Lipid signalling modulates neuroendocrine cell excitability and cation channel function
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Authors
Sturgeon, Raymond
Date
Type
thesis
Language
eng
Keyword
Aplysia , Electrophysiology , Cation channel , Neuroendocrine cells , TRP Channels
Alternative Title
Abstract
Ion channel function and modulation determine the strength and duration of periods of enhanced neuronal activity and excitability that control and shape behaviour. The bag cell neurons of the hermaphroditic sea snail, Aplysia californica, provide a model to examine the regulation of ion channel function. Reproduction is initiated when bag cell neurons secrete egg-laying hormone during a protracted depolarization and afterdischarge. Early in the afterdischarge, phospholipase C (PLC) hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP2) into inositol trisphosphate (IP3) and diacylglycerol (DAG). In cultured bag cell neurons, activating PLC with N-(3-trifluoromethylphenyl)-2,4,6-trimethylbenzenesulfonamide (m-3M3FBS), or adding a DAG analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG), induced depolarization and spiking, mediated by a large, prolonged, Ca2+-permeable, inward current (IOAG) through a non-selective, voltage-independent cation channel that reversed near -20 mV and was enhanced by intracellular IP3. Furthermore, IOAG required basal PKC activity prior to the current, though OAG failed to trigger PKC. Molecular biology revealed a transient receptor potential (TRP) channel in bag cell neurons with secondary structure conserved in other TRPC5 channels. Designated ApTRPC5, when expressed in HEK cells it was a voltage-independent, non-selective cation current gated by PLC-coupled receptors or OAG. A separate source of depolarization for the afterdischarge is a voltage-gated, Ca2+-activated, non-selective cation channel. The effect of exogenous OAG and IP3, as well as PLC activation via m-3M3FBS, was investigated at the single channel level. OAG transiently elevated open probability (PO), and co-application of IP3 boosted and prolonged this response. Exposure to OAG and IP3 also left-shifted channel voltage-dependence; moreover, PLC activation using m-3M3FBS increased PO in excised patches, suggesting PLC may be physically localized to the channel. The Ca2+-activation of this channel desensitized during continued exposure to high Ca2+, which is reminiscent of PIP2-mediated desensitization seen in some TRPM channels. Accordingly, addition of a PIP2 analogue, di-C8-PIP2, ablated Ca2+ desensitization, while inhibiting the hydrolysis of PIP2 by PLC, with U73122, mirrored that effect. The phosphoinositol signalling cascade regulates multiple TRP-like channels during the afterdischarge, providing a means to facilitate prolonged depolarization and reproductive behaviour. Because TRP channels are modulated by phosphoinositols in numerous organisms, these results have implications for neuroendocrine function throughout the animal kingdom.
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ProQuest PhD and Master's Theses International Dissemination Agreement
Intellectual Property Guidelines at Queen's University
Copying and Preserving Your Thesis
This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.