General objective. The objective of this interdisciplinary project between electroengineering and electrophysiology is twofold: i) to develop a force-controlled patch clamp (pc-FluidFM) starting from the recent invention of the FluidFM; and ii) to carry out original experiments with cardiac cells as demonstration that the pc-FluidFM can contribute to answer relevant electrophysiology questions.
State of the art. Patch clamping represents a powerful methodology both in fundamental biology research (investigation of ion-channel-related questions) and in applied medicine research (test of membrane-protein associated drugs). Although already invented in the seventies and although indispensable in drug screening to study potential adverse effects of drug candidates on ion channels, this technique is hardly finding its place outside re-search laboratories mainly because it is a very low throughput technique and very demanding in personnel resources. Patch clamping is carried out by addressing a cell with a glass micropipette while monitoring the approach process with optical microscopy. Be-cause of its limited resolution, the ultimate moment as the pipette touches the cell membrane cannot be controlled making this procedure unreliable and difficult, so that only experienced and skillful scientists can cope with it. Since about a decade, many efforts have been deployed towards automated patch-clamp based on glass chips to increase the throughput of conventional patch clamp by a factor of 1’000 to 10’000. Nevertheless, one major disadvantage of the current automated patch clamp systems is that they are only suitable for recording from cell lines with stable, high expression of the ion channel of interest while academic researchers rely heavily on transient expression of ion channels and their accessory and regulatory subunits. Another major disadvantage for academic applications is the lack of protocol flexibility of automated patch clamp systems.
Working strategy. With this project, we propose a new approach toward a new patch-clamp solution offering a higher throughput with the flexibility required for academic an d industrial research. The starting point is our recent invention of the FluidFM, an atomic force microscope (AFM) modified for microinjection experiments with individual cells in their environment by means of microchanneled AFM cantilevers. On the one hand, the accurate force feedback of the AFM allows for a reliable and automated approach of the cantilever tip onto or into the cell membrane. On the other hand, such microchanneled cantilevers are characterized by a much smaller (at least one order of magnitude) access impedance compared to standard glass micropipettes. The combination of these two advantages should allow for a drastic transformation of the patch-clamp from a “hit and miss” machine to a reliable and simple one. We hence postulate that this approach will allow to in-crease significantly the throughput for recording current and action potential values from native cells such as freshly isolated cardiac myocytes.
Accordingly, the specific aims of this pc-FluidFM research proposal are:
• Different strategies for force-controlled formation of gigaseal in whole-cell mode;
• Serial patch clamping with the same FluidFM tip;
• Investigation of the regionalization of sodium current at the surface of cardiac cells;
• Study of the cardiac action potential properties following drug application.
Added value of the project. With this new patch-clamping approach we will provide a system that is more reliable, more stable, and which should thus permit to obtain more in-formation by allowing for patch-clamping of cells with at least a double rate. Therefore, such technological breakthrough will be of great interest for the electrophysiological and pharmaceutical community.