NANIONLIGAND · Development of a new method for brief compound exposures to ion channels proteins using an automated patch clamp platform

Development of a new method for brief compound exposures to ion channels proteins using an automated patch clamp platform

Advanced fluidics for compound applications in drug testing In modern drug development, the typical approach for finding a new active substance is screening of a large number of compounds against a specific drug target. Upon application of the compound, any modulation of the drug targets activity, e.g. increase or decrease of a proteins activity, is monitored to judge the compounds effect and potency. For this approach, sophisticated methods for drug application are needed, as some receptors tend to desensitise quickly and hence need fast and brief compound application to be activated. Within this project, the focus was on ion channels as drug targets and their pharmacological modulation. Ion channels are transmembrane proteins residing in virtually all cell types in animals, fungicides, plants and bacteria. Ion channels are directly or indirectly involved in chronic and acute disease such as cholera, hypertension, deafness, startle disease and have been linked to immuno- and inflammatory reactions and acute and chronic pain. Ion channels as such, are highly drugable targets, since they are relatively easily modulated by small molecules, reflected in the fact that 15 of the 100 top selling drugs target ion channels. Ion channels are classified based upon the stimuli required for activation. The stimuli can be a change in membrane voltage (voltage gated), binding of a ligand (ligand gated) or mechanical stress (mechano sensitive). When investing ligand gated ion channels there are a few important parameters to be considered for obtaining relevant data. Firstly, continuous recording should be possible before, during and after the application to be able to determine the initial peak current response on the addition of the drug, the following steady state current, and the eventual wash out of the compound. Receptor desensitisation is a phenomenon occurring at almost every ligand gated ion channel, causing the ion channel to enter a closed, inactive state despite the constant stimulation. A rapid solutions exchange is therefore essential, so that the cell is not exposed to a concentration gradient before reaching the highest concentration. The gold standard technique for investigating ion channel function is the patch clamp technique. Here, a glass microelectrode is used to record ionic currents passing through ion channels of a single cell. Minute trans-membrane currents can be resolved with high sensitivity in the sub-pA range with µs time resolution, thus capable of resolving single channel events. Unfortunately, being the best method for extracting information dense data on ion channels and their effectors, patch clamp is an extremely labour intensive technique, requiring a spacious and expensive experimental set up, operated by a person with high and lengthy education. The throughput is ultralow (about 10 cells per day) which is insufficient for screening and validation purposes in drug development. The limitations of conventional patch clamping have spurred the development of automated patch clamp robots, to simplify the patch clamp procedure and increase the data throughput. Within the project work, a method has been developed for brief compound application to cells patch clamped on a planar patch clamp substrate. The developed method utilises microfluidic channels for liquid administration, where two solutions were aspirated in sequence by a robotic pipettor, so that they form a stack of two distinct zones of solutions inside the pipette. When added to microfluidic channels, the patch-clamped cells will rapidly be exposed to the stacks, in sequence, so that the exposure time to the first solution of the stack can be very short. In this way, ligand gated ion channels can correctly be investigated without the risk of deleterious effects caused by receptor desensitisation. By combining planar patch clamping with microfluidic channels and using the stacked application approach, only minute volumes of solutions, compounds and cells are required. The dimensions and volume of the channel allows for rapid exchange of solutions present in the channel, which is important for data accuracy and data quality. The entire process of liquid handling and patch clamping a cell is automated. The developed methodology is extremely useful for efficient investigation of protein analysis and beneficial for basic research of ion channel function and pharmacology. It also serves as a valuable platform in drug development searching for drug candidates acting on ligand gated ion channels. It indeed allows for the fast development of better drugs targeting this class of ion channels and in ion channel research, it helps gaining more knowledge of a constantly growing family of proteins.

Data: CORDIS, © European Union