Activation of TRPC6 channels contributes to (+)-conocarpan-induced apoptotic cell death in HK-2 cells

(+)-Conocarpan (CNCP), a neolignan frequently found in many medicinal and edible Plants displays a broad spectrum of bioactivity. Here, we demonstrated that CNCP induced apoptotic cell death in human kidney-2 (HK-2) cells in a concentration-dependent manner (IC₅₀ = 19.3 μM) and led to the sustained elevation of intracellular CA²⁺ ([CA²⁺]_i). Lower extracellular CA²⁺ concentrations from 2.3 mM to 0 mM significantly suppressed the CNCP-induced CA²⁺ response by 69.1%. Moreover, the depletion of intracellular CA²⁺ stores using thapsigargin normalized CNCP-induced CA²⁺ release from intracellular CA²⁺ stores, suggesting that the CNCP-induced CA²⁺ response involved both extracellular CA²⁺ influx and CA²⁺ release from intracellular CA²⁺ stores. SAR7334, a TRPC3/6/7 channel inhibitor, but neither Pyr3, a selective TRPC3 channel inhibitor, nor Pico145, a TRPC1/4/5 inhibitor, suppressed the CNCP-induced CA²⁺ response by 57.2% and decreased CNCP-induced cell death by 53.4%, suggesting a critical role for TRPC6 channels in CNCP-induced CA²⁺ influx and apoptotic cell death. Further electrophysiological recording demonstrated that CNCP directly activated TRPC6 channels by increasing channel open probability with an EC₅₀ value of 6.01 μM. Considered together, these data demonstrate that the direct activation of TRPC6 channels contributes to CNCP-induced apoptotic cell death in HK-2 cells. Our data point out the potential risk of renal toxicity from CNCP if used as a therapeutic agent.