Electrophysiological Characterization of the Two-Pore Channel 2

Modulating of cytoplasmic calcium (Ca2+) concentration through the activation of intracellular Ca2+ release channels elicits signal that control numerous cellular processes. The characterization of the regulation of these intracellular Ca2+ release channels is therefore the elementary to understand the generation and modulation of intracellular Ca2+ signaling events. In human, three Ca2+ mobilizing messengers have been identified. The inositol trisphosphate (InsP3) and cyclic ADP-ribose (cADPR) have been demonstrated to mobilize Ca2+ from the endoplasmic reticulum (ER) by activating the InsP3 receptor (InsP3R) and ryanodine receptor (RyR), respectively, whereas the nicotinic acid adenine dinucleotide phosphate (NAADP) mobilizes Ca2+ from the endosome and lysosome. However, the receptor responsible for NAADP activation was mystery until recently that several research groups have proposed a novel lysosomal cation channel, two-pore channel 2 (TPC2), is the putative candidate for NAADP. Although the intracellular localization of TPC2 hindered the electrophysiological study by conventional patch-clamp technique, the biophysical properties of TPC2 have been investigated by incorporating the purified protein into planar lipid bilayer or by targeting the channel proteins to plasma membrane. However, these techniques require tedious experimental procedures or genetic modification and therefore the channel properties recorded by these techniques might not totally reflect their physiological properties as they are in the native environment. Furthermore, recent articles challenged that the activation of TPC2 channel is mediated by phosphoinositide urging the development of novel methodology for characterizing the TPC2 channel. In this regard, we employed human TPC2 retrovirus to transduce a cell line that lacking both endogenous InsP3R and RyR (DT40TKO). In combination with the nuclear membrane electrophysiology, we developed a cell model that enabled us to rigorously investigate the biophysical properties of the TPC2 channel with minimal manipulations. Using this newly developed approach, we successfully detected NAADP-activated TPC2 single channel currents in the isolated nuclei from TPC2 virus transduced DT40TKO cells.

In this study, we aim to address the most elemental question that whether the TPC2 is a NAADP-mediated Ca2+ channel. If so, the conductive properties and the ligands regulations of the TPC2 channels will also be investigated. Furthermore, we will investigate the effects of phosphorylation on TPC2 channel activities as we have identified putative PKA and Akt phosphorylation sites in the human TPC2 protein. Elucidation of the biophysical properties of the TPC2 channels and its regulation by phosphorylation will provide information for the physiological regulations of the lysosomal Ca2+ release and its role in lysosome functions.