Karnik, Rucha (2010) Trafficking motifs in potassium channels. PhD thesis, University of Leeds.
Available under License Creative Commons Attribution-Noncommercial-Share Alike 2.0 UK: England & Wales.
The pancreatic ATP-sensitive potassium (KATP) channels couple glucose metabolism to excitability of the pancreatic β-cells to regulate insulin secretion. The channel subunits, Kir6.2 and SUR1, are encoded by the KCNJ11 and ABCC8 genes respectively. Genetic polymorphisms in these genes, which reduce channel activity, cause congenital hyperinsulinism (CHI) characterized by insulin hyper-secretion and hypoglycemia. The hERG (human ether-a-go-go related gene) potassium channels,encoded by the KCNH2 gene, contribute to the rapidly activating delayed rectifier K+ current (IKr), which is responsible for rapid repolarisation of the cardiac action potential. Decreased hERG channel function causes the Long QT syndrome 2 (LQTS2) and life threatening cardiac arrhythmias. Several mutations in these two clinically important potassium ion channels alter their surface density leading to disease. Therefore, it is of fundamental importance to investigate the trafficking mechanisms that regulate the surface density of these channels. Techniques in cell biology, molecular biology and biochemistry were employed to identify the molecular basis of Sar1-GTPase dependent ER exit of the KATP and hERG channels in COPII vesicles. Blocking the cargo binding sites on the Sec24 protein of the COPII coat with membrane-permeable synthetic peptides prevented ER exit of both these channels. While the diacidic 280DLE282 sequence on the Kir6.2 subunit of KATP channels was found to be the ER exit motif required for entry of the channels into COPII vesicles at the ER exit sites, such a motif was found to be absent on hERG Cterminus. Further, endocytic trafficking mechanism of hERG channels was studied in recombinant (HEK MSRII and HeLa) and native (neonatal rat cardiac myocytes) systems using cell biological and pharmacological tools. hERG channels were found to be internalised by a dynamin-independent, raft-mediated, and ARF6-dependent pathway. A prolonged block of this pathway revealed that the channels could also undergo internalisation by an alternate dynamin-mediated pathway. Internalised hERG channels were found to recycle back to the cell surface and undergo lysosomal degradation. Degradation of the channels was enhanced when Rab11a-GTPase function was disrupted leading to reduced surface density indicating that recycling is crucial to maintain cell surface density of the channels. Thus this study investigated and compared the previously unknown mechanisms of biosynthetic and endosomal trafficking of the KATP and hERG potassium channels with a conclusion that these processes play an important role in maintaining surface density and thereby in the function of these channels in physiological and patho-physiological conditions.
|Item Type:||Thesis (PhD)|
|Academic Units:||The University of Leeds > Faculty of Biological Sciences (Leeds) > Institute of Membrane and Systems Biology (Leeds)|
|Depositing User:||Ethos Import|
|Date Deposited:||16 Mar 2011 10:23|
|Last Modified:||08 Aug 2013 08:46|