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Molecular determinants of different gating mechanisms in inwardly-rectifying potassium channels

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dc.contributor.author Huang, Xinyang
dc.date.accessioned 2012-06-20T17:42:07Z
dc.date.available 2012-06-20T17:42:07Z
dc.date.copyright 2012 en_US
dc.date.issued 2012-06-20
dc.identifier.uri http://hdl.handle.net/2429/42519
dc.description.abstract Inwardly-rectifying potassium (Kir) channels comprise a transmembrane domain (TMD) that makes up the conducting pore and a large cytoplasmic domain (CTD) that controls channel gating via ligand-binding. Tissue-specific expression and diverse gating mechanisms endow each Kir family with distinct physiological roles. To better understand channel function and improve treatments for Kir channel-associated diseases, we set out to determine the molecular mechanisms underlying channel gating and block. Inward rectification via voltage-dependent polyamine block is important for maintaining a negative resting membrane potential and permitting the generation of action potentials in excitable cells. Polyamine block was studied in Kir2.1, a prototypical strong rectifier. Using inside-out patch clamp electrophysiology and sterically expanded polyamine blockers, our findings suggest that steep voltage-dependence and external K⁺-dependence of high-affinity block do not require extensive polyamine interactions with the selectivity filter. Furthermore, by introducing MTS moieties into the inner cavity, as well as utilizing a hydrogen bond-deficient blocker and the disease mutant D172N, we determined that hydrogen bonding is crucial to high-affinity binding between spermine and the rectification controller (D172 in Kir2.1). It is important to note that spermine is structurally unique and binds very deep in the inner cavity. This is likely not the case for diamine blockers, which warrants caution when drawing conclusions from them about spermine. A second gating mechanism in Kir channels is regulation by ligand-binding in the CTD. Ligand transduction to the channel gate is key to many physiological roles of Kir channels and can be demonstrated in KATP channels, which are responsible for transducing cellular metabolism to insulin secretion. Using inside-out patch clamp electrophysiology, we screened Kir6.2 slide helix mutants for ATP-sensitivity and voltage-dependent gating kinetics with a “forced gating” background mutation F168E that functionally rescues non-expressing mutants. We found that a highly conserved aspartate in the Kir slide helix is essential for ligand transduction. Further screening of nearby CTD residues revealed several potential interacting partners for this slide helix “aspartate anchor”. However, specific interactions between the aspartate anchor and the CTD remain unclear. en_US
dc.language.iso eng en_US
dc.publisher University of British Columbia en
dc.rights Attribution-NonCommercial 2.5 Canada *
dc.rights.uri http://creativecommons.org/licenses/by-nc-nd/3.0/ *
dc.title Molecular determinants of different gating mechanisms in inwardly-rectifying potassium channels en_US
dc.type Electronic Thesis or Dissertation en
dc.degree.name Master of Science - MSc en_US
dc.degree.discipline Pharmacology and Therapeutics en_US
dc.degree.grantor University of British Columbia en
dc.date.graduation 2012-11 en_US
dc.degree.campus UBCV en_US
dc.description.scholarlevel Graduate en


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