The London Consortium for High-Throughput Electrophysiology

Every biological cell in humans, animals, plants, or of bacterial origin is surrounded by a hydrophobic envelope. The membrane coat is essential for maintaining the function of cells. It helps to keep water-soluble nutrients and essential components such as DNA inside the cell. Similarly, membranes block toxic substances from entering and damaging the cellular interior.

A controlled exchange of ions/chemicals across the membrane is, however, key to support life and achieve higher function. Fresh nutrients have to be able to enter and support growth, while molecular cargo (e.g. antibodies) has to be exported from the production site inside human immune cells across the membrane to fight pathogens.

Control over cellular entry and exit is executed by ion channels and membrane transporters. Ion channels are remarkable gatekeepers which play a vital role in nerve and muscle cells. They decide which types of ion pass across the membrane, and also open or close in response to chemical and physical stimuli. Dysfunction of the molecular structure can lead to dramatic health consequences such as cognitive impairment or neurological or muscular diseases. Ion channels in horticulture-damaging insects, by comparison, are of great agricultural interest as they represent a molecular 'Achilles heel' to be exploited in the development of insecticides. Furthermore, membrane transporters in bacteria can spread resistance-conferring genes to other pathogens. Finally, membrane-spanning pores are of interest in biotechnology where they are key components in next-generation sequencing devices for better and cheaper diagnosis of cancers.

A detailed understanding of ion channels and membrane transporters is hence of tremendous significance to health, agriculture and the economy.

Our proposal aims to achieve a step-change in the characterisation of the membrane channels. Conventional analysis techniques are slow due to their serial nature. The methods also require a considerable amount of manual involvement which can increase experimental variability and lead to unreliable scientific results. These bottlenecks will be overcome by new equipment to characterise ion channels and transporters in a highly parallel and automated fashion. Scientific data will be generated at high speed and unprecedented volume.

The output of our study will be manifold. Firstly, the new equipment will improve the scientific infrastructure of the partner universities and indeed all universities in Greater London as the devices will be the first in London. In addition, the equipment will have benefits in physiology and health by examining ion channels from the brain and heart to understand their molecular function and interaction with drugs. Similarly, bacterial translocation machines for DNA will be studied to understand the molecular mechanism for the spread of antibiotic resistance. In agriculture, the study of insect channels will help explain why different candidate insecticides are either activating or inhibiting. Finally, novel membrane pores will be developed for analytical and diagnostic devices.

Analysing ion channels and membrane proteins is vital for improving our understanding as to how ions and substrates transport across biological membranes. Ion channels play a crucial role in signal propagation in neurons and muscle cells while membrane pores transport large biomolecular cargo such as antibiotic resistance genes to other cells.

Electrophysiology is the method of choice for characterising ion channels but its time-consuming nature calls for simplified tools to close the performance gap to modern high-throughput genetics and proteomic techniques.

This project will address the demand for high throughput, automatised, standardised, and easy-to-use electrophysiology equipment. The requested state-of-the-art kits for parallel nanopore recording will (i) speed up research and match other high-throughput methods, (ii) increase the generation of high-quality data, and (iii) open up completely new fields of investigation.

Three different electrophysiology kits are requested: Kit 1 is for parallel semi-automated analysis of in vitro reconstituted membrane proteins such as those from prokaryotes. Kit 2 is for the fully automated yet experimentally flexible electrophysiological characterization of ion channels expressed in cells. Kit 3 is also for the in vivo analysis but constitutes a high-throughput screening platform under pre-selected conditions.

Our team of world-leading researchers at UCL, Birkbeck, and Queen Mary will capitalise on the new research equipment and conduct high-impact science on biologically and biotechnologically important membrane proteins from prokaryotes and eukaryotes. This equipment will enable new types of experiment and significantly increase the number of questions that can be addressed which would be difficult or impossible to do otherwise. Furthermore, although additional users within the greater London area not included within this proposal they will gain access to high-throughput electrophysiology facilities.

The potential academic, economic, educational and public impact of the proposed research are numerous, because receptors and ion channels, studied in this proposal, are essential components in human physiology and health, in agriculture, and are present in most tissues.
The beneficiaries of this project will include:

(i) the academic community covering many disciplines by obtaining new equipment to analyse ion channels with unprecedented speed, volume throughput and controlled data quality, as described in the previous section.

(ii) the commercial private sector and economy, by enabling a deeper understanding of the physiological roles of ion channels and nanopores, and offering novel molecular targets with the ability to probe drug-target interactions.

(iii) students and post-doctoral RAs by widening participation in specialist research techniques, allowing training on next-generation scientific equipment

(iv) the public by enhancing the quality of life through new research.

With regard to beneficiaries (ii), the project will profit from established collaborations with industry, such as Pfizer Neusentis, Bayer Animal Health, Xention, and Oxford Nanopore Technologies. The collaborations with Pfizer and Bayer have the ultimate goal of contributing to rational drug and insecticide/mitocide design, which are of interest economically to the pharmaceutical and agricultural industries. The new equipment will also be useful for planned drug development at UCL with the aim of linking to industry and most important, using industrial- (e.g., Eisai) and UCL-based chemical libraries. As exemplars of the industrial links, the collaboration with Xention aims to develop inhibitors of potassium channels for the treatment of atrial fibrillation. The collaboration with Oxford Nanopore Technology aims to develop new nanopore sensors for the lab-free and electrical sensing approach particularly for larger protein analytes that cannot be sensed easily with existing pores. The project is also open to commercial exploitation with other partners in order to develop new pharmaceutical/agricultural agents.

(iii) Postgraduate (MSc, PhD) and post-doctoral research assistants will benefit as they will be introduced to electrophysiological methods with devices that can be operated with minimal training thereby enabling students to rapidly characterise the ion channels they have isolated.

(iv) The public will benefit as our science will have an impact on the lives of people in terms of living standards and health. For example, ion channels are critical for numerous physiological processes and the channels studied in our projects are integrally involved in regulating heart rate, pain sensation, and also in neural processing, and the causation of neurological diseases. In addition, the ability to more efficiently produce crops not destroyed by insects, using more specifically-targeted insecticides that do not harm either humans, plants or non-targeted insects, would also have major impacts on quality of life with the ability to produce crops more effectively without harmful side-effects to farmers, the human population at large, and the environment.