Date of Award

7-15-2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Brij B. Singh

Abstract

Transient receptor potential canonical 3 (TRPC3), a non-selective Ca 2+-permeable cation channel, is a member of the TRP superfamily of ion channels. The mode of activation of TRPC3 is governed by a cascade of Phospholipase C (PLC) mediated signaling events, following G-protein coupled receptor (GPCR) or tyrosine kinase receptor activation, resulting in endoplasmic reticulum (ER) Ca2+ store depletion. This very store depletion triggers the activation of plasma membrane TRPC3 and hence they are termed as receptor operated or store operated cation channels. In addition to the receptor mediated activation of TRPC3, an alternate mode of channel activation has been described, which is store-depletion independent and is mediated by the lipid messenger DAG (diacylglycerol). This study describes a critical role of TRPC3 cation channel in the regulation of neuronal function. Using OAG (a DAG analogue), to specifically activate TRPC3 we studied the physiological importance of the channel in neurosecretion. For this study we employed PC12 cells in culture system and the vasopressin and oxytocin secreting supraoptic nucleus (SON) of rat brain as the in vivo system, both of which are well established models for studying neuro-endocrine cell function. Previous research has demonstrated that TRPC3 is highly expressed in the brain and is also associated with SNARE complex proteins like synaptobrevinNAMP2 (Vesicle associate membrane protein 2). However, the biological importance of such interaction has not been deciphered. Towards this end, we studied the expression of TRPC3 and the consequence of its activation in the above mentioned model systems. Using biochemical and cell biological tools we have identified vesicular association of TRPC3, a previously unrecognized finding. Following OAG mediated activation there was enhanced interaction of TRPC3 with the core proteins of the vesicle fusion machinery - VAMP2, SNAP25, and syntaxin1A. These interactions were dependent on Ca2+ influx, as exclusion of extracellular Ca 2+ or inhibition of TRPC3 channel function significantly impeded the activation dependent association of SNARE complex proteins with TRPC3. Vesicular expression and its association with SNARE proteins provided a strong plausibility for TRPC3 to influence neuronal function. As Ca 2+ influx is one of the prerequisites during the final steps of vesicle fusion, we reasoned that activation of TRPC3 would regulate neurosecretion. Indeed, OAG mediated activation of TRPC3 resulted in release of human growth hormone from cultured PC12 cells as well as the in vivo release of vasopressin from rat supraoptic neurons. Pharmacological inhibition or genetic manipulation of TRPC3 further substantiated its role in OAG evoked neurosecretion. Thus, this study identifies an as of yet uncovered modality of exocytosis wherein vesicular expression of TRPC3 and its activation-dependant physical association with the proteins of vesicle fusion machinery regulate cells' secretory events in concert.

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