Microsystems for coupled expression and electrophysiology of ion channels

Single-channel electrophysiology is the gold standard for investigating the function of ion channel proteins and their modulation by pharmaceutical drugs, but requires the expression and purification of these channels from cell cultures, which is a notoriously difficult and low-yield process. As an alternative approach, cell-free expression of channel proteins is relatively straightforward, but because of the high cost of the required cell extracts, it is only feasible to obtain very small amounts of expressed protein, insufficient for purification. This proposal aims to develop a new platform for microscale electrophysiology that enables the characterization of ion channels from microliters of a cell-free expression reaction without any purification step.The project will systematically work towards proof of concept, i.e. extensive electrophysiological characterization of a cell-free expressed voltage-gated potassium channel. The main objectives are: 1) the fabrication of elastomeric microwells for the formation of an interdroplet membrane in which ion channels can be incorporated, 2) systematic optimization of the stability of this membrane so that it can be in direct contact with cell-free reaction mixtures, and 3) membrane incorporation of cell-free expressed KvAP for electrophysiological characterization, including drug screening.Ion channels play a central role in many diseases, including seizures, cystic fibrosis, myasthenia gravis, and generalized epilepsy. Many of these channelopathies are chronic conditions which impact heavily on the quality of life. New drugs to treat these diseases or alleviate their symptoms are hence of great value. This project directly addresses a major bottleneck in the drug discovery process by developing a novel method for obtaining pure ion channels that can be directly investigated with microscale electrophysiology.This technology has the clear potential to accelerate ion channel research and drug screening and will benefit biomedical researchers, pharmaceutical companies and the general public. Maximum impact of the research will be realized by professional press releases and by actively approaching the major electrophysiology companies, which have the resources and expertise to commercialize the technology, at specific national and international conferences with an established industry presence.

This project seeks to realize single-channel electrophysiology with cell-free expressed ion channels without any protein purification, which has the potential to dramatically increase the rate and scope of ion channel research, including the screening for new pharmaceutical drugs. As a technology development, it is of immediate interest to the electrophysiological companies who have already successfully commercialized high-throughput patch clamp instruments. Single-channel electrophysiology is a powerful complementary approach; the major companies develop both types of instruments. Given the rapid uptake by the pharmaceutical industry of the high-throughput patch clamp systems that have recently come on the market, we anticipate that our coupled expression and electrophysiology approach for single-channel recordings, which is easy to scale up, will be of considerable interest to the commercial sector. At an intermediate time scale, several years after completion of the project, the pharmaceutical industry could benefit from a powerful and cost-effective commercialized platform for the functional drug screening of ion channels. The route from the first identification of a potential new drug to approval by the relevant health authorities can take up to 15 years but the potential societal benefits are enormous, especially in view of an ageing population and an increasing socio-economical drive towards early intervention. The platform developed in this project will enable pharmaceutical companies and public sector biomedical laboratories to substantially increase the throughput and widen the scope of their drug screening programmes, hence offering the prospect of an increase in the amount of approved drugs for the many diseases that have an ion channel basis. In particular, our project aims to obtain single-channel recordings of the large voltage-gated potassium channel KvAP, which represents human voltage-gated potassium channels, and to demonstrate the effects of known drugs on KvAP activity. We will systematically work towards the development of protocols for the cell-free expression and membrane incorporation of KvAP, and we expect that these will also be applicable to the biomedically important human analogs of this channel. The pharmaceutical market for specific and highly efficacious channel inhibitors/modulators is enormous, because ion channel-related diseases such as seizures, cystic fibrosis, myasthenia gravis, and generalized epilepsy effect a large number of people. It is for this reason that most of the big pharmaceutical companies, as well as many smaller biotech companies, have active programmes for the development of channel-targeting drugs. In summary, the economic benefits of our novel platform for single-channel electrophysiology concern electrophysiological and pharmaceutical companies, which form a substantial part of the UK economy. At a longer time scale, societal benefits could arise from an increased availability of new drugs, reducing the impact of (chronic) disease and enhancing the quality of life of an ageing population. A wider range of manageable diseases may in turn reduce the cost of the healthcare system and lead to further economic benefits. Realization of this potential requires commercialization of the developed technology after proof of concept has been demonstrated. Maximum impact of the research will be realized by professional press releases and by actively approaching the major electrophysiology companies, which have the resources and expertise to commercialize the technology, at specific national and international conferences with an established industry presence.