The role of two pore channels (TPCs) in pancreatic beta cell stimulus-secretion coupling

Thesis

This thesis presents an investigation into the role of the recently identified two pore channels (TPCs) in beta-cell stimulus-secretion coupling. TPCs are the receptors for calcium mobilising messenger nicotinic acid adenine dinucleotide phosphate (NAADP) located in the membrane of acidic intracellular calcium stores. It is proposed that they are responsible for the ATP-sensitive potassium channel (KATP channel) independent pathway of stimulus-secretion coupling; and that this pathway is not subordinate to the KATP channel dependent pathway; but an alternative explanation of stimulus-secretion coupling in its own right.

The first section of this thesis presents a characterisation of sub-membrane calcium signals observed in primary mouse beta-cells in response to glucose and the membrane-permeable acetoxymethyl ester form of NAADP (NAADP-AM) using the non-ratiometric fluorescent calcium indicator fluo-4 and total internal reflection (TIRF) microscopy. These are compared to global cytosolic calcium changes observed with epifluorescence microscopy. Factors affecting the shape and time course of responses are investigated, and pharmacological tools used to provide evidence for the role of intracellular calcium release from acidic stores mediated by NAADP.

Having characterised the calcium responses of beta-cells using TIRF; the second part of the thesis examines the effects of knocking out TPC2 (single KO), or both TPC1 and TPC2 (DKO) on these responses; after an initial assessment of pancreatic islet and beta-cell morphology using electron microscopy. Gender differences in beta-cell responses to glucose and NAADP are assessed in both wild type and knockout animals.

Finally, the third section presents the discovery of elementary calcium release events in pancreatic beta-cells. The current project visualises what are likely the triggering events for the global calcium signals examined in sections one and two. They take the form of localised calcium release in response to NAADP-AM and glucose; akin to sparks and puffs observed by stimulation with cADPR and IP3. Optical quantal analysis demonstrates the quantal nature of the events and estimates the size of the unitary calcium release unit (CRU) for NAADP.

Authors: Heister, P

• Publication date: 2012
• DOI: 10.5287/ora-j0zzzjgwx
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: Calcium Signalling; Stimulus-Secretion Coupling; NAADP; Pancreatic Beta Cells; TPC; Two Pore Channels
• Subjects: Physiology; Diabetes; Pharmacology; Biology (medical sciences)