CELL SURFACE EXPRESSION OF ACID SENSITIVE K2P CHANNELS: DISSECTING THE ENDOCYTIC AND RECYCLING PATHWAYS

All cells maintain different concentrations of ions (e.g. sodium, potassium, chloride) across their cell membrane and results in a difference in charge (called the resting membrane potential; RMP) between inside and outside the cell. This membrane potential is of critical important to every cells function. Excitable cells (e.g. heart (or cardiac), brain (or neuronal) or muscle cells) could not function without a RMP. The RMP is maintained by controlling the movement of ions between the inside and outside of the cell. Ions need to travel through specialised channels called ion channels to move from one side of the cell membrane to the other. This proposal focuses on an ion channel that is key to maintaining the RMP of cells, these channels are called two-pore domain potassium (K2P) channels. K2P channels allow potassium ions to pass from the inside of the cell to the outside thus helping the cell maintain its appropriate RMP. These channels have been implicated in playing roles in the response of cardiac and neuronal cells to substances such as drugs and anaesthetics as well as natural substances like hormones, neurotransmitters and changes in oxygen tension.
An important feature of K2P channels is that once at the surface of the cell these channels are active and allow potassium to move from areas of high potassium concentration (inside the cell) to areas of low potassium concentration (outside the cell). Because these channels allow potassium to leak out of the cell once they are inserted into the cell membrane it is critical to closely regulate the number of K2P channels on the cell surface at any given time. Similarly it is important to understand how the cell increases or decreases the number of the channels at the cell surface in response to different environmental messages or needs.
The work in my laboratory focuses on the different ways a cell can control the production and delivery of ion channels to the correct location within the cell. The focus of this proposal is on the mechanisms the cell uses to retrieve K2P channels from the cell surface and how this process can be disrupted or regulated. This is important as understanding the balance between channel delivery to and recovery from the cell surface will provide critical understanding of the mechanism by which cells control not only their RMP but their ability to respond to their environment and perform their cellular functions.

Two-pore domain potassium (K2P) channels underlie the leak currents which contribute to the resting membrane potential of both excitable and non-excitable cells. Hence these channels are key components in a range of physiological processes. This proposal focuses on the acid sensitive K2P (or TASK) channels, K2P3.1 and K2P9.1, which have been implicated in cardiac and nervous tissue responses to hormones, neurotransmitters and drugs, as well as having roles in ventilatory regulation and responses to volatile anaesthetics. As K2P channels are active at physiological resting membrane potentials, changes in channel number at the plasma membrane drastically alters the electrical properties of the cell. For this reason, control of cell surface expression of K2P channels is of paramount importance to cell function. The applicant's previous work has provided valuable information on the regulation of forward transport of these channels. To date, no information is available regarding the removal of these channels from the cell surface. Using a combined confocal microscopy, flow cytometry and electrophysiological approach, this proposal will assess the importance of different endocytic pathways to channel internalisation. It will determine the fate of TASK channels following endocytosis and will identify regulators of TASK channel internalisation and recycling. Understanding the balance between channel delivery to and recovery from the cell surface will provide critical understanding of the mechanisms by which cells control not only their innate excitability and cellular function but also their responses to external stimuli.

The research outputs from this study will advance our basic knowledge of how acid-sensitive K2P (TASK) channels, an important class of ion channels, function at the molecular level. This project is predominantly science led and driven by a desire to understand the molecular basis of ion channel surface expression. The long-term potential impact of understanding the regulation of the synthesis, degradation and turnover of TASK and other ion channels is significant. In general terms, cells adjust their protein composition in response to specific cellular and environmental cues. Determining how the cell surface expression of K2P channels fluctuates and how these fluctuations are controlled will provide valuable information about the many physiological and pathophysiological processes to which K2P3.1 and K2P9.1 have been linked. Furthermore, knowledge gained through this work may impact the future design of novel therapeutic strategies to target these channels.

Academic impact will be achieved from enhancing the knowledge of not only ion channel regulation but also cell trafficking and endocytosis mechanisms, with the result of connecting both physiological and cell biological knowledge. This will have impact on a number of disparate research fields and has the potential in crossing these fields to advance the knowledge of both.

As techniques to study membrane protein endocytosis and recycling are currently being developed, the studies outlined here and their expected optimisation have the potential to advance current research and to develop novel approaches to the study of membrane protein cellular localisation.

Closer to home, the PDRA employed on this grant will also receive training in new areas to enhance her career development. She is already highly skilled in fluorescence microscopy and the project will provide her with an opportunity to extend these skills and also to attend training to develop her electrophysiological skills.

Economic and societal impact will be achieved immediately by our continued work to increase public awareness and understanding of science and societal issues. Both the PI (IO) and the PDRA are committed and have a track record for engaging in public awareness projects. IO visits local schools on a regular basis and has run interactive sessions with students (5-18 years) and spoken on both her research and also scientific careers. IO also participates in forums which focus on promoting women in science and acts as a mentor to postdoctoral workers endeavouring to make the transition from PDRA to PI. IO is a strong advocate of encouraging young scientists, and as well as speaking at schools, taking part in Science week events, IO also host workshops within her lab and has provided individual students with an opportunity to work within her lab both on summer vacation studentships or longer-term basis.

In a longer timeframe, Economic and Societal impact will be achieved due to the varied roles TASK channels play in normal physiology and pathophysiology and the potential of attracting research and development investment from pharmaceutical companies. TASK channels have been proposed as sensors for acidosis, hypercapnia and hypoxia and are key in chemoreception. Furthermore, TASK channels have roles within the cardiovascular and immune systems as well as being important in aldosterone secretion and production. Changes in protein expression of TASK channels have been identified in Birk Barel mental retardation syndrome as well as prostate, breast and colon cancers. Significantly, in terms of potential clinically relevant targets, TASK channels have been proposed to mediate the effects of anaesthetics. Novel findings could be exploited for commercialisation either in partnership with a pharmaceutical company or as a spin out to create new products or adaptation of current products to target TASK channels with significant potential societal and economic benefits.