Functional and structural subdomains of the intracellular loop domain of the GABAAR α1 subunit Open Access

O'Toole, Kate Kristen (2011)

Permanent URL: https://etd.library.emory.edu/concern/etds/9019s332h?locale=en
Published

Abstract

The GABAAR functions as a ligand gated ion channel, which permits the flow of anions across the cell membrane, to mediate inhibitory signaling in the central nervous system. To date little is known of the functional importance of the intracellular loop domain of this critical neuronal protein. Recent studies in homologous proteins suggest a role for the intracellular loop domain in controlling the amplitude of channel conductance; therefore, residues within the intracellular loop domain of the GABAAR α1 subunit were hypothesized to define a portion of the ion channel pore. If charged residues define the permeation pathway, then mutation of integral positions will perturb the electrostatic landscape of the pore to influence ion permeation. First, deletions within the C-terminus of the α1 subunit enhanced the amplitude of macroscopic currents and decreased the apparent affinity of the receptor for GABA, which confirmed that the intracellular loop domain must be intact for proper channel function. Second, a mutagenic screen was conducted of all amino acids harboring ionizable side chains within the intracellular loop domain of the α1 subunit to investigate the functional contribution of individual charged residues. Using whole-cell and single channel voltage clamp recording techniques, charged residues within this domain were shown to control channel gating and anion permeation in a subdomain dependent manner. Third, the importance of the direction of anion flux was investigated. Results showed that currents through the GABAAR exhibited outward rectification that was inversely related to the open probability of the channel. Finally, secondary structure predictions suggested that the intracellular loop domain is composed of two membrane associated α helices, denoted MA3 and MA4, which are separated by a β strand. Comparison of theoretical and empirical results showed that the predicted functional and structural subdomains overlap. Residues within both MA3 and MA4 control current amplitude and anion permeation, while residues within MA3 determine agonist dependent channel gating and residues within MA4 mediate the rate of desensitization. In sum, these results define the role of the intracellular loop domain in GABAAR function and expand our knowledge of inhibitory neurotransmission.

Table of Contents

Table of Contents

Distribution Agreement
Approval Sheet
Abstract Cover Page
Abstract
Cover Page
Dedication
Table of Contents
List of Figures
List of Tables
List of Equations

Chapter 1: Introduction…………………………………………………………………………1
1.1: Overview
1.2: The Pentameric Ligand Gated Ion Channel Superfamily
1.3: The γ-aminobutyric acid type A receptor
1.3.1: Subunit Diversity
1.3.2: Structure and Homology Models
1.3.3: Function
1.3.3A: Binding and Gating
1.3.3B: Ion Permeation and Selectivity
1.3.3C: Trafficking and Phosphorylation
1.3.4: Clinical Target
1.3.4A: Modulation
1.3.4B: Dysfunction
1.4: Conclusion

Chapter 2: Experimental Methods and Materials……………………………………………36
2.1: Overview
2.2: Cell Culture
2.2.1: HEK293 cells
2.2.2: Resuscitation of cells
2.2.3: Preparation of Frozen Stocks
2.2.4: Passage and Plating
2.2.5: Vector Expression System
2.2.6: Mutagenesis
2.2.7: cDNA Preparation
2.2.8: Calcium Phosphate Transfection
2.3: Surface Expression
2.3.1: Confocal Microscopy
2.3.2: Luminescent Assay
2.4: Electrophysiology
2.4.1: Solutions

2.4.2: Electrophysiology Circuitry
2.4.3: The Electrophysiology Rig
2.4.4: Achieving the Patch Configurations
2.4.5: The Perfusion System
2.4.6: Whole-Cell Recordings
2.4.7: Whole-Cell Analysis
2.4.8: Single Channel Recordings
2.4.9: Single Channel Analysis
2.5: Statistical Analysis
2.6: Bioinformatics
2.6.1: Sequence Alignment
2.6.2: Secondary Structure Prediction
2.6.3: Homology Model
2.7: Conclusion


Chapter 3: The ILD facilitates channel gating and hinders ion permeation………………...81
3.1: Overview
3.2: Introduction
3.3: Results
3.3.1: Deletion of the α1 ILD and C-terminus
3.3.2: Deletion of the α1 ILD did not alter ion selectivity
3.3.3: Truncation of the α1 subunit decreased surface expression
3.4: Discussion
3.4.1: The ILD controls current magnitude
3.4.2: The ILD controls channel gating
3.4.3: The ILD controls ion permeation but not charge selectivity
3.4.4: Which domains are necessary for surface expression?
3.5: Conclusion

Chapter 4: Functional Subdomains of the ILD……………………………………………...100
4.1: Overview
4.2: Introduction
4.3: Results
4.3.1: Charged residues of the α1 ILD control ion permeation
4.3.1A: Point mutations increased whole-cell current magnitude
4.3.1B: Surface expression
4.3.1C: Single channel currents
4.3.1D: Ion replacement solutions confirmed anion selectivity of the WT pore
4.3.1E: Point mutations did not alter charge selectivity
4.3.1F: Point mutations shifted the relative reversal of anions
4.3.2: Charged residues of the α1 ILD control channel gating
4.3.2A: ILD charge switch point mutations decreased GABA apparent affinity
4.3.2B: The α1(K312E) mutation caused a gating impairment
4.3.2C: ILD charge switch point mutations increased the speed of desensitization

4.4: Discussion
4.4.1: Charge switch mutations revealed functional subdomains of the ILD
4.4.1A: Defining the Subdomains
4.4.1B: The MA3 Subdomain
4.4.1C: The MA4 Subdomain
4.4.2: ILD residues define an anion selectivity filter
4.5: Conclusion

Chapter 5: The GABA
AR exhibits outward rectification at low PO………………………..135
5.1: Overview
5.2: Introduction
5.3: Results
5.3.1: Channel gating is voltage dependent
5.3.1A: Membrane potential determined current magnitude and desensitization
5.3.1B: IV relationship was dependent on the effective concentration of GABA
5.3.1C: Asymmetry of currents was independent of Goldman rectification
5.3.1D: Chloride dependence of gating
5.3.2: Outward rectification of currents occurs at low PO
5.3.2A: Extrasynaptic receptors are outwardly rectifying
5.3.2B: Decreasing PO enhanced rectification
5.3.2C: GABAAR modulation by general anesthetics was voltage dependent
5.3.3: Rectification can be used as a tool to identify ion sensor sites with α1 ILD
5.4: Discussion
5.4.1: Channel open probability is inversely related to degree of outward rectification
5.4.1A: Rectification is not limited to extrasynaptic GABAARs
5.4.1B: PO modulates degree of rectification
5.4.1C: The efficacy of drugs that modulate PO is voltage dependent
5.4.2: Probable causes of rectification
5.4.2A: Goldman rectification
5.4.2B: Permeability of chloride
5.4.2C: Chloride concentration
5.4.3: Gating elements
5.4.3A: Desensitization
5.4.3B: Chloride dependence
5.4.4: Ion sensor sites within the permeation pathway
5.4.5: PO mediated rectification is a universal property of GABAARs
5.5: Conclusion

Chapter 6: Secondary Structure of the ILD………………………………………………….179
6.1: Overview
6.2: Introduction
6.3: Results
6.3.1: Sequence alignment revealed conservation of domains
6.3.2: Secondary structure predicted from primary amino acid sequence
6.3.3: Homology model of the α1 subunit
6.4: Discussion
6.5: Conclusion

Chapter 7: Discussion………………………………………………………………………....202

7.1: Subdomains of the ILD
7.2: Permeation Pathway
7.3: Gating elements
7.4: Conclusion

Appendices……………………………………………………………………………………..215
A: Solution Recipes
B: Pipette Programs
C: pClamp Acquisition Parameters
D: Matlab Scripts
E: QuB Protocol

References
Acknowledgements



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