Tembo, Maiwase
(2020)
Uncovering the Signaling Mechanisms of TransMEMbrane 16A.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
The Ca2+-activated Cl- channel, TransMEMbrane member 16A (TMEM16A), regulates diverse physiological functions including smooth muscle contraction, mucosal secretion and signal transduction. TMEM16A is an essential protein that we cannot live without. In fact, changes that enhance or reduce TMEM16A’s activity result in diseases such as hypertension and inflammatory airway diseases, respectively. Despite its importance, we are just beginning to understand how TMEM16A channel activity is regulated. To study TMEM16A’s regulation, I used electrophysiology techniques. Specifically, I recorded endogenous TMEM16A currents from Xenopus laevis oocytes using the inside-out configuration of the patch clamp technique. I observed that TMEM16A-conducted currents diminished within seconds of patch excision despite the continued presence of Ca2+. Current rundown is common amongst channels regulated by the fatty acid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). I demonstrated that TMEM16A current depletion in the membrane patch was due to loss of PI(4,5)P2, thereby revealing that PI(4,5)P2 is required for TMEM16A to conduct Cl- currents. Because PI(4,5)P2 is just one of eight cell membrane phosphoinositides that share a common backbone but differ in phosphate group number and location on the inositol ring, I sought to determine which features of PI(4,5)P2 enabled the lipid-channel interaction. I found that PI(4,5)P2 recovered the most current compared to the other phosphoinositides, and phosphoinositides containing at least one phosphate group on position 4 were capable of recovering intermediate levels of current. The extent of recovery was dependent on whether or not the phospholipid included a phosphate at position 4 rather than the number of negatively charged phosphates. In a separate line of experimentation, I determined that the rate of current rundown was influenced by the concentration of Ca2+ applied to excised patches in a way that activates a Ca2+-sensitive phospholipase C (PLC) to then deplete PI(4,5)P2. Taken together, my data reveal that PI(4,5)P2 regulates TMEM16A, and that the phosphate at position 4 is key in this interaction. Overall, my work revealed key mechanisms in how signals other than intracellular Ca2+ alter opening and closing of these critical channels. Expanding our understanding of TMEM16A’s regulatory mechanisms may lay the foundation for developing novel therapeutics for TMEM16A-associated diseases.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
16 September 2020 |
| Date Type: |
Publication |
| Defense Date: |
14 April 2020 |
| Approval Date: |
16 September 2020 |
| Submission Date: |
6 April 2020 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
112 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Biological Sciences |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
na |
| Date Deposited: |
16 Sep 2020 15:08 |
| Last Modified: |
16 Sep 2020 15:08 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/38564 |
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