Persistent Link:
http://hdl.handle.net/10150/193686
Title:
Understanding the Function of Bestrophin
Author:
Kinnick, Tyson Rand
Issue Date:
2007
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
The purpose of this study was to investigate the function of the protein bestrophin-1 (best-1). Previous studies have suggested that best-1 is either a Ca++-activated Cl- channel (ClCa) and/or that it is a regulator of Ca++ induced responses in the retinal pigment epithelium (RPE).Experiments were performed on whole monolayers of cultured primary fetal human retinal pigment epithelium (fhRPE). The amount and polarity of the transduced proteins exposed at the cell surface, as assessed by domain selective biotinylation, was not altered by overexpression of best-1 or the best-1 mutants W93C or R218C. Best-1 (Wt) transduction of fhRPE increased baseline short circuit current (Isc) over Controls and best-1 mutant experimental groups during electrophysiology on whole monolayers. Cl-ion substitution in the Wt group caused a reduction in Isc that was reversible upon re-introduction of Cl- to the bathing solution. Cl-ion substitution did not significantly alter Isc in any other experimental group. This data supports a regulatory function for best-1 for Cl- transport across the epithelium.Application of the Ca++-ionophore ionomycin resulted in a biphasic response in Control monolayers, which was reduced in monolayers overexpressing best-1. Following exposure to ionomycin, NFA was used to block ClCa currents. NFA resulted in a decrease in the TEP of Controls and Wt monolayers to approximately half the value observed at the maximal response to ionomycin (Peak 2 or P2). The Isc response of Control and Wt monolayers mimicked the TEP response, resulting in an Isc at the end of exposure to NFA that was the same as the Isc at Peak 1 (P1) of the biphasic response to ionomycin. This suggests that ClCa currents are at most responsible for P2 of the ionomycin response, but this is not exclusive of other channels sensitive to NFA being involved. R218C expressing cells showed little response to Ca++ and NFA, and W93C cells were not responsive.In conclusion, with the cellular expression and polarity of best-1 and best-1 mutants the same, the data from the present study support a Ca++ regulatory role for best-1 that functions to prime the RPE to respond to changes in intracellular Ca++.
Type:
text; Electronic Dissertation
Keywords:
Physiological Sciences
Degree Name:
PhD
Degree Level:
doctoral
Degree Program:
Physiological Sciences; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Marmorstein, Alan D.
Committee Chair:
Marmorstein, Alan D.

Full metadata record

DC FieldValue Language
dc.language.isoENen_US
dc.titleUnderstanding the Function of Bestrophinen_US
dc.creatorKinnick, Tyson Randen_US
dc.contributor.authorKinnick, Tyson Randen_US
dc.date.issued2007en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractThe purpose of this study was to investigate the function of the protein bestrophin-1 (best-1). Previous studies have suggested that best-1 is either a Ca++-activated Cl- channel (ClCa) and/or that it is a regulator of Ca++ induced responses in the retinal pigment epithelium (RPE).Experiments were performed on whole monolayers of cultured primary fetal human retinal pigment epithelium (fhRPE). The amount and polarity of the transduced proteins exposed at the cell surface, as assessed by domain selective biotinylation, was not altered by overexpression of best-1 or the best-1 mutants W93C or R218C. Best-1 (Wt) transduction of fhRPE increased baseline short circuit current (Isc) over Controls and best-1 mutant experimental groups during electrophysiology on whole monolayers. Cl-ion substitution in the Wt group caused a reduction in Isc that was reversible upon re-introduction of Cl- to the bathing solution. Cl-ion substitution did not significantly alter Isc in any other experimental group. This data supports a regulatory function for best-1 for Cl- transport across the epithelium.Application of the Ca++-ionophore ionomycin resulted in a biphasic response in Control monolayers, which was reduced in monolayers overexpressing best-1. Following exposure to ionomycin, NFA was used to block ClCa currents. NFA resulted in a decrease in the TEP of Controls and Wt monolayers to approximately half the value observed at the maximal response to ionomycin (Peak 2 or P2). The Isc response of Control and Wt monolayers mimicked the TEP response, resulting in an Isc at the end of exposure to NFA that was the same as the Isc at Peak 1 (P1) of the biphasic response to ionomycin. This suggests that ClCa currents are at most responsible for P2 of the ionomycin response, but this is not exclusive of other channels sensitive to NFA being involved. R218C expressing cells showed little response to Ca++ and NFA, and W93C cells were not responsive.In conclusion, with the cellular expression and polarity of best-1 and best-1 mutants the same, the data from the present study support a Ca++ regulatory role for best-1 that functions to prime the RPE to respond to changes in intracellular Ca++.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectPhysiological Sciencesen_US
thesis.degree.namePhDen_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplinePhysiological Sciencesen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorMarmorstein, Alan D.en_US
dc.contributor.chairMarmorstein, Alan D.en_US
dc.contributor.committeememberFuglevand, Andrew J.en_US
dc.contributor.committeememberLevine, Richard B.en_US
dc.contributor.committeememberMcKay, Brian S.en_US
dc.identifier.proquest2046en_US
dc.identifier.oclc659747324en_US
All Items in UA Campus Repository are protected by copyright, with all rights reserved, unless otherwise indicated.