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 Oc TPC1 > Oc TPC3 > Oc TPC2, only Oc TPC2 displayed a strict dependence on PI(3,5)P 2 . Both Oc TPC1 and Oc TPC3 were activatable by PI3P and Oc TPC3 was also activated by additional phosphoinositide species. While Oc TPC2 was voltage-independent, Oc TPC1 and Oc TPC3 showed voltage dependence with Oc TPC3 depending on high positive voltages. Finally, while Oc TPC2 preferred a luminal pH of 4.6-6.0 in endolysosomes, Oc TPC1 was strongly inhibited by extracytosolic pH 5.0 in both voltage-dependent and -independent manners, and Oc TPC3 was inhibited by pH 6.0 but potentiated by pH 8.0. Thus, the three Oc TPCs 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.