The Three Two-Pore Channel Subtypes from Rabbit Exhibit Distinct Sensitivity to Phosphoinositides, Voltage, and Extracytosolic pH

Two pore channels (TPCs) are implicated in vesicle trafficking, virus infection, and autophagy regulation. As Na(+)- or Ca(2+)-permeable channels, TPCs have been reported to be activated by NAADP, PI(3,5)P(2), and/or high voltage. However, a comparative study on the function and regulation of the three mammalian TPC subtypes is currently lacking. Here, we used the electrophysiological recording of enlarged endolysosome vacuoles, inside-out and outside-out membrane patches to examine the three TPCs of rabbit (Oryctolagus cuniculus, or Oc) heterologously expressed in HEK293 cells. While PI(3,5)P(2) evoked Na(+) currents with a potency order of OcTPC1 > OcTPC3 > OcTPC2, only OcTPC2 displayed a strict dependence on PI(3,5)P(2). Both OcTPC1 and OcTPC3 were activatable by PI3P and OcTPC3 was also activated by additional phosphoinositide species. While OcTPC2 was voltage-independent, OcTPC1 and OcTPC3 showed voltage dependence with OcTPC3 depending on high positive voltages. Finally, while OcTPC2 preferred a luminal pH of 4.6–6.0 in endolysosomes, OcTPC1 was strongly inhibited by extracytosolic pH 5.0 in both voltage-dependent and -independent manners, and OcTPC3 was inhibited by pH 6.0 but potentiated by pH 8.0. Thus, the three OcTPCs form phosphoinositide-activated Na(+) channels with different ligand selectivity, voltage dependence, and extracytosolic pH sensitivity, which likely are optimally tuned for function in specific endolysosomal populations.