Transient receptor potential mucolipin 1 (TRPML1) and two-pore channels are functionally independent organellar ion channels

NAADP is a potent second messenger that mobilizes Ca²⁺ from acidic organelles such as endosomes and lysosomes. The molecular basis for Ca²⁺ release by NAADP, however, is uncertain. TRP mucolipins (TRPMLs) and two-pore channels (TPCs) are Ca²⁺-permeable ion channels present within the endolysosomal system. Both have been proposed as targets for NAADP. In the present study, we probed possible physical and functional association of these ion channels. Exogenously expressed TRPML1 showed near complete colocalization with TPC2 and partial colocalization with TPC1. TRPML3 overlap with TPC2 was more modest. TRPML1 and to some extent TRPML3co-immunoprecipitated with TPC2 but less so with TPC1. Current recording, however, showed that TPC1 and TPC2 did not affect the activity of wild-type TRPML1 or constitutively active TRPML1(V432P). N-terminally truncated TPC2 (TPC2delN), which is targeted to the plasma membrane, also failed to affect TRPML1 and TRPML1(V432P)channel function orTRPML1(V432P)-mediated Ca²⁺ influx. Whereas overexpression of TPCs enhanced NAADP-mediated Ca²⁺ signals, overexpression of TRPML1 did not, and the dominant negative TRPML1 (D471K) was without affect on endogenous NAADP-mediated Ca²⁺ signals. Furthermore, the single channel properties of NAADP-activated TPC2delN were not affected by TRPML1. Finally, NAADP-evoked Ca²⁺ oscillations in pancreatic acinar cells were identical in wild-typeand TRPML1^-/- cells. We conclude that although TRPML1 and TPC sare present in thesamecomplex, they function astwoindependent organellar ion channels and that TPCs, not TRPMLs, are the targets for NAADP.