Phosphoinositide regulates dynamic movement of the S4 voltage sensor in the second repeat in two-pore channel 3

Kiichi Hirazawa,Michihiro Tateyama,Yoshihiro Kubo,Takushi Shimomura

doi:10.1016/j.jbc.2021.101425

Abstract

The two-pore channels (TPCs) are voltage-gated cation channels consisting of single polypeptides with two 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 fourth helix in the second repeat (second S4) plays a major role in detecting membrane voltage, whereas the first 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 remain unknown. Here, we found that PI binding to the first repeat in TPC3 regulates the movement of the distally located second 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, measurement of gating charges, and Cys-accessibility analysis, we observed that PI binding significantly potentiates the voltage dependence of the movement of the second S4 helix. Notably, voltage clamp fluorometry analysis revealed that the voltage-dependent movement of the second 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 second 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.

Highlights

Voltage-gated cation channels are membrane proteins, which permeate specific ions in response to the change of the membrane voltage [1]
It was reported that TPC1 and TPC2 are important for the maintenance of the ionic homeostasis of the intracellular organelle [8], autophagy [12, 13], nutrient sensing [14], and Ebola virus infection [15]
To investigate how the PI[3,4]P2 binding to the first repeat affects the second repeat, we examined the dynamic structural rearrangement of the second the fourth helix (S4), which governs the voltagedependent gating of TPC3, by cysteine residue (Cys)-accessibility analysis, voltage clamp fluorometry (VCF) and measurement of the gating charges

Summary

Introduction

Voltage-gated cation channels are membrane proteins, which permeate specific ions in response to the change of the membrane voltage [1]. The inter-repeat interaction site is located between the first S6 and the second S6 in the pore region of TPC3, involving hydrophilic and electrostatic interactions of Tyr293 (first S6), Arg297 (first S6), and Glu665 (second S6) Disruption of this interaction impairs the transmission of the signal of the PI[3,4] P2 binding, resulting in a largely weakened potentiation of the voltage dependence of TPC3 by PI[3,4]P2 [25]. This interrepeat transmission must affect the dynamic structural rearrangement of the second repeat. It was shown that the signal of the PI[3,4]P2 binding is transmitted to the distally located second S4, revealing tight coupling across the whole molecule of TPC3

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Conclusion