There is a significant knowledge gap in understanding how multi-subunit membrane proteins are inserted, folded, and assembled in the membrane of the endoplasmic reticulum (ER), a cellular organelle for protein quality control with the assistance of molecular chaperones. We use ?- aminobutyric acid type A (GABAA) receptors to answer this question. The GABAA receptors, the primary inhibitory neurotransmitter-gated ion channels in the mammalian central nervous systems, are a pentameric protein complex. Despite extensive research on GABAA receptors physiology on the plasma membrane and their role in controlling the inhibition-excitation balance in neural circuits, little effort has been made to investigate how the protein homeostasis (proteostasis) network regulates the folding and assembly of GABAA proteins in the ER. This brings a significant barrier for the treatment of genetic epilepsies because numerous epilepsy-associated mutations in GABAA receptor subunits cause subunit protein misfolding in the ER and/or disrupt assembly of the pentameric complex, leading to decreased cell surface localization of the receptor complex and imbalanced neural circuits. Here, specifically, our affinity purification-mass spectrometry-based proteomics analysis identified a potential Membrane Protein Assembly Chaperone Complex (MPACC) that interacts with endogenous GABAA receptors, consisting of heat shock protein 47 (Hsp47) in the ER lumen and the ER membrane protein complex (EMC) in the ER membrane.
In Aim 1, we will test our hypothesis that Hsp47 positively regulates the assembly of endogenous GABAA receptors in the ER and their functional surface expression.
In Aim 2, we will test our hypothesis that the EMC positively regulates the assembly of GABAA receptors in the ER membrane and thus their functional surface expression.
In Aim 3, we will manipulate the assembly chaperone complex in the ER to correct the function of pathogenic GABAA receptors.
Loss of function of GABAA receptors, the primary inhibitory ion channels in the human brain, leads to neurological diseases, including epilepsy. Numerous mutations in the receptors cause inefficient subunit assembly, resulting in reduced receptor function and thus epilepsy. Here, we propose to elucidate how the proteostasis network regulates the multi-subunit assembly process and use the principles acquired to correct the function of pathogenic mutant receptors.
