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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

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 (Unpublished)

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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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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Aldrich, Stephanie Beatricesba21@pitt.edusba210000-0002-0210-9339
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairMeriney, Stephen D.meriney@pitt.edumeriney
Committee MemberJacob, Tija C.tcj11@pitt.edutcj11
Committee MemberBahar, Ivetbahar@pitt.edubahar
Committee MemberLipscombe, Dianediane_lipscombe@brown.eduN/A
Committee MemberJohnson, Jon W.jjohnson@pitt.edujjohnson
Committee MemberSchlüter, Oliverschluter@pitt.eduschluter
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


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