Regulation of endocytic trafficking of KATP channels by protein kinase C

Lay summary: Type-2 diabetes is characterized by an increase in blood glucose. Prolonged elevation of plasma glucose levels could result in kidney, heart and nerve damage, often leading to blindness, kidney failure, amputation of the extremities and, almost certainly, premature death. In normal people, such increased levels are avoided by the regulated release of the chemical messenger insulin from the pancreas. This is because insulin helps removal of excess glucose by muscle, liver and fat tissues. However, in diabetes, the tissues become resistant to insulin and fail to remove excess glucose from the blood. Although pancreas initially responds to this by pumping more insulin into the blood, it gradually loses this ability. As a result, insulin levels drop resulting in severe diabetes. The reasons for the secretion failure are not entirely clear, but research has demonstrated that a protein, known as the KATP channel, plays a crucial role. This channel sits in the membrane which surrounds the insulin containing pancreatic cell. Whenever, blood glucose levels rise, for example after a meal, the channel sends out electrical signals to insulin storage sites within the cell and trigger insulin release. Genetic screening studies revealed that mutations in the genes encoding the channel can predispose individuals to type-2 diabetes. The mechanisms by which the channel sense rise in blood glucose levels and triggers insulin release are not fully understood. The aim of this proposal is to improve our understanding of the mechanisms and exploit the understanding to develop novel strategies for treatment or prevention of insulin secretion disorders including type-2 diabetes, the incidence of which is reaching endemic proportions. The work will employ the state-of-the art techniques in modern biology and will be carried out by a team of expert scientists whose major goal is to make discoveries that lead to safer and effective disease treatments.

Pancreatic ?-cells secrete insulin in response to increase in blood sugar, a process known as glucose stimulated insulin secretion (GSIS). This ability can be progressively lost by the ?-cells leading to type-2 diabetes. Large scale genome-wide association studies have established a strong link between type-2 diabetes and polymorphic mutations in KCNJ11. KCNJ11 encodes Kir6.2, which together with SUR1, forms the KATP channel, well known for its role in GSIS. Some genetic mutations in Kir6.2 and SUR1 cause severe diseases, such as congenital hyperinsulinism and neonatal diabetes by affecting the trafficking of the channel to, and out of, the plasma membrane. This raises the previously unrecognized possibility that cellular signals may alter channel trafficking and thus play a physiological role in GSIS. Using a pancreatic ?-cell line, we found that acute pharmacological activation of protein kinase C (PKC) stimulates KATP channel activity, but chronic activation decreases its cell surface density by reducing the rate of recycling of internalised channels. The effect on recycling is significant because glucose and other insulin secretagogues stimulate PKC and PKC activation enhances insulin secretion. Thus we propose that PKC induced downregulation of channel density stimulates insulin secretion.

Our hypothesis is that kinetic retention from recycling provides a mechanism for tightly tuning the abundance of KATP channels at the plasma membrane by shifting the steady-state equilibrium between intracellular compartments and the plasma membrane and that this is linked to GSIS. We will test the hypothesis using molecular, cell biological, biochemical and physiological approaches. Specifically, we will aim to:
1. identify the PKC isoform(s) responsible for activation and downregulation of KATP channels,
2. determine the PKC target/ site responsible for downregulation,
3. investigate the mechanism of recycling and how PKC reduces recycling, and
4. use the information and molecular tools (developed in aims 1-3) to understand the role of downregulation of channel density in insulin secretion.
This integrated, multidisciplinary approach will provide novel molecular information on how the function and trafficking of KATP channels are regulated by PKC and the role of regulated trafficking of the channel in GSIS, a fundamentally important aspect that has not been hitherto addressed. The studies may reveal novel therapeutic opportunities for treatment of insulin secretion disorders. Although our focus is the pancreatic KATP channel, the results will be applicable to KATP channels in other tissues, including the heart and brain, where PKC and KATP channels play a protective role in ischaemia.