Conformational rearrangements in 2nd voltage sensor domain switch PIP2- and voltage-gating modes in two-pore channels

Two-pore channels (TPCs) are activated by 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 the distinct conformations of DII-S4 in the voltage-sensor domain determine the two gating modes. DII-S4 takes an intermediate conformation, and forced stabilization in this conformation was found to give or maintain a high PIP 2 -dependence in primarily voltage- dependent TPC3 or in PIP 2 -gated and non-voltage-dependent TPC2, respectively. We also found in TPC2 that a tricyclic antidepressant desipramine induces the DII-S4 based voltage-dependence and that a flavonoid naringenin 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. This will pave the way for drug development by targeting specific gating modes of TPCs. Significance statement Membrane voltage and PIP 2 are different types of signals on endosomal and lysosomal membranes. The two signals are integrated into two-pore channels (TPCs) whose two repeating domains, DI and DII, play roles in PIP 2 binding and voltage sensing, respectively. We showed that the conformation of the S4 helix in DII determines the voltage-dependent or PIP 2 -dependent gating mode, which explains the different preferences of the two signals between TPC subtypes. The preference for these two gating modes can be changed by a flavonoid, naringenin. Our findings on the molecular mechanism of the two gating modes in TPCs provide a clue to the understanding and pharmacological manipulation of the signaling by PIP 2 and voltage in intracellular organelles.