The two-pore channels (TPCs) are voltage-gated cation channels consisting of single polypeptides with 2 repeats of a canonical 6-transmembrane unit. TPCs are known to be regulated by various physiological signals such as membrane voltage and phosphoinositide (PI). The 4^th helix in the 2^nd repeat (2^nd S4) plays a major role in detecting membrane voltage, while the 1^st repeat contains a PI binding site. Therefore, each of these stimuli is detected by a unique repeat to regulate the gating of the TPC central pore. How these various stimuli regulate the dynamic structural rearrangement of the TPC molecule remains unknown.

　Here, we found that PI binding to the 1^st repeat in TPC3 regulates the movement of the distally located 2^nd S4 helix, showing that the PI binding signal is not confined to the pore gate but also transmitted to the voltage sensor. Using voltage clamp fluorometry (VCF), measurement of gating charges, and Cys-accessibility analysis, we observed that PI binding significantly potentiates the voltage dependence of the movement of the 2^nd S4 helix. Notably, VCF analysis revealed that the voltage-dependent movement of the 2^nd S4 helix occurred in two phases, of which the second phase corresponds to the transfer of the gating charges. This movement was observed in the voltage range where gate-opening occurs, and was potentiated by PI. In conclusion, this regulation of the 2^nd S4 helix by PI indicates a tight inter-repeat coupling within TPC3, a feature which might be conserved among TPC family members to integrate various physiological signals.

Funding

KAKENHI