K_ATP channels may partake in NOD2-derived effects.