Pharmacologically targeting the GABA-A receptors in neurological disease Embargo

Moody, Olivia Ann (Fall 2017)

Permanent URL: https://etd.library.emory.edu/concern/etds/0c483j381?locale=es
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Abstract

Altering GABAA receptor activity can shift the balance of inhibition and excitation in the brain, leading to neurological diseases. Two examples where GABAA receptor activity can be impaired are the daytime sleepiness characteristic of idiopathic hypersomnia (IH) and the increased seizure susceptibility in epilepsy. In both diseases, drugs that target the benzodiazepine site on the GABAA receptor have been used to modulate symptoms, but further study of this site could help develop novel drugs treatments with fewer side effects. The mechanisms by which GABAA receptor activity is altered in IH and by rare mutations of the GABR genes in epilepsy remain incompletely understood.

In the first part of this thesis, I examined the structure-function relationship of the benzodiazepine binding site on GABAA receptors. Mutations were created in loop A, loop B, and loop C of the benzodiazepine site across the six different α subunits. Effects were measured using midazolam. Results from whole-cell patch clamp recording of mutated αxβ2γ2s receptors revealed that mutating loop A dramatically conferred or abolished the efficacy of midazolam. Surprisingly, mutating loop C also moderately altered the efficacy of midazolam depending on the α subunit mutated.

Second, I assessed the role of the high-affinity benzodiazepine site in mediating the positive allosteric modulator (PAM) actions of cerebrospinal fluid (CSF) taken from hypersomnia patients experiencing IH. Whole-cell patch clamp recording found that hypersomnolent CSF samples enhanced the activity of αxβ2γ2s GABAA receptors, even in receptors without a functional benzodiazepine site. Furthermore, CSF enhanced whole-cell current responses for extrasynaptic αxβ2δ receptors that are generally insensitive to benzodiazepines. Overall, the CSF results were not consistent with the active component of hypersomnolent CSF acting primarily through the high-affinity benzodiazepine site of the GABAA receptors.

Third, three rare, novel GABR mutations identified in pediatric patients with severe early-onset epilepsy were characterized. Whole-cell patch clamp recording showed that the mutations in the M2 and M2-M3 linker domains can alter the gating, desensitization and GABA apparent-affinity of receptors. Results offer insight into which GABAergic treatments may or may not be beneficial to patients with rare variants.

Understanding the pharmacology of GABAA receptors as they relate to neurological diseases will offer new insights for better treating diseases.

Table of Contents

Table of contents:

 

1.    Chapter 1: Introduction …………………………………………………………….    2

1.1.  GABAA receptors and GABAergic neurotransmission in the brain …………    3

1.2.  Modulators of GABAA receptors …………………………………………..……  12

1.3.  Benzodiazepines act at GABAA receptors …………………………………….  15

1.3.1.    Benzodiazepines …………………………………………………………  15

1.3.2.    Genetic knock-in mice & benzodiazepines ……………………………  17

1.3.3.    Positive and negative benzodiazepines ……………………………….  19

1.3.4.    The benzodiazepine binding site on the GABAA receptor ……………  22

1.3.5.    Subunit composition affects benzodiazepine modulation ……………  22

1.3.6.    The high-affinity site is made up of structural loops A-F ……………..  23

1.3.7.    Midazolam ………………………………………………………………… 32

1.3.8.    Therapeutics of benzodiazepines ………………………………………  34

1.4.  GABAA receptors and neurological disease ………………………………….   36

1.4.1.    Altered GABAA receptor activity in neurological disease …………….  36

1.4.2.    Idiopathic hypersomnia ………………………………………………….  37

1.4.3.    Epilepsy …………………………………………………………………… 47

1.5.  Summary of background information and rationale ………………………….  51

 

2.    Chapter 2: Methods …………………………………………………………………   54

2.1.  Plasmids and mutagenesis ……………………………………………………    55

2.2.  HEK293T cell properties and origin …………………………………………..    62

2.3.  Cell culture and transfection …………………………………………………..    64

2.4.  Theory and circuits of whole-cell patch clamp electrophysiology …………    68

2.5.  Bath and drug perfusion system ………………………………………………   73

2.6.  Whole-cell patch clamp electrophysiology …………………………………..    76

2.6.1.    Pharmacology patch clamp rig setup …………………………………   76

2.6.2.    Patch clamp rug used for CSF assays ……………………………….   78

2.6.3.    Whole-cell voltage clamp recordings …………………………………   81

2.6.4.    GABA concentration-response assay protocol ……………………...   81

2.7.  Whole-cell analysis ……………………………………………………………..   84

2.7.1.    Analyzing whole-cell recordings ……………………………………....   84

2.7.2.    Analyzing GABA concentration-response curves ……………………  84

2.7.3.    Interpretation of changes in Hill parameters …………………………   85

2.8.  Statistics …………………………………………………………………………   87

 

3.    Chapter 3: The molecular pharmacology of midazolam at Synaptic GABAA receptors  …………………………………………………………………………….   88

3.1.  Introduction ………………………………………………………………………   89

3.2.  Methods ………………………………………………………………………….   93

3.2.1.    Cell culture

3.2.2.    Mutagenesis

3.2.3.    In vitro electrophysiology

3.2.3.1.        Recording  

3.2.3.2.        GABA concentration-response assays    

3.2.3.3.        Selecting the EC10 GABA concentration for midazolam experiments    

3.2.3.4.        Midazolam concentration-response assays  

3.2.3.5.        GABA concentration-response + 1 μM midazolam  

3.2.3.6.        1μM midazolam + saturating GABA.  

3.2.4.    Whole-cell Analysis

3.2.4.1.        GABA concentration-response curves

3.2.4.2.        Midazolam concentration-response curves 

3.2.4.3.        GABA concentration-response + 1 μM midazolam  

3.2.4.4.        1μM midazolam + saturating GABA

3.2.5.    Statistics

3.3.  Results  ………………………………………………………………………..    99

3.3.1.    GABA concentration-response curves with loop A-C mutations

3.3.2.    Exposure protocol affects the degree of midazolam potentiation   measured

3.3.3.    Midazolam concentration-response curves for loop A-C mutations

3.3.3.1.        Loop A mutations 

3.3.3.2.        Loop B mutations

3.3.3.3.        Loop C mutations

3.3.3.4.        Wildtype αxβ2γ2s receptors 

3.3.3.5.        Summary of results

3.3.4.    Effects of midazolam on the GABA concentration-response relationship for α1β2γ2 receptors

3.4.  Discussion …………………………………………………………………….   133

3.4.1.    Mutation of single residues in loops A-C can alter the efficacy of midazolam

3.4.2.    Midazolam shifts the GABA concentration-response relationship leftwards, inconsistent with conventional benzodiazepine theory ..

3.4.3.    Conclusions and future directions …………………………………..  

 

4.    Chapter 4: The allosteric modulation of GABAA receptors by cerebrospinal fluid from patients suspected of having idiopathic hypersomnia  ……...   145

4.1.  Introduction ……………………………………………………………………   147

4.2.  Methods  ………………………………………………………………………   151

4.2.1.    Cell culture, cDNA plasmids and transfections

4.2.2.    Cerebrospinal fluid sample preparation

4.2.3.    In vitro electrophysiology

4.2.3.1.        In vitro electrophysiology

4.2.3.2.        CSF potentiation assays

4.2.4.    Whole-cell analysis

4.2.5.    Statistics

4.3.  Results …………………………………………………………………………   157

4.3.1.    Measuring CSF potentiation at α1β2γ2s GABAA receptors

4.3.2.    Ruling out the high-affinity benzodiazepine binding site as a site of action

4.3.3.    Alpha- and Delta- subunit specificity of CSF potentiation

4.3.4.    CSF shifts GABA concentration-response curve leftwards

4.4.  Discussion  ………………………………………………………………………   174

4.5.  Limitations and future directions ………………………………………………   180

 

5.    Chapter 5: Functional consequences of missense mutations in the GABR gene linked to early-onset epilepsy ……………………………………………………   183

5.1.  Introduction  ……………………………………………………………………..   185

5.2.  Methods  …………………………………………………………………………   189

5.2.1.    Whole-cell patch clamp recording

5.2.2.    Whole-cell analysis

5.2.3.    Statistics

5.3.  Results  …………………………………………………………………………..  192

5.3.1.    Identification of GABR mutations from patients with epilepsy

5.3.2.    Functional characterization of α2(T292K) mutation

5.3.3.    Functional characterization of the α5(V294L) mutation

5.3.4.    Functional characterization of β3(P301L) mutation

5.4.  Discussion  ………………………………………………………………………  203

5.5.  Conclusions and Future Directions  …………………………………………..  215

 

6.    Chapter 6: Discussion  …………………………………………………………….  218

6.1.  Summary of findings  …………………………………………………………..   219

6.2.  Implications of findings for pharmacology and neurological disease  …….   223

6.3.  Final conclusions  ………………………………………………………………   230

 

Appendices:

Appendix A: pClamp drug perfusion protocols  ……………………………….  233

Appendix B: Matlab Scripts ……………………………………………………..  240

Appendix C: Pipette pulling program …………………………………………..  251

Appendix D: Example sequencing results of GABAA receptor cDNA ……...  252

 

References    ……………………………………………………………………..  253

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