Abstract
Ca(2+) mobilization is central to many cellular processes, including stimulated exocytosis and cytokine production in mast cells. Using single cell stimulation by IgE-specific Ag and high-speed imaging of conventional or genetically encoded Ca(2+) sensors in rat basophilic leukemia and bone marrow-derived rat mast cells, we observe Ca(2+) waves that originate most frequently from the tips of extended cell protrusions, as well as Ca(2+) oscillations throughout the cell that usually follow the initiating Ca(2+) wave. In contrast, Ag conjugated to the tip of a micropipette stimulates local, repetitive Ca(2+) puffs at the region of cell contact. Initiating Ca(2+) waves are observed in most rat basophilic leukemia cells stimulated with soluble Ag and are sensitive to inhibitors of Ca(2+) release from endoplasmic reticulum stores and to extracellular Ca(2+), but they do not depend on store-operated Ca(2+) entry. Knockdown of transient receptor potential channel (TRPC)1 and TRPC3 channel proteins by short hairpin RNA reduces the sensitivity of these cells to Ag and shifts the wave initiation site from protrusions to the cell body. Our results reveal spatially encoded Ca(2+) signaling in response to immunoreceptor activation that utilizes TRPC channels to specify the initiation site of the Ca(2+) response.