Type
Text
Type
Dissertation
Advisor
White, Thomas W, Brink, Peter R | Mathias, Richard | Musil, Linda.
Date
2013-05-01
Keywords
Connexin, Deafness, Gap Junction, Hemichannel, Ichthyosis, Keratitis | Physiology--Biophysics--Biology
Department
Department of Physiology and Biophysics
Language
en_US
Source
This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree.
Identifier
http://hdl.handle.net/11401/71059
Publisher
The Graduate School, Stony Brook University: Stony Brook, NY.
Format
application/pdf
Abstract
Gap junctions are intercellular channels formed by connexin (Cx) proteins which create a pathway for the transport of ions and small metabolites between two adjacent cells that are in contact. In individual cells connexin proteins can form hemichannels, single ion channels, which interact with the extracellular solution. Connexin proteins are found ubiquitously throughout the body and naturally occurring mutations within their genes are associated with a range of diseases. Connexin related diseases can manifest from a variety of differing functions of the resulting protein. Mutations in the Cx26 gene (GJB2) are a major cause of non-syndromic deafness and also can result in syndromic deafness associated with skin disease. While the function of the mutations which cause non-syndromic deafness can be related to a complete loss of channel functionality, the role of mutations associated with syndromic deafness is not understood. It has been theorized that this complication may result from aberrantly active hemichannels which disrupt normal homeostasis within the epidermis. It was also theorized that dominant negative inhibition or trafficking defects could be responsible for syndromic disorders where mutations do not lead to an increase in hemichannel activity. Six naturally occurring mutations (Gly12Arg, Asn14Lys, Asn14Tyr, Ser17Phe, Asp50Asn, and Asp50Tyr) were selected to investigate what differences the proteins may have compared to wild-type Cx26 activity. Gap junction activity was quantified using the Xenopus oocyte expression system via dual whole-cell voltage clamp on paired cells, and hemichannel activity was determined by single whole-cell voltage clamp in unpaired cells. Immunofluorescent staining in cultured and transfected HeLa cells was used to detect the trafficking patterns of the mutants and wild-type protein. All tested mutations resulted in single cells with altered hemichannel activity compared to wild-type Cx26 cells. Three tested mutations (Gly12Arg, Asn14Lys, and Asp50Asn) saw robust outward currents several fold larger than wild-type, and three mutations (Asn14Tyr, Ser17Phe, and Asp50Tyr) resulted in cells with negligible currents compared to wild-type injected cells. Cells injected with mutations associated with larger aberrant hemichannels also resulted in an increased rate of cell death. All but one mutation (Asn14Lys, equivalent) tested had severely lowered gap junction conductance in paired oocytes compared to wild-type Cx26 pairs. Four of these mutations (Asn14Lys, Asn14Tyr, Asp50Asn, and Asp50Tyr) were selected to determine trafficking patters of Cx26 mutant proteins in HeLa cells. All cells transfected with mutant proteins showed altered trafficking patterns compared to wild-type Cx26 transfected cells. These findings lead to the conclusion that while some mutations may operate through robust and aberrant hemichannels as originally hypothesized, there also seems to a second pathway related to protein trafficking which creates the same phenotype associated with Syndromic Deafness. | 157 pages
Recommended Citation
Lee, Jack, "The Role of Connexin26 Mutations in Keratitis-Ichthyosis-Deafness Syndrome" (2013). Stony Brook Theses and Dissertations Collection, 2006-2020 (closed to submissions). 266.
https://commons.library.stonybrook.edu/stony-brook-theses-and-dissertations-collection/266