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Functional characterization of recombinant Kv1 channel subtypes in HEK-293 cells for screening of selective inhibitors

Kumar Kaza, Seshu (2016) Functional characterization of recombinant Kv1 channel subtypes in HEK-293 cells for screening of selective inhibitors. PhD thesis, Dublin City University.

Abstract
Shaker-related voltage activated K+ channels or Kv1 channels occur in neurons as homo and hetero-tetramers of α subunits (Kv1.1-1.7), associated with auxiliary β subunits, and control their excitability. The aim of this project was to create recombinantly homo- and hetero-meric Kv1 channel constructs resembling those in neurons, using a novel concatenation strategy, and to evaluate their expression and function in HEK-293 cells. This involved either altering the number and arrangement of each subunit or mutating their genes to change the sensitivities to tetraethylammonium (TEA) or other blockers. α subunit genes encoding Kv1.1, 1.2, 1.4 and 1.6 were amplified and concatenated with unique restriction enzymes for cassette cloning and sub-cloned in different combinations, generating homo- and hetero-tetramers of known composition to facilitate their expression as single proteins. Restriction enzyme digestion and DNA sequencing of extracted plasmid from E. coli transformed with the concatenated genes established that the tetramers have the correct integrity and composition. Biotinylation and Western blotting confirmed the expression of intact tetrameric channels on the surface of transfected HEK-293 cells. Repositioning of α Kv1.6 subunit in a hetero-tetramer revealed that N-type rapid inactivation, a characteristic feature of Kv1.4-containing channels, can be over-ridden by an adjacent Kv1.6 via the latter’s N-type inactivation prevention (NIP) domain. Mutation of critical residues in the NIP restored the fast inactivation in Kv1.4-containing channels. In another objective to understand and raise the TEA sensitivity of Kv1.1/1.2-containing hetero-tetrameric channels, increasing the ratio of Kv1.1 to 1.2 in a Kv1.2-1.2-1.1-1.2 or mutagenesis of the critical residue in the first Kv1.2, placed adjacent or in close proximity to a Kv1.1 subunit in Kv1.2-1.2-1.2-1.1, made the channels more sensitive to TEA. Additionally, increasing the number of Kv1.1 subunits in a tetrameric channel decreases the voltage threshold and accelerates the activation kinetics. Overall, these findings assessed the possible consequences of α subunits being precisely arranged in Kv1 channels, by examining recombinantly created variants with known composition, an important criterion in designing drugs selective for Kv1 oligomeric subtypes. Screening of small inhibitor compounds for Kv1.1 allowed us to identify 2,2’-((5,5’-(di-p-topyldipyrromethane)bis(2,2’-carbonyl)bis(azanediyl))diethaneamine.2HCl as a potential therapeutic drug for use in the treatment of multiple sclerosis (MS). It potently (IC50 = ~15 μM) and selectively blocks Kv1.1 channels recombinantly expressed in mammalian cells. This novel inhibitor also induces a positive shift in the voltage-dependency of K+ current activation and slows the activation kinetics. Importantly, this new non-photo-toxic compound inhibited Kv1 heteromeric channels containing 2 or more Kv1.1 subunits, regardless of their positioning in concatenated tetramers, though Kv1.3-mediated K+ current was reduced to a lesser extent.
Metadata
Item Type:Thesis (PhD)
Date of Award:November 2016
Refereed:No
Supervisor(s):Dolly, Oliver, Wang, Jiafu and Lawrence, Gary
Uncontrolled Keywords:HEK-293; tetraethylammonium; TEA; multiple sclerosis; MS;
Subjects:Biological Sciences > Neuroscience
Biological Sciences > Biochemistry
DCU Faculties and Centres:DCU Faculties and Schools > Faculty of Science and Health > School of Biotechnology
Research Initiatives and Centres > International Centre for Neurotherapeutics (ICNT)
Use License:This item is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 License. View License
ID Code:21303
Deposited On:16 Nov 2016 13:59 by Oliver Dolly . Last Modified 29 Jul 2018 03:30
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