Agonist-specific voltage-dependent gating of lysosomal two-pore Na+ channels

Abstract

Mammalian two-pore-channels (TPC1, 2; TPCN1, TPCN2) are ubiquitously- expressed, PI(3,5)P2-activated, Na+-selective channels in the endosomes and lysosomes that regulate luminal pH homeostasis, membrane trafficking, and Ebola viral infection. Whereas the channel activity of TPC1 is strongly dependent on membrane voltage, TPC2 lacks such voltage dependence despite the presence of the presumed ‘S4 voltage-sensing’ domains. By performing high-throughput screening followed by lysosomal electrophysiology, here we identified a class of tricyclic anti-depressants (TCAs) as small-molecule agonists of TPC channels. TCAs activate both TPC1 and TPC2 in a voltage-dependent manner, referred to as Lysosomal Na+ channel Voltage-dependent Activators (LyNa-VAs). We also identified another compound which, like PI(3,5)P2, activates TPC2 independent of voltage, suggesting the existence of agonist-specific gating mechanisms. Our identification of small-molecule TPC agonists should facilitate the studies of the cell biological roles of TPCs and can also readily explain the reported effects of TCAs in the modulation of autophagy and lysosomal functions.

Targeting Two-Pore Channels: Current Progress and Future Challenges

“…Amitriptyline clomipramine, desipramine, imipramine, and nortriptyline were the five LyNa-VA1.x, and were categorised as tricyclic antidepressants (TCAs). Chlorpromazine and triflupromazine were the two LyNa-VA2.x identified, and were also recognised as tricyclic antipsychotics [ 71 ]. All seven identified molecules could act as agonist voltage-dependent gating modulators for both TPC1 and TPC2 [ 71 ].…”

“…Chlorpromazine and triflupromazine were the two LyNa-VA2.x identified, and were also recognised as tricyclic antipsychotics [ 71 ]. All seven identified molecules could act as agonist voltage-dependent gating modulators for both TPC1 and TPC2 [ 71 ]. Conversely, the study observed no significant activation of TPCs with carbamazepine (an anticonvulsant) and phenothiazine (an antipsychotic), which share a similar chemical structure with TCAs (the tricyclic ring).…”

“…Conversely, the study observed no significant activation of TPCs with carbamazepine (an anticonvulsant) and phenothiazine (an antipsychotic), which share a similar chemical structure with TCAs (the tricyclic ring). This distinct function could be attributed to the chemical structures of these compounds lacking the aliphatic chain, which might be responsible for TPC activation [ 71 ]. In addition, the study showed that riluzole, a drug approved by the US FDA in 1995 for the treatment of amyotrophic lateral sclerosis (ALS) [ 72 ] and classified as a glutamate receptor blocker, can act as a specific voltage-independent TPC2 agonist [ 71 , 72 ].…”

Discovery of lipophilic two‐pore channel agonists

“…None of these compounds was found to activate TRPML1. Clomipramine (38) and desipramine (39) also activate TPC1 in a voltage-dependent manner, while chlorpromazine (43) inhibits TPC1 [14]. Hence, additional analysis of structure-activity relationships is needed, as slight changes in structures seemingly can reverse the activity on two-pore channel isoforms.…”

Evolutionary Aspects of TRPMLs and TPCs

“…It has also been postulated that mammalian TPCs are not Ca 2+ release channels activated by NAADP, but rather Na + release channels activated by PI(3,5)P 2 [23,31]. The discovery of novel lipophilic small molecule agonists for TPCs in two independent high-throughput calcium imaging campaigns [127,128] partially reconciled this debate. Both campaigns revealed that TPC agonists can be identified in calcium imaging experiments, suggesting that TPCs are indeed permeable to Ca 2+ .…”