Our recent study has shown that activation of transient receptor potential A1 channel (TRPA1) by pungent chemicals such as allyl-isothiocyanate (AITC) requires an unidentified cytosolic factor whose action can be mimicked by inorganic polyphosphates. Thus, AITC and other pungent chemicals fail to activate TRPA1 in excised patches. It is unclear whether TRPA1 switches to a conformation that is insensitive to the pungent chemicals, or whether TRPA1 simply becomes completely non-functional and insensitive to all activators when the cytosolic factor is absent. To help distinguish between these possibilities, the effects of Delta(9)-tetrahydrocannabinol (THC) and Ca(2+) that are structurally different from pungent chemicals were tested on AITC-sensitive and AITC-insensitive states of TRPA1. In HeLa cells transiently expressing mouse TRPA1, activation of TRPA1 by THC was slow and weak from the extracellular side (cell-attached; K(1/2) >20 microM), but was faster and more potent from the intracellular side (inside-out; K(1/2), approximately 0.7 microM), and this did not require the presence of a polyphosphate. Similar results were observed in rat trigeminal neurons. Increasing the extracellular [Ca(2+)] from approximately 0-1-3 mM activated TRPA1 in cell-attached patches. Elevation of cytosolic [Ca(2+)] using thapsigargin (inhibitor of Ca(2+)-ATPAse) and histamine (that elevates IP(3)) also activated TRPA1 in cell-attached patches. Similar to pungent chemicals, Ca(2+) (1-5 microM) failed to activate TRPA1 in inside-out patches, unless polyphosphates were present. These results show that TRPA1 can exist in different functional states: a native state (cell-attached patch) and a non-native state (excised patch). THC can activate TRPA1 even in the absence of polyphosphates, whereas pungent chemicals and Ca(2+) require it for activation.