Figure 6. Participation of TRPC channels in Ag stimulated initiation of the Ca²⁺ response.

RBL cells were co-transfected with GCaMP2 and shRNA plasmids targeting TRPC1, 3, 5, 7 or mock sequence (for each condition n>29). Cells sensitized with anti-DNP IgE were stimulated with puff from pipette containing 1.7 ng/ml DNP-BSA in BSS. A) Percentage of cells from multiple experiments showing a measurable Ca²⁺ response corresponding to elevation of intracellular Ca²⁺ cells. Dark blue bars include cells that only include Ca²⁺ puffs in the count, and light blue bars exclude this population. B) Average lag time before initiation of Ca²⁺ wave with specified shRNA. Error bars correspond to SEM. *P < 0.05, **P < 0.01 vs. cells expressing shGFP. C) Percentage of cells responding to stimulus with Ca²⁺ waves with indicated shRNA. Bar height shows total % waves; orange portion represents % waves originating in protrusions. D) Model for initiation of Ca²⁺ mobilization in extended protrusions. Ag crosslinks IgE/FcεRI complexes, activating Ca²⁺ influx via TRPC1 channels to cause local increase in cytoplasmic Ca²⁺ at the tip of a cell protrusion. This potentiates IP₃-mediated Ca²⁺ release from the nearby ER store that propagates via Ca²⁺-induced Ca²⁺ release to generate a Ca²⁺ wave that initiates Ca²⁺ oscillations and cell activation. Two possible mechanisms for wave initiation at these protrusions are indicated.