Bailey, Mark Andrew
(2010)
ROLE OF THE SIXTH TRANSMEMBRANE DOMAIN IN THE CALCIUM-DEPENDENT GATING OF THE INTERMEDIATE CONDUCTANCE CALCIUM-ACTIVATED POTASSIUM CHANNEL, KCa3.1.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
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 gatingequilibrium 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.
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Details
| Item Type: |
University of Pittsburgh ETD
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| Status: |
Unpublished |
| Creators/Authors: |
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| ETD Committee: |
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| Date: |
23 November 2010 |
| Date Type: |
Completion |
| Defense Date: |
23 September 2010 |
| Approval Date: |
23 November 2010 |
| Submission Date: |
19 October 2010 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Medicine > Cell Biology and Molecular Physiology |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
cysteine modification; electrophysiology; intermediate conductance calcium-activated potassi; ion channel gating; potassium channel |
| Other ID: |
http://etd.library.pitt.edu/ETD/available/etd-10192010-105034/, etd-10192010-105034 |
| Date Deposited: |
10 Nov 2011 20:03 |
| Last Modified: |
15 Nov 2016 13:50 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/9491 |
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