Electronic Thesis and Dissertation Repository

Degree

Master of Science

Program

Physiology and Pharmacology

Supervisor

Dr. Peter B. Stathopulos

Abstract

A major intracellular calcium (Ca2+) uptake pathway in excitable and non-excitable eukaryotic cells is store-operated Ca2+ entry (SOCE). Stromal interaction molecule-1 (STIM1) is the key regulator of SOCE and responds to changes in endoplasmic reticulum (ER)-stored Ca2+ through luminal sensing machinery composed of EF-hand and SAM domains (EFSAM). EFSAM can undergo N-glycosylation at Asn131 and Asn171 sites; however, the exact molecular and functional effects of N-glycosylation are unclear. By establishing a site-specific chemical approach to covalently linking glucose to EFSAM and subsequently examining EFSAM biophysical properties, I found that this modification enhances STIM1 activation through localized structural perturbations, decreased Ca2+ binding affinity, reduced stability and enhanced oligomerization. Further, Ca2+ influx via SOCE in HEK293 cells co-expressing Orai1 and STIM1 was diminished when N-glycosylation was blocked. Collectively, my data suggests that N-glycosylation enhances EFSAM response to ER Ca2+ depletion thereby augmenting the role of STIM1 as the ON/OFF regulator of SOCE.


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