Ion channels are the molecular units that underlie electrical signaling in cells. Many physiological processes are dependent upon this signaling mechanism, as dysregulation often leads to severe pathophysiological consequences. The intermediate conductance calcium-activated potassium channel (KCa3.1) functions as heteromeric complexes with calmodulin (CaM), which is constitutively bound to the calmodulin-binding domain (CaMBD) of KCa3.1 located in the C-terminus, just distal to the sixth transmembrane domain (S6). This arrangement enables CaM to function as an intracellular Ca2+-sensor, coupling changes in the intracellular Ca2+ concentration to the regulation of channel activity. Understanding how channels gate or transition from the closed to the open conformation is a fundamental question in the field of ion channel biophysics. A chemomechanical gating model was proposed to explain how Ca2+-binding causes the channel to transition from a non-conducting to a conducting configuration. However, this model lacks a specific mechanism explaining how the conformational change in the CaMBD is coupled to the activation gate. Therefore, the goal of this dissertation was to investigate the role of S6 in the activation mechanism of KCa3.1. Specifically, I tested the hypothesis that the non-luminal residues in the C-terminal portion of S6 function as an interacting surface to couple CaM to the activation gate. Biochemical perturbation and site directed mutagenesis targeting predicted non-luminal residues in S6 act to shift the gating
equilibrium toward the open state by increasing the apparent Ca2+ affinity and dramatically slowing the deactivation process. Kinetic modeling using a 6-state gating scheme showed these perturbations act to slow the transition between the open state back to the closed state. The modification in the steady-state and kinetic behavior of the channel in combination with the kinetic analysis indicate the shift in gating equilibrium is caused by slowing the closing transition, suggesting the non-luminal surface of S6 is allosterically coupled to the activation gate. Therefore, in addition to being a structural component of the pore; S6 is also a dynamic component of the activation mechanism. Continuing to identify regions of the channel participating in the activation mechanism is critical to understand how Ca2+ binding leads to channel opening.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-10192010-105034 |
| Date | 23 November 2010 |
| Creators | Bailey, Mark Andrew |
| Contributors | Thomas Kleyman, Daniel Devor, Michael Grabe, Peter Drain, Raymond Frizzell |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-10192010-105034/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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