Membrane trafficking of ATP-sensitive potassium channels

ATP-sensitive potassium (KATp) channels are known to play a vital role in the regulation of insulin secretion from pancreatic 0-cells. Changes in the ratio of [ATP]/[ADP] within the cell are known to regulate the activity of channels, but very little is known how the number of channels at the cell surface is regulated. The number of channels in the plasma membrane could be regulated in two ways; firstly by regulating the overall population of channels within the cells by increasing/decreasing the rates of channel synthesis or degradation, and secondly by regulating the insertion and removal of channels from the plasma membrane. The aim of the current study is to investigate the involvement of both of these mechanisms in regulating the cell surface density of KATp channels. It is shown that a sudden decrease in glucose concentration causes a rapid stimulation of KATp channel synthesis as shown by both immunocytochemistry and protein chemistry in both INS-le and isolated mouse pancreatic ß-cells. The intensity of fluorescence associated with Kir6.2 and SUR1 was - 2.5 fold greater in cells incubated with 3 mM compared to 25 mM glucose. This sudden increase in channel numbers is due to an increase in the rate of translation of pre-existing mRNA and may be mediated by the activation of AMP-activated protein kinase. Despite the - 2.5 fold increase in channel numbers only a small, but non significant, difference in cell surface density was observed as determined by patch-clamp. The internalisation of KATp channels with an extracellular HA-epitope was also investigated in stably transfected HEK293 cells. Channels were seen to internalise rapidly from the cell surface into a perinuclear compartment. The trafficking itinerary of these channels has been found to include the sorting endosome, late endosome and elements of the trans-Golgi network. Upon inhibition of protein kinase-C activity the internalised channels are redirected into a pathway which allows rapid recycling of the channels. Trafficking and function of KATP channels has also been shown to be disrupted by mutations of Kir6.2 known to cause congenital hyperinsulinism. In summary, it has been demonstrated that both regulated expression and trafficking are likely to be involved in determining the cell surface density of pancreatic KATP channels.

Name dc:type.qualificationname

Ph.D

Level dc:type.qualificationlevel

doctoral

Grantor dc:publisher.institution

University of Leeds

Year dc:date.issued

2005

Author dc:creator

• Smith, Andrew James

Identifier

uk.bl.ethos.416843

OAI identifier oai:identifier

oai:etheses.whiterose.ac.uk:365