Date of Award

7-15-2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

First Advisor

Brij B. Singh

Abstract

Caveolae are cholesterol-rich plasma-membrane (PM) microdomains which foster wide-range of signal-transduction events including Ca2+-influx. Caveolae contain Caveolin1 (Cav1) as their major structural-protein. Transient receptor potential canonical 1 (TRPC1) channels constitute an integral-component of store-operated Ca2+ channels (SOCC) which initiate store-operated Ca2+ entry (SOCE), in response to endoplasmic reticulum (ER) Ca2+-store depletion. Recently, Stromal interaction molecule 1 (STIM1) was identified as the ER Ca2+-sensor which, following store-depletion, forms clusters and translocate to peripheral-ER at sub-plasma membrane region and communicates with PM-SOCC activating SOCE. This study identifies a significant role of Cav1 and STIM1 in regulating TRPC1 function. In salivary epithelial cells, TRPC1 interacted with Cav1 and partitioned into caveolar raft fractions as opposed to its largely non-raft association in Cav1 knockout tissues. Silencing of Cav1-expression, in cultured cells, severely reduced the PM expression of TRPC1 whereas over-expression of Cav1 increased the PM-TRPC1, suggesting that the PM expression of TRPC1 is dependent on Cav1. Contrary to an amplification of SOCE, due to increased PM-TRPC1 in Cav1 over-expressing cells, there was a significant reduction in SOCE. Thus, it was reasoned that, Cav1 scaffolds PM-TRPC1 primarily in an inactive-state and the dissociation of TRPC1-Cav1 complex would be obligatory for TRPC1 activation. A dynamic interaction between TRPC1-STIM1, occurring preferentially at membrane raft/caveolar microdomains was also observed. The integrity of these domains was a determining factor for TRPC1-STIM1 interactions and SOCE function. Following store-depletion, a unique molecular re-arrangement in the 'TRPC1-Cav1-STIM1' protein complex was found. Store-depletion resulted in an increased association of 'TRPC1-STIM1' with a concomitant decrease in 'TRPC1-Cav1' association, which was STIM1 dependant, resulting in SOCE activation. This study puts forward a novel regulatory-paradigm for cellular Ca 2+-signal transduction. Thus, it is proposed that, the association of TRPC1-Cav1 is required for targeting TRPC1 to specific PM-compartments, however, following store-depletion, the STIM1-mediated dissociation of the pre-assembled, caveolae-associated 'TRPC1-Cav1' complex is imperative for SOCE activation. Identification of such an elaborate molecular-choreography involved in the activation of TRPC1-mediated SOCE Will be invaluable to understand and fine-tune Ca2+-responsive signaling processes such as gene regulation, cell proliferation, cell death and hormone and fluid secretion in normal and pathological context.

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