Activation of vanilloid receptor type I in the endoplasmic reticulum fails to activate store-operated Ca2+ entry

To evaluate interaction of vanilloid receptor type 1 (TRPV1) with endogenous Ca2+ signalling mechanisms, TRPV1 was expressed in Spodoptera frugiperda (Sf9) insect cells using recombinant baculovirus. Stimulation of TRPV1-expressing cells, but not control Sf9 cells, with resiniferatoxin (RTX), capsaicin or anandamide, produced an increase in cytosolic free Ca2+ concentration ([Ca2+), with EC50 values of 166 pM, 24.5 nM and 3.89 muM respectively. In the absence of extracellular Ca2+, both capsaicin and RTX caused an increase in [Ca2+], with EC50 values of approx. 10 muM and 10 nM respectively. This TRPV1-induced release of Ca2+ from intracellular stores was not blocked by U73122, suggesting that phospholipase C was not involved. Substantial overlap was found between the thapsigargin-and RTX-sensitive internal Ca2+ pools, and confocal imaging showed that intracellular TRPV1 immunofluorescence colocalized with the endoplasmic reticulum targeting motif KDEL. To determine if TRPV1-induced mobilization of intracellular Ca2+ activates endogenous store-operated Ca2+ entry, the effect INTRODUCTION of 2-aminoethoxydiphenyl borate (2-APB) on Ba2+ influx was examined. 2-APB blocked thapsigargin-induced Ba2+ influx, but not RTX-induced Ba2+ entry. In the combined presence of thapsigargin and a store-releasing concentration of RTX, the 2-APB-sensitive component was essentially identical with the thapsigargin-induced component. Similar results were obtained in HEK-293 cells stably expressing TRPV1. These results suggest that TRPV1 forms agonist-sensitive channels in the endoplasmic reticulum, which when activated, release Ca2+ from internal stores, but fail to activate endogenous store-operated Ca2+ entry. Selective activation of intracellular TRPV1, without concomitant involvement of plasmalemmal Ca2+ influx mechanisms, could play an important role in Ca2+ signalling within specific subcellular microdomains.