Functional dynamics of the KATP channel

Ion channels are essential for all life on Earth. These tiny gated pores sit in the membrane which surrounds every one of our cells, and their opening and closing underlies everything that we do. Your ability to read this page, to move your limbs, to think and speak is down to the activity of ion channels. They govern every aspect of our lives, from conception to the grave, controlling fertilization, the beating of our hearts, our ability to fight infection, even consciousness itself. A multitude of medicinal drugs and many poisons work by regulating the activity of these minute molecular machines, and impaired ion channel function is responsible for many human and animal diseases. Their important functional roles are explained in the book 'The Spark of Life' by one of the applicants of this grant (Frances Ashcroft).

This project is focused on an ion channel known as the KATP channel. It plays a very important role in the regulation of blood glucose levels because it controls the release of the hormone insulin from the beta-cells of the pancreas. Insulin is essential for ensuring blood glucose levels do not rise too high and an insufficiency of insulin results in diabetes. Chronic elevation of blood glucose is deleterious to many cells, and gives rise to kidney disease, eye disease, heart disease and loss of sensation in the peripheral limbs (which often leads to unrecognized trauma, necessitating amputation). Understanding KATP channel function is therefore of high priority.

We have shown that when the KATP channel is pore is open, insulin is not released and when the pore is shut insulin is secreted. Both glucose and the sulphonylurea drugs used to treat type 2 diabetes stimulate insulin release by closing the channel. We have also shown that mutations in KATP channel genes cause a rare inherited form of diabetes (neonatal diabetes or ND), which presents within the first six months of life. The mutant channels are no longer closed properly by glucose, impairing insulin release. However, sulphonylurea drugs are still effective. This finding has enabled most ND patients to switch from insulin injections to oral tablet therapy, with considerable improvement in their clinical condition and quality of life.

One aim of the current grant is to understand more precisely how glucose closes the KATP channel. We know this requires breakdown (metabolism) of the sugar but we still don't fully understand how metabolites - such as the nucleotides ATP and MgADP - interact with the channel to influence its opening and closing. Nor do we fully understand how many of the ND mutations impair this process. A second aim is to identify the binding site for sulphonylurea drugs on the channel, and determine how drug binding promotes channel closure. This should facilitate the design of new and potentially better drugs to treat diabetes. To address these aims, we are developing a novel approach to studying the binding of ligands (drugs and nucleotides) to their receptors that has high spatial and temporal resolution. This should result in a new tool for studying other membrane proteins (such as ion channels, transporters and receptors) many of which cause common human diseases, such as cystic fibrosis, or are major drug targets. Thus our project will have important general, as well as KATP-channel-specific, outcomes.

Advances in structure determination via cryo-EM have led to an explosion in the number of membrane protein structures. What is now required are methods that complement these high resolution snapshots by providing information on the dynamics of proteins expressed in their native membranes. We have developed two novel techniques that address this fundamental problem: ligand-binding FRET (lbFRET) and transition metal FRET (tmFRET).

We will use these methods to study the regulation of the ATP-sensitive K channel (KATP) by anti-diabetic sulphonylurea (SU) drugs and nucleotides. This octameric complex, comprising 4 pore-forming Kir6.2 and 4 regulatory SUR subunits, has 3 types of nucleotide-binding sites (NBS) (12 in total). ATP/ADP binding to Kir6.2 inhibits KATP activity, and MgATP/ADP binding to the NBS1 and NBS2 of SUR, activates KATP. LbFRET measures ligand binding to channels in native membranes with high spatial and temporal resolution by measuring FRET between a fluorescent unnatural amino acid (ANAP), engineered into the protein, and a fluorescent ligand. This will allow us to discriminate ATP/ADP binding to each NBS independently and study their affinity, specificity, and requirement for co-factors (Mg). We will also use this method to define the binding site for SU on KATP, and the allosteric interplay between SU and nucleotide binding. tmFRET uses coloured transition metal ions (Ni, Co, Fe) as acceptors, enabling measurement of very small distances (8-30 Å). This method, coupled with molecular dynamics, will allow us to study ligand-induced conformational changes in KATP, and thus determine how long-range communication between different proteins in the KATP complex is accomplished. The recent cryo-EM KATP structures provide an excellent starting point for our studies. We will combine our new techniques with high-resolution electrophysiological analysis, so that binding/structural changes can be directly correlated with changes in functional state.

The aim of the project is to reveal how nucleotides and drugs interact with the two subunits of the KATP channel and how this is translated into changes in channel opening/closing. A detailed mechanistic understanding of these processes is needed to underpin therapeutic strategies targeted at the KATP channel, which plays a key role in insulin secretion. Impaired nucleotide regulation of the channel leads to diabetes (or its converse, hyperinsulinism). Its regulatory subunit, SUR1, is also the target for the anti-diabetic sulphonylurea drugs, which are in widespread clinical use. Yet despite the scientific and clinical importance of the KATP channel, our current understanding of the interaction between drugs, nucleotides and channel activity is poor, in part because it is extremely complex. To our knowledge, sulphonylureas are unique in that their efficacy is modulated by metabolism. Understanding how drugs and nucleotides modulate KATP channel activity at the molecular level may help with the rational design of new therapeutic agents. These are urgently needed to treat type-2 diabetes, which affects >4 million people in the UK (>350 million worldwide) and consumes 10% of the NHS budget.

We have a track record of successfully translating the fruits of our basic science studies into the clinic. Over 90% of patients with neonatal diabetes caused by activating KATP channel mutations have now transferred from insulin to oral SU drugs (which close their open KATP channels). This has resulted in dramatic improvements in their clinical condition and quality of life. It also costs less. There is evidence the KATP channel also contributes to the aetiology of type 2 diabetes, although the molecular mechanism(s) involved are not well understood. A better understanding of how the channel is regulated by glucose metabolism (ATP, ADP etc), will be invaluable in this respect.

SUR1 is a member of the extensive ABC transporter family, which includes PgP, CFTR and many other ABC proteins of major physiological and pathological importance. All members of this family share common nucleotide-binding domains and nucleotide binding/hydrolysis regulates their function. Our results will therefore shed light on nucleotide interactions with other ABC proteins. They may also help explain why, uniquely, SUR does not act as an ATP-driven transporter, but rather as an ion channel regulator. Our data will therefore be of considerable significance to a wide scientific community.

The long-term socio-economic benefits of this study will include improvements in the design and development of drugs targeted at other membrane protein receptors, as our approach should be easily transferrable to other proteins. This is expected to have a major impact upon the rational design of drugs targeted at many other membrane receptors, particularly ABC transporters. This will impact the early stages of drug design and development and has the potential to reduce both the time and the overall costs involved in drug development.

Finally, as the general public has a tremendous curiosity about science, we intend to host an extensive outreach programme of public engagement. This will engage with a wide section of the community and is expected to have a major impact on the public perception of science and public trust in UK-based science. Our previous experience is that it will also have the added benefit of stimulating interest in STEM subjects within the next generation of potential leaders in both science and health-related sectors. This has especially been the case for FMA's popular science books. Our collaboration with the dance company Motionhouse is uniquely significant in this respect as it will reach a far more diverse audience than most public engagement activities.