Characterization of mouse two-pore channels (TPCs) in NAADP-mediated Ca(2+) signalling

Thesis

Recent studies have identified Two-Pore Channels (TPCs) as the channels activated by NAADP. To date, most studies that characterized these channels have employed heterologous expression or overexpression systems. The research reported here has focused principally on endogenous TPC activity by using single and dual gene knockout (KO) in a mouse system and has yielded insights into TPC expression levels, subcellular localisation, NAADP binding, and channel function.

Mouse models that had been generated by both the “gene-trapping” and the “genetargeting” techniques were obtained and validated. These included a knock-down strain (“hypomorph”). Surprisingly, all TPC mutant mice showed no gross phenotypes. In addition to the two known isoforms in mouse, TPC1 and TPC2, the expression of a shorter variant of TPC1 was discovered; this has an alternative (truncated) N-terminus, and has been termed (DELTA)N-TPC1. All TPC variants/isoforms were widely expressed in all mouse tissue types tested. Overexpression of mouse TPCs in mouse embryonic fibroblasts showed that (DELTA)N-TPC1 and TPC2 were expressed primarily in late endosomes/lysosomes while TPC1 was expressed in both endosomes and lysosomes. Dileucine sorting motifs target TPCs to late endosomes/lysosomes; it was shown that truncation or mutation of dileucine motifs significantly reduced localization in late endosomes/lysosomes. Furthermore, TPCs were shown not to be the direct binding target of NAADP, as the high affinity NAADP binding was retained in hepatic membranes from TPC double KO (DKO) mice. It is concluded that NAADP binds to an (as yet, unidentified) accessory protein.

The functional role of TPCs was studied in depth using mouse pancreatic acinar cells. NAADP is known to release Ca²⁺ from the acidic stores in response to the stimulation by the hormone cholecystokinin (CCK). In all TPC mutant mice, CCK was still able to evoke Ca²⁺ oscillations, but with slower and attenuated oscillations in the TPC1 hypomorph, and with slower oscillations in TPC DKO. In all TPC KOs, oscillations were disrupted by known inhibitors of the NAADP-signalling pathway (Ned-19, GPN and bafilomycin A1), indistinguishable from the responses with wild-type cells. This suggests that TPCs are not involved in CCK signalling, although it is possible that functional compensation masked the phenotype arising from the impaired signalling.

Authors: Chuang, KT

• Publication date: 2011
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: Ca(2+); NAADP; Two-Pore Channels; calcium signalling; TPC
• Subjects: Biology (medical sciences); Pharmacology