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Discrete M3-M4 intracellular loop subdomains control specific aspects of ?-aminobutyric acid type A receptor function.

ABSTRACT: The GABA type A receptor (GABA(A)R) is a member of the pentameric ligand gated ion channel (pLGIC) family that mediates ionotropic neurotransmission. Residues in the intracellular loop domain (ILD) have recently been shown to define part of the ion permeation pathway in several closely related members of the pentameric ligand gated ion channel family. In this study, we investigated the role the ILD of the GABA(A)R ?1 subunit plays in channel function. Deletion of the ?1 ILD resulted in a significant increase in GABA EC(50) and maximal current amplitude, suggesting that the ILD must be intact for proper receptor function. To test this hypothesis, we conducted a mutagenic screen of all amino acids harboring ionizable side chains within this domain to investigate the contribution of individual charged residues to ion permeation. Using macroscopic and single channel voltage-clamp recording techniques, we found that mutations within a subdomain of the ?1 ILD near M3 altered GABA apparent affinity; interestingly, ?1(K312E) exhibited reduced partial agonist efficacy. We introduced point mutations near M4, including ?1(K383E) and ?1(K384E), that enhanced receptor desensitization. Mutation of 5 charged residues within a 39-residue span contiguous with M4 reduced relative anion permeability of the channel and may represent a weak intracellular selectivity filter. Within this subdomain, the ?1(K378E) mutation induced a significant reduction in single channel conductance, consistent with our hypothesis that the GABA(A)R ?1 ILD contributes directly to the permeation pathway.

SUBMITTER: O'Toole KK

PROVIDER: S-EPMC3207432 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Discrete M3-M4 intracellular loop subdomains control specific aspects of γ-aminobutyric acid type A receptor function.

O'Toole Kate K KK Jenkins Andrew A

The Journal of biological chemistry 20110908 44

The GABA type A receptor (GABA(A)R) is a member of the pentameric ligand gated ion channel (pLGIC) family that mediates ionotropic neurotransmission. Residues in the intracellular loop domain (ILD) have recently been shown to define part of the ion permeation pathway in several closely related members of the pentameric ligand gated ion channel family. In this study, we investigated the role the ILD of the GABA(A)R α1 subunit plays in channel function. Deletion of the α1 ILD resulted in a signifi ...[more]

PMID: 21903587

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Project description:Computational approaches to tune the activation of intracellular signal transduction pathways both predictably and selectively will enable researchers to explore and interrogate cell biology with unprecedented precision. Techniques to control complex nonlinear systems typically involve the application of control theory to a descriptive mathematical model. For cellular processes, however, measurement assays tend to be too time consuming for real-time feedback control and models offer rough approximations of the biological reality, thus limiting their utility when considered in isolation. We overcome these problems by combining nonlinear model predictive control with a novel adaptive weighting algorithm that blends predictions from multiple models to derive a compromise open-loop control sequence. The proposed strategy uses weight maps to inform the controller of the tendency for models to differ in their ability to accurately reproduce the system dynamics under different experimental perturbations (i.e. control inputs). These maps, which characterize the changing model likelihoods over the admissible control input space, are constructed using preexisting experimental data and used to produce a model-based open-loop control framework. In effect, the proposed method designs a sequence of control inputs that force the signaling dynamics along a predefined temporal response without measurement feedback while mitigating the effects of model uncertainty. We demonstrate this technique on the well-known Erk/MAPK signaling pathway in T cells. In silico assessment demonstrates that this approach successfully reduces target tracking error by 52% or better when compared with single model-based controllers and non-adaptive multiple model-based controllers. In vitro implementation of the proposed approach in Jurkat cells confirms a 63% reduction in tracking error when compared with the best of the single-model controllers. This study provides an experimentally-corroborated control methodology that utilizes the knowledge encoded within multiple mathematical models of intracellular signaling to design control inputs that effectively direct cell behavior in open-loop.

Dataset Information

Discrete M3-M4 intracellular loop subdomains control specific aspects of ?-aminobutyric acid type A receptor function.

Publications

Discrete M3-M4 intracellular loop subdomains control specific aspects of γ-aminobutyric acid type A receptor function.

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