(A) A PIP₂-dependent pathway is involved in the SL-NH2–induced potentiation of AITC-activated currents. AITC was reapplied 60 seconds after exposure to bath solution with SL-NH2 or LR-NH2. Currents were normalized to the values first induced by AITC application in the absence of SL-NH2 or LR-NH2. The potentiation by SL-NH2 was inhibited when a water-soluble PIP₂ (10 μM) was added to pipette solution. ^#P < 0.05 versus SL-NH2; unpaired Student’s t test. *P < 0.05 versus LR-NH2. (B) Representative traces show hTRPA1 currents produced by repeated applications of AITC (100 μM) with the PIP₂ antibody (right; 1:100) or boiled PIP₂ antibody (boiled antibody was added to the pipette solution as a control) (left) in the pipette solution. A dialyzed mouse monoclonal PIP₂ antibody (see Methods) was included in the pipette solution. V_h was –60 mV. (C) The intracellular PIP₂ antibody potentiated the AITC-evoked hTRPA1 response. Currents were normalized to values first induced by AITC application. *P < 0.05 versus boiled PIP₂ antibody; unpaired Student’s t test. (D) Intracellular application of polylysine (3 μg/ml), a PIP₂ scavenger, also potentiated AITC-activated currents in transfected HEK cells. *P < 0.05 versus control; unpaired Student’s t test. (E) A PIP₂-dependent pathway is involved in the bradykinin-induced potentiation of AITC activated currents in HEK cells transfected with B2R and hTRPA1. AITC was reapplied 60 seconds after exposure to bath solution with or without bradykinin. Currents were normalized to the values first induced by AITC application in the absence of bradykinin. The potentiation by bradykinin was inhibited when pretreated with the PLC inhibitor ET-18-OCH₃ (2 μM) for 120 seconds before bradykinin (20 nM) application or a water-soluble PIP₂ (10 μM) was added to pipette solution. **P < 0.005 versus control; ^†P < 0.05 versus bradykinin (BK); ^##P < 0.005 versus BK; unpaired Student’s t test. Numbers in parentheses indicate cells tested.