Membrane potential regulates Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) dependence of the pH- and Ca²⁺-sensitive organellar two-pore channel TPC1

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent second messenger that mobilizes Ca²⁺ from the acidic endolysosomes by activation of the two-pore channels TPC1 and TPC2. The channel properties of human TPC1 have not been studied before, and its cellular function is not known. In the present study, we characterized TPC1 incorporated into lipid bilayers. The native and recombinant TPC1 channels are activated by NAADP. TPC1 activity requires acidic luminal pH and high luminal Ca²⁺. With Ba ²⁺ as the permeable ion, luminal Ca²⁺ activates TPC1 with an apparent K_m of 180 μM. TPC1 operates in two tightly coupled conductance states of 47 ± 8 and 200 ± 9 picosiemens. Importantly, opening of the large conductance markedly increases the small conductance mean open time. Changes in membrane potential from 0 to -60 mV increased linearly both the small and the large conductances and NP_o, indicating that TPC1 is regulated by voltage. Intriguingly, the apparent affinity for activation of TPC1 by its ligand NAADPis not constant. Rather, hyperpolarization increases the apparent affinity of TPC1 for NAADP by 10 nM/mV. The concerted regulation of TPC1 activity by luminal Ca²⁺ and by membrane potential thus provides a potential mechanism to explain NAADP-induced Ca²⁺ oscillations. These findings reveal unique properties of TPC1 to explain its role in Ca²⁺ oscillations and cell function.