Tyrosine Kinase and Protein Kinase A Modulation of α7 Nicotinic Acetylcholine Receptor Function on Layer 1 Cortical Interneurons

Date

2014-12-18

Authors

Komal, Pragya

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Abstract

Nicotinic acetylcholine receptors (nAChRs) are a major class of ligand-gated ion channels in the brain, with the α7 subtype of nAChRs playing an important role in attention, working memory and synaptic plasticity. Alterations in expression of α7 nAChRs are observed in neurological disorders including schizophrenia and Alzheimer’s disease. Therefore, understanding the fundamentals of how α7 nAChRs are regulated will increase our comprehension of how α7 nAChRs influence neuronal excitability, cognition and the pathophysiology of various neurological disorders. The purpose of this thesis was to investigate how protein kinases modulate the function and trafficking of α7 nAChRs in CNS neurons. In chapter 2, I describe a novel fast agonist applicator that I developed to reliably elicit α7 nAChR currents in both brain slices and cultured cells. In chapter 3, I examined whether an immune protein in the brain, the T-cell receptor (TCR), can modulate α7 nAChR activity. Activation of TCRs decreased α7 nAChR whole-cell recorded currents from layer 1 prefrontal cortical (PFC) neurons. TCR attenuated α7 nAChR currents through the activation of Fyn and Lck tyrosine kinases, which targeted tyrosine 442 in the M3-M4 cytoplasmic loop of α7. The mechanisms of the attenuated α7 current were contributed by a TCR mediated decrease in surface receptor expression and an attenuation of the α7 single-channel conductance. TCR stimulation also resulted in a decrease in neuronal excitability by negatively modulating α7 activity. In chapter 4, I tested whether PKA can modulate α7 nAChR function in CNS neurons. The pharmacological agents PKA agonist 8-Br-cAMP and PKA inhibitor KT-5720, as well as over-expressing dominant negative PKA and the catalytic subunit of PKA, demonstrated that activation of PKA leads to a reduction of α7 nAChR currents in HEK 293T cells and layer 1 cortical interneurons. Serine 365 of the M3-M4 cytoplasmic domain of α7 was necessary for the PKA modulation of α7. The mechanism of down-regulation in α7 receptor function was due to decreased surface receptor expression but not alterations in single-channel conductance nor gating kinetics. The results of this thesis demonstrate that α7 nAChRs constitute a major substrate for modulation via TCR activated tyrosine kinases and the cyclic AMP/PKA pathway.

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Keywords

nicotinic acetylcholine receptors, ion channels, protein kinases, modulate, neurons

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