Year: 2002
Type: dissertation
Abstract: Endocytosis is a mechanism used by all eukaryotic cells as a way to replenish and regulate the expression of cell-surface constituents. As the maintenance of distinct voltage-dependent ion channel distributions governs overall nerve excitability, differential rates of ion channel endocytosis would be a powerful mechanism for shaping neuronal electrophysiology. However, nothing is known about voltage-dependent ion channel internalization, or its role in determining channel membrane density. The underlying goal was to investigate voltage-dependent potassium channel (VDPC) internalization, using a model system featuring Kv1.4. The central hypothesis was: Voltage-dependent ion channels contain endocytic signals whose modulation can regulate their stabilization at the cell surface. The sub-hypotheses were: (1) Ion channel endocytosis signals exist as entities intrinsic to the channel proteins, (2) these signals are unique, removable, and exchangeable, (3) channel clustering by membrane-associated guanylate kinases, such as PSD-95, leads to regional stabilization of surface proteins by internalization suppression, and (4) Kv? subunits of VDPCs modulate channel internalization rates. An anti-kv1.4 polyclonal antibody was developed and characterized, to detect transfected channels in HEK293 cells, confirmed by 125I-dendrotoxin binding and electrophysiology. A cell-surface biotinylation assay was devised to measure channel internalization, confirmed by assessing rates of HEK293 membrane protein internalization, and that of Kv1.4. The suppression of Kv1.4 internalization by PSD-95 was governed by the ability of PSD-95 to self associate and duster Kv1.4 channels directly. Kv1.4 internalization was enhanced by its interaction with Kv?2, but not KvP1.2. Both Kvp subunits were able to surmount the ability of PSD-95 to suppress Kv1.4 internalization, with an overall trend between the amount of Kv?2 expressed and degree of hierarchical control. The identification of the Kv1.4 internalization signal was pursued by comparing the rates of Kv1.4 internalization to those of Kv1.2 and Kv1.3, and a Kv1.4/Kv4.2 carboxy-terminal (C-term) chimera, implicating the Kv1.4 C-term. A battery of large-scale deletion mutants mapped a novel ubiquitination internalization signal to the Kv1.4 C-term. These data provide novel evidence suggesting that the differential cell surface residency of ion channels may, in part, be conferred by a highly regulated mechanism that differentially controls their rates of internalization.
Source: TSpace (University of Toronto)
Author: Jugloff, Denis George Mano
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7133003590