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Date of Award


Document Type

Dissertation - Pacific Access Restricted

Degree Name

Doctor of Philosophy (Ph.D.)


Pharmaceutical and Chemical Sciences

First Advisor

David Thomas

First Committee Member

Roshanak Rahimian

Second Committee Member

Timothy Smith

Third Committee Member

Miki Park

Fourth Committee Member

Goeff Lin Cereghino


Generation of Ca 2+ signals in cells involves regulation by multiple components controlling Ca 2+ release from the internal stores, Ca 2+ influx across the plasma membrane (PM), elicitation of Ca 2+ sensitive processes and finally the removal of Ca 2+ from the cell. One such mode of facilitating Ca 2+ entry is called store-operated Ca 2+ entry (SOCE) mediated by the store operated Ca 2+ channels (SOCs). SOCE, wherein the depletion of internal Ca 2+ stores triggers the influx of Ca 2+ across the PM, not only plays a vital role in refilling the Ca 2+ stores, but also regulates a multitude of downstream Ca 2+ regulated signalling events. Despite recent advances in elucidating the entry pathway, its molecular identity, biophysical properties and store-depletion signal remains undefined. The most potent inducer of SOCE, thapsigargin (TG), fails to induce Ca 2+ influx in the NG115-401L (401L) cells. This unusual phenotype of the cell makes it a useful model to study the mechanisms and components underlying the SOCE pathway. Although TG failed to induce SOCE in the 401L cells, we report that the activation of intracellular release channels such as the inositol-1,4,5-trisphosphate (fP3Rs) and ryanodine receptors (RyRs) were able to activate Ca 2+ influx upon store depletion. This is in keeping with mechanisms proposed to explain SOCE, namely the conformational coupling hypothesis, wherein depletion of the ER stores signals the release channels to physically interact with the PM SOCs. We found that disrupting the communication between the ER and the PM channels induced by actin disassembly affected both Ca 2+ release and influx. Our study shows that Ca 2+ release and influx is dependent on cortical actin organization and that the RyR mediated release is less regulated by cortical actin than the IP3R induced Ca 2+ release. Studies conducted using 2-aminoethoxy diphenylborate (2-APB), a commonly used SOC blocker, revealed that 2-APB stimulated Ca 2+ release in the 401L cells. This release of Ca 2+ was also found to be dependent on the conformational coupling between the ER and PM SOCs. We also studied the effect of overexpressing various isoforms of the transient receptor potential (TRP) channels. We found that protein kinase C (PKC) differentially regulated the activity of the TRP channels in the 401L cells. PKC activation prolonged the Ca 2+ influx in the wild type cells while attenuating the same in the TRP transfected cells. We also found that influx of surrogate cations (Ba 2+ ) is augmented in the TRPC transfected cells. Our studies reveal that the activation of ER release channels followed by conformational coupling with the PM channels may be a mechanism by which the 401L cells or neurons in general maintain a rigid control over intracellular Ca 2+ concentrations and thus regulate Ca 2+ homeostasis.



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