Original Article

Potentiation of Fc Receptor I–activated Ca²⁺ Current (I_CRAC) by Cholera Toxin: Possible Mediation by ADP Ribosylation Factor

Correspondence to: Michael A. McCloskey, Department of Zoology and Genetics, Iowa State University, Ames, IA 50011-3223. Tel:(515) 294-5925 Fax:(515) 294-8457 E-mail:drmike{at}iastate.edu.

Antigen-evoked influx of extracellular Ca²⁺ into mast cells may occur via store-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 ⁴⁵Ca²⁺ through cAMP-independent means. Here, we have used perforated patch clamp recording at physiological temperature to test whether cholera toxin or its substrate, Gs, directly modulates the activity of CRAC channels. Cholera toxin dramatically amplified (two- to fourfold) the Ca²+ release–activated Ca²⁺ current (I_CRAC) elicited by suboptimal concentrations of antigen, without itself 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 thapsigargin, an inhibitor of organelle Ca²⁺ pumps, or by intracellular dialysis with low Ca²⁺ pipette solutions. Thus, the activity of CRAC channels is not directly controlled by cholera toxin or Gs. Nor was the potentiation of I_CRAC due to enhancement of phosphoinositide hydrolysis or calcium release. Because Gs and the A subunit of cholera toxin bind to ADP ribosylation factor (ARF) and could modulate its activity, we tested the sensitivity of antigen-evoked I_CRAC to brefeldin A, an inhibitor of ARF-dependent functions, including vesicle transport. Brefeldin A blocked the enhancement of antigen-evoked I_CRAC without inhibiting ADP ribosylation of Gs, 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 Fc receptor I–triggered Ca²⁺ influx, and they suggest that ARF may modulate the induction of I_CRAC by antigen.

Key Words: mast cells, patch clamp, Ca²⁺ imaging, Gs, brefeldin A

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