Quality control of heteromultimeric membrane proteins includes mechanisms to ensure that only properly assembled complexes reach the cell surface. ER retrieval signals that are recognised by the COPI vesicle coat play a central role in such trafficking checkpoints. In many cases R-based ER retrieval signals are presented on one or all subunits of the heteromultimeric complexes. ATP-sensitive potassium (KATP) channels provide an example of a heteromultimeric cargo protein presenting eight R-based signals (one in each Kir6.X and SUR subunit of the 4+4 heterooctamer). KATP channels are metabolic sensors that couple cellular energy metabolism to electrical excitability by controlling the membrane potential at the plasma membrane. By this mechanism they are involved in glucose-stimulated insulin secretion, regulation of skeletal muscle excitability, cardioprotective ischemic preconditioning, neurotransmitter release and smooth muscle relaxation. In this thesis, we have established Blue Native Poly-Acrylamide Gel Electrophoresis (BN PAGE) as a method to characterize KATP channel complexes from different rodent tissues. Knock-out mice lacking individual subunits were employed as controls to probe the composition of the observed macromolecular complexes. This approach was complemented by density gradient centrifugation, subcellular fractionation and glycosylation analysis. We demonstrate the existence of distinct macromolecular complexes containing the pore-forming subunit Kir6.2 in pancreatic islets, the brain and the heart. Furthermore, we confirm assembly-dependent forward transport of channel subunits for endogenously expressed KATP channels. We report that the steady-state levels of Kir6.2 are drastically reduced in the brains of SUR1 knock-out mice, whereas we identify both fully assembled KATP channels and putative biogenetic intermediates in the hearts of SUR1 knock-out mice. Our results also suggest that SUR1, in native neuronal and cardiac KATP channels, is differentially glycosylated. Finally we have analyzed COPI-based recognition of Arg-based signals and KATP channel assembly in a mouse model (nur17) with a point mutation in the gene encoding the delta (delta)-subunit of COPI, in close vicinity to the reported binding site of Arg-based signals. We demonstrate that both the recognition of Arg-based signals and the assembly of several multimeric cargo proteins, including the KATP channel, are unaffected in the nur17 mouse. When analyzing the subunit composition of the nur17 COPI coat by two-dimensional BN SDS PAGE we discovered the presence of distinct COPI complexes in the mutant. We have also provided evidence that the association of COPI with a specific membrane compartment is compromised in the nur17 mouse. In conclusion, we have extended the study of a model COPI cargo, the KATP channel complex, to the physiologically relevant context. Furthermore, we have also provided initial insights into the molecular mechanism underlying the physiological consequences of the nur17 delta-COP mutation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:764204 |
| Date | January 2011 |
| Creators | Arakel, Eric |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/copibased-quality-control-of-native-atpsensitive-potassium-channels(ee92d5cf-54e2-4d0e-8c56-7475cb016b64).html |
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