Characterization of Novel Poly(lipid) BLMs for Long-Term Ion Channel Scaffolds Towards the Development of High-Throughput Screening Devices

Persistent Link:
http://hdl.handle.net/10150/196024
Title:
Characterization of Novel Poly(lipid) BLMs for Long-Term Ion Channel Scaffolds Towards the Development of High-Throughput Screening Devices
Author:
Heitz, Benjamin Arthur
Issue Date:
2010
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:
Suspended lipid bilayers, or black lipid membranes (BLMs), have been used to study the electrophysiological properties of ion channels (ICs); however, BLMs assembled from natural, non-polymerizable lipids are inherently unstable due to the non-covalent associations on which they are based. Lifetimes of several hours are commonly observed in BLMs until rupture due to mechanical, thermal, or chemical insults. One potential improvement is the use of polymerizable phospholipids (poly(lipids)). BLMs prepared using dienoyl functionalized poly(lipids) and binary mixtures of fluid, non-polymerizable lipids with poly(lipids) were investigated for IC recordings.poly(BLMs) exhibited enhanced lifetimes from several hours to upwards of 4 weeks while maintaining IC functionality for one week. Activity of ICs that require membrane fluidity was retained using binary phospholipid mixtures of fluid and polymeric phospholipids. IC activity was retained by inducing domain formation, wherein ICs incorporated into the fluid domains. The binary membranes exhibited marked enhancement in stability resulting from fractional poly(lipids) polymerization. Additionally, ICs can be reconstituted into the fluid domains following photopolymerization and subsequent domain formation, a key requirement when UV-sensitive ICs are utilized. Here, the electrical properties, stability, and incorporation of pore-forming ICs, including hemolysin, alamethicin, and gramicidin, into poly(lipid) membranes are reported. Potential applications developing ligand-gated IC based sensors for high throughput screening are being investigated.In parallel to the characterization of poly(lipids) for potential long-term IC membranes, a model ligand-gated IC was expressed, characterized, and reconstituted into non-polymerizable lipids. Mutant K<sub>ATP</sub> channels were expressed in mammalian and yeast systems. The orientations of mutant K<sub>ATP</sub> channels were studied using electrophysiological and immunohistochemical techniques. Large quantities were expressed and purified from <italic>Pichia pastoris</italic> and functionally reconstituted into BLMs. ATP and long-chaing coenzyme A ester sensitivity was maintained in reconstituted in BLMs. K<sub>ATP</sub> channels will serve as a model system for testing the effect of poly(lipid) BLMs on IC function. Future utilization of poly(lipid) BLMs in combination with ligand-gated ICs offer major advancements to potential increased throughput for IC screening.
Type:
text; Electronic Dissertation
Keywords:
Biosensors; BLMs; Ion channels; Polymerizable lipids
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Chemistry; Graduate College
Degree Grantor:
University of Arizona
Committee Chair:
Saavedra, Steven Scott; Aspinwall, Craig A.

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleCharacterization of Novel Poly(lipid) BLMs for Long-Term Ion Channel Scaffolds Towards the Development of High-Throughput Screening Devicesen_US
dc.creatorHeitz, Benjamin Arthuren_US
dc.contributor.authorHeitz, Benjamin Arthuren_US
dc.date.issued2010en_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.abstractSuspended lipid bilayers, or black lipid membranes (BLMs), have been used to study the electrophysiological properties of ion channels (ICs); however, BLMs assembled from natural, non-polymerizable lipids are inherently unstable due to the non-covalent associations on which they are based. Lifetimes of several hours are commonly observed in BLMs until rupture due to mechanical, thermal, or chemical insults. One potential improvement is the use of polymerizable phospholipids (poly(lipids)). BLMs prepared using dienoyl functionalized poly(lipids) and binary mixtures of fluid, non-polymerizable lipids with poly(lipids) were investigated for IC recordings.poly(BLMs) exhibited enhanced lifetimes from several hours to upwards of 4 weeks while maintaining IC functionality for one week. Activity of ICs that require membrane fluidity was retained using binary phospholipid mixtures of fluid and polymeric phospholipids. IC activity was retained by inducing domain formation, wherein ICs incorporated into the fluid domains. The binary membranes exhibited marked enhancement in stability resulting from fractional poly(lipids) polymerization. Additionally, ICs can be reconstituted into the fluid domains following photopolymerization and subsequent domain formation, a key requirement when UV-sensitive ICs are utilized. Here, the electrical properties, stability, and incorporation of pore-forming ICs, including hemolysin, alamethicin, and gramicidin, into poly(lipid) membranes are reported. Potential applications developing ligand-gated IC based sensors for high throughput screening are being investigated.In parallel to the characterization of poly(lipids) for potential long-term IC membranes, a model ligand-gated IC was expressed, characterized, and reconstituted into non-polymerizable lipids. Mutant K<sub>ATP</sub> channels were expressed in mammalian and yeast systems. The orientations of mutant K<sub>ATP</sub> channels were studied using electrophysiological and immunohistochemical techniques. Large quantities were expressed and purified from <italic>Pichia pastoris</italic> and functionally reconstituted into BLMs. ATP and long-chaing coenzyme A ester sensitivity was maintained in reconstituted in BLMs. K<sub>ATP</sub> channels will serve as a model system for testing the effect of poly(lipid) BLMs on IC function. Future utilization of poly(lipid) BLMs in combination with ligand-gated ICs offer major advancements to potential increased throughput for IC screening.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectBiosensorsen_US
dc.subjectBLMsen_US
dc.subjectIon channelsen_US
dc.subjectPolymerizable lipidsen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineChemistryen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.chairSaavedra, Steven Scotten_US
dc.contributor.chairAspinwall, Craig A.en_US
dc.contributor.committeememberBrown, Michael F.en_US
dc.contributor.committeememberPemberton, Jeanneen_US
dc.identifier.proquest11245en_US
dc.identifier.oclc752261090en_US
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