Aldrich, Stephanie Beatrice
(2024)
Mutations in the Inner Pore of a CaV2.1 Voltage-Gated Calcium Channel Differentially Affect the Efficacy, Potency, and Binding Kinetics of an (R)-Roscovitine-Derived Positive Allosteric Gating Modifier.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Insufficient release of neurotransmitter from motor neurons is a cause of debilitating muscle weakness in multiple types of neuromuscular disease, including Lambert-Eaton Myasthenic Syndrome and Spinal Muscular Atrophy. Neurotransmitter release from motor neurons is controlled by the CaV2 family of voltage-gated Ca2+ channels (VGCCs), which open in response to an action potential, causing an influx of Ca2+ that triggers vesicle fusion. The drug GV-58, an analog of the CDK inhibitor (R)-roscovitine, has been found to rescue neuromuscular transmission in animal disease models by selectively prolonging the opening of CaV2 VGCCs, thereby enhancing Ca2+ influx during action potentials and increasing the probability of vesicle fusion. Other analogs of (R)-roscovitine have been shown to have similar effects on VGCCs, with varying degrees of selectivity, potency, efficacy, and speed of action.
Understanding where (R)-roscovitine analogs bind to VGCCs and the mechanism by which they prolong channel opening would aid in the design of new analogs with improved therapeutically-relevant characteristics, as well as providing new insight into the structural basis of VGCC gating. To investigate these questions, my colleagues and I used homology models of the structure of the VGCC subtype CaV2.1 to guide mutagenesis experiments aimed at identifying specific amino acid residues that may interact with (R)-roscovitine analogs.
In Chapter 3, we describe our in silico predictions of GV-58 binding, which support the interpretation that the (R)-roscovitine analog binding site is accessible from the plasma membrane through a fenestration in the channel protein. In Chapter 4, we present whole-cell patch clamp electrophysiology data from channels in which we had individually mutated amino acid residues that are unique to CaV2 channels and were predicted in silico to bind (R)-roscovitine analogs. We found multiple mutations that affect the drug’s ability to bind the channel and/or the extent to which drug binding prolongs channel opening. In chapter 5, we show how mutating key residues implicated in VGCC gating affects (R)-roscovitine analog action. Finally, in Chapter 6, we propose a mechanistic hypothesis of (R)-roscovitine analog action in VGCCs.
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Details
| Item Type: |
University of Pittsburgh ETD
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| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
14 February 2024 |
| Date Type: |
Publication |
| Defense Date: |
23 November 2021 |
| Approval Date: |
14 February 2024 |
| Submission Date: |
9 December 2021 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
243 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Neuroscience |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
voltage-gated calcium channels
electrophysiology
mutagenesis
mutation
homology modeling
GV-58
roscovitine
agonist
modifier
neuromuscular disease
CaV2.1
CaV2 |
| Date Deposited: |
14 Feb 2024 17:22 |
| Last Modified: |
14 Feb 2024 17:22 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/42039 |
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