Potentiation of Fc epsilon receptor I-activated Ca2+ current (I-CRAC) by cholera toxin: Possible mediation by ADP ribosylation factor

Antigen-evoked influx of extracellular Ca2+ into mast cells may occur via s tore-operated Ca2+ channels called calcium release-activated calcium (CRAC) channels. In mast cells of the rat basophilic leukemia cell line (RBL-2H3) , cholera toxin (CT) potentiates antigen-driven uptake of Ca-45(2+) through cAMP-independent means. Here, we have used perforated patch clamp recordin g at physiological temperature to test whether cholera toxin or its substra te, Gs, directly modulates the activity of CRAC channels. Cholera toxin dra matically amplified (two- to fourfold) the Ca2+ release-activated Ca2+ curr ent (I-CRAC) elicited by suboptimal concentrations of antigen, without itse lf inducing I-CRAC and this enhancement was not mimicked by cAMP elevation. In contrast, cholera toxin did not affect the induction of I-CRAC by thaps igargin, an inhibitor of organelle Ca2+ pumps, or by intracellular dialysis with low Ca2+ pipette solutions. Thus, the activity of CRAC channels is no t directly controlled by cholera toxin or Gs alpha. Nor was the potentiatio n of I-CRAC due to enhancement of phosphoinositide hydrolysis or calcium re lease. Because Gs and the A subunit of cholera toxin bind to ADP ribosylati on factor (ARF) and could modulate its activity, we tested the sensitivity of antigen-evoked I-CRAC, to brefeldin A, an inhibitor of ARF-dependent fun ctions, including vesicle transport. Brefeldin A blocked the enhancement of antigen-evoked I-CRAC without inhibiting ADP ribosylation of Gs alpha, but it did not affect I-CRAC induced by suboptimal antigen or by thapsigargin. These data provide new evidence that CRAC channels are a major route for F c epsilon receptor I-triggered Ca2+ influx, and they suggest that BRF may m odulate the induction of I-CRAC by antigen.