Defense Date

11-1-2019

Graduation Date

Fall 12-20-2019

Availability

Immediate Access

Submission Type

dissertation

Degree Name

PhD

Department

Chemistry and Biochemistry

Committee Chair

Michael Cascio

Committee Member

Stephanie Wetzel

Committee Member

Skip Kingston

Committee Member

Jana Patton-Vogt

Abstract

The glycine receptor (GlyR) belongs to a superfamily of pentameric ligand-gated ion channels (pLGICs) that mediate fast neurotransmission. GlyR typically modulates inhibitory transmission by antagonizing membrane depolarization through anion influx. Allosteric interactions between the receptor and its lipid surroundings affect receptor function, and cholesterol is essential for pLGIC activity. Human α1 GlyR was purified from baculovirus infected insect cells and reconstituted in unilamellar vesicles at cholesterol: lipid ratios below and above the cholesterol activity threshold with aliquots of azi-cholesterol. State-dependent crosslinking studies of receptors primarily in its resting (no glycine), desensitized (10mM glycine) and open (F207A/A288G, 30nM ivermectin) states were then performed at elevated cholesterol levels necessary for activity. After photoactivation, covalently crosslinked cholesterol-GlyR were trypsinized, mass fingerprinted by tandem mass spectrometry (MS-MS), and sites of cholesterol crosslinks in peptides were refined by targeted MS-MS. Within the GlyR apo state, cholesterol interactions differed as a function of membrane cholesterol concentration correlating to the chemical activity of cholesterol, suggesting two distinct conformations. Differential cholesterol crosslinking patterns between resting, desensitized, and open states were observed, highlighting state-dependent differences in GlyR lipid accessibility. Distinct state-dependent crosslinking patterns indicative of alterations in either the lipid environment and/or channel structure were observed throughout GlyR, most prominently observed in the M4 transmembrane helix, extracellular domain loops and regions nearing the bilayer interface, and the large intracellular M3-M4 loop. The changes in M4 accessibility (transition from surface-mapped crosslinking to regions of the helix less exposed when mapped) suggest an outward twisting motion and translocation towards the bilayer/lipids as GlyR allosterically transitions. Strikingly, crosslinking patterns within the M3-M4 loop offer insight into the generalized structure of this unresolved region in all current pLGIC structural models, by suggesting the crosslinked regions of this intracellular loop are intimately associated or buried within the lipid bilayer. Taken together, crosslinking coupled with MS-MS has the capability to accurately probe and define physiological protein frameworks which can aid in the refinement of allosteric modulation and current structural models.

Language

English

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