Thesis (Ph.D. in Human Medical Genetics) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 140-146).

The epithelial sodium channel (ENaC) is a key regulator of salt homeostasis. The classic ENaC consists of three subunits: α, β and γ, which are highly expressed in the kidney and colon where they mediate electrogenic Na⁺ influx into cells under the tight hormonal regulation of aldosterone. A fourth ENaC subunit named [delta]ENaC also generates Na⁺ influx with the β- and γENaC subunits in Xenopus oocytes. However [delta]ENaC differs to the other subunits in its channel properties and tissue distribution, suggesting that [delta]ENaC may possess a physiological role other than salt regulation. A copper-toxicosis related protein called COMMD1/Murr1 was previously identified to directly interact with [delta]ENaC and downregulate [delta]ENaC activity. COMMD1 is linked with multiple ubiquitination pathways, therefore we hypothesised that COMMD1 directly interacts with [delta]ENaC through novel protein-protein interaction motifs and promotes internalisation of [delta]ENaC from the cell surface through enhanced ubiquitination. With the use of GST pulldown assays and coimmunoprecipitation, it was found that the binding of COMMD1 to [delta]ENaC is mediated by the COMM domain of COMMD1, primarily through amino acids 120-150 of COMMD1. Immunocytochemical studies showed that the intracellular interaction between [delta]ENaC and COMMD1 predominantly occurred in the early and recycling endosomes, suggesting that COMMD1 may promote the retrieval of [delta]ENaC from the cell surface to the intracellular pool. COMMD1 mediated a decrease in the [delta]ENaC cell surface population, as shown by a biotinylation surface labelling assay. This may be driven by an ubiquitin-regulated endocytosis, as COMMD1 increased ubiquitination, but not proteasomal/lysosomal degradation, of [delta]ENaC. COMMD1 may promote [delta]ENaC ubiquitination through the action of the ubiquitin ligase Nedd4-2 as coexpression with Nedd4-2 enhanced the COMMD1-mediated decrease in surface [delta]ENaC expression. This is abolished by the addition of the Nedd4-2 downregulator kinase sgk1, suggesting that COMMD1 may downregulate [delta]ENaC through the Nedd4-2/sgk1 pathway. Surface levels of [delta]ENaC may also be affected by XIAP, a RING domain ubiquitin ligase which is able to decrease the levels of COMMD1. Coimmunoprecipitation of endogenous [delta]ENaC and COMMD1 proteins, and the enhanced colocalisation of endogenous [delta]ENaC in the recycling endosomes with transfected COMMD1, indicate that interaction between transfected [delta]ENaC and COMMD1 reflect the intracellular interactions of the endogenous proteins. Taken together, these findings suggest that COMMD1 downregulates [delta]ENaC activity by promoting the internalisation of surface [delta]ENaC into early and recycling endosomes and this may be mediated by enhanced [delta]ENaC ubiquitination via the ubiquitin ligase Nedd4-2.

Thesis (M.S.)--University of Delaware, 2007. / Principal faculty advisor: Robert A. Sikes, Dept. of Biological Sciences. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 128-138). Available also in a digital version from Dissertation Abstracts.

published_or_final_version / Medicine / Master / Master of Research in Medicine

No description available.

Thesis (Ph.D.)--University of Delaware, 2008. / Principal faculty advisor: Robert A. Sikes, Dept. of Biological Sciences. Includes bibliographical references.

Thesis (Ph. D.)--University of Virginia, 1999. / Includes bibliographical references (p. 104-113). Also available online through Digital Dissertations.

Thesis (M.Res.(Med.))--University of Hong Kong, 2003. / Includes bibliographical references (leaves 76-92) Also available in print.

The nodes of Ranvier are short, periodical interruptions in the myelin sheath of myelinated axons, at which voltage-gated sodium channels are highly concentrated. The correct targeting of sodium channels to the nodes of Ranvier permits rapid propagation of action potentials in myelinated axons. The nodes of Ranvier contain a unique set of ion channels, cell-adhesion molecules, and cytoplasmic adaptor proteins. Neurofascins are cell adhesion molecules of the immunoglobulin superfamily and previous work has shown they are involved in the assembly of the node of Ranvier. The Neurofascin (Nfasc) gene is subject to extensive alternative splicing. RT-PCR studies have suggested that there were several different Neurofascin (Nfasc) transcripts. Thus far, research on the Neurofascins has concentrated on two isoforms, Nfasc186 and Nfasc155, which are expressed in neurons and glia respectively. A third Neurofascin isoform, Nfasc140, lacking the Mucin domain and two of the fibronectin repeats was originally identified in the laboratory of V. Bennett. However, neither the location nor function of this protein was known. By RT-PCR I successfully cloned the Nfasc140 cDNA and determined its domain composition, which was confirmed by a series of Western blots using domain-specific antibodies. The developmental expression of Nfasc140 revealed that it is the predominate isoform of Neurofascin during the embryonic stage. Using cell-type-specific conditional Neurofascin knock-out mice, I have also found that Nfasc140 is a neuronal isoform, like Nfasc186. I have used transgenic mouse lines to characterize the location and function of Nfasc140. Like Nfasc186, Nfasc140 is targeted to the nodes of Ranvier and axonal initial segment. Also Nfasc140 alone can reconstitute the nodal complex in Neurofascin knock-out mice in CNS and PNS in the absence of Nfasc186 and Nfasc155. It can also partially restore the electrophysiological function of PNS nerves. In order to address the role of the paranodes in sodium channel clustering, I generated a new neuronal-Cre-expressing transgenic line which, when bred with floxed Nfasc mice, generated early neuronal Neurofascin knock-out mice. Using those animals I have shown that after the ablation of all neuronal Neurofascins, when only glial Nfasc155 is presented, sodium channels can still target to the nodes of Ranvier in both PNS and CNS. These conditional knock-out mice have a longer life span than pan-Neurofascin knock-out mice. This indicates the importance of paranodal junctions, in addition to nodal neuronal Neurofascins, in clustering sodium channels at the node.