Student Theses and Dissertations

Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Simon Laboratory


Excitability of nerve and muscle cells depends on the number and the types of ion channels expressed at the plasma membrane. This work examines aspects of biogenesis and traffic of the Shaker voltage-gated potassium channel. Shaker is an oligomeric, polytopic membrane protein and, as such, its biogenesis begins at the endoplasmic reticulum (ER). I have studied (i) targeting of Shaker to the ER and stable integration into the lipid bilayer, (ii) N-linked glycosylation, assembly and folding Shaker in the ER, and (iii) export of Shaker from the ER and subsequent traffic to the surface. Targeting to the endoplasmic reticulum, integration into the lipid bilayer and assembly into tetramers occurs efficiently for Shaker translated in vitro. The first transmembrane domain (TM1) is most likely the earliest ER targeting signal on the growing Shaker polypeptide. TM1 that has adequately emerged from the ribosome is sufficient to initiate targeting to the ER in the absence of additional transmembrane domains. Further, efficient integration of Shaker into the bilayer is promoted by a glycoprotein fraction of ER microsomes, in which the active component was the translocon associated membrane protein (TRAM). Shaker is N-glycosylated on two consensus sites in the first extracellular loop. The importance of glycosylation at this location for Shaker biogenesis has not been previously studied. Elimination of the two consensus sites for N-linked glycosylation yields a channel that targets to the ER, integrates and tetramerizes normally, but is transported at a reduced rate to the surface the cell. This is due at least in part to a retardation of the unglycosylated channel early (i.e. pre-Golgi) step in its secretory traffic. Lastly, we attempted to develop assays to determine the efficiency at which the Shaker channel acquires its final, folded, conduction-competent state in the endoplasmic reticulum.


A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

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