Two-pore channels (TPCs) are activated by phosphatidylinositol bisphosphate (PIP ₂ ) binding to domain I and/or by voltage sensing in domain II (DII). Little is known about how these two stimuli are integrated, and how each TPC subtype achieves its unique preference. Here, we show that distinct conformations of DII-S4 in the voltage-sensor domain determine the two gating modes. DII-S4 adopts an intermediate conformation, and forced stabilization in this conformation was found to result in a high PIP ₂ -dependence in primarily voltage-dependent TPC3. In TPC2, which is PIP ₂ -gated and nonvoltage-dependent, a stabilized intermediate conformation does not affect the PIP ₂ -gated currents. These results indicate that the intermediate state represents the PIP ₂ -gating mode, which is distinct from the voltage-gating mode in TPCs. We also found in TPC2 that the tricyclic antidepressant desipramine induces DII-S4-based voltage dependence and that naringenin, a flavonoid, biases the mode preference from PIP ₂ -gating to desipramine-induced voltage gating. Taken together, our study on TPCs revealed an unprecedented mode-switching mechanism involving conformational changes in DII-S4, and its active role in integrating voltage and PIP ₂ stimuli.