Ion channels are a large diverse group of integral membrane proteins, which play a central and key role in signaling, transport, and maintaining electrophysiological potential in all cells. Abnormalities in ion channels cause many diseases such as: cardiovascular dysfunction (Long QT syndrome), neurological disorders (multiple sclerosis, epilepsy), deafness and blindness. Biomedical researchers and pharmacologists are increasingly looking at ion channels as the source of disease and as targets for therapies based on novel drugs which can address previously misunderstood medical challenges. Because of this major paradigm shift, it is now estimated that approximately 15% of all new drugs being screened are generally classified as """"""""ion channel modifiers."""""""" To date new technologies for high throughput screening (HTS) at an early stage of the drug discovery process for compounds with activities as ion channel modifiers have been marginally successful. Because of the large number of compounds available for screening, the fast kinetics of ion channels and the need to generate >10,000 data points per day, rapid and autonomous methods to dynamically measure ionic current or fluxes from these ion channels are needed. Additionally, in mammalian cells Na+, K+, Ca2+ and Cl- channels operate synchronously to maintain a precarious status, thus requiring the monitoring of multiple ionic activities to further our understanding. Conventional electrophysiology technology, based on the patch clamp approach, provides the most direct and detailed method of studying ion channels. However the approach does suffer from some basic limitations, most notably low throughput (<100 data points/day) and inability to identify ionic composition of electrophysiological events. In spite of recent progress towards automated patch clamp and patch-on-a-chip, the invasive nature of this technique, the need of highly trained and skilled scientists and its inability to resolve and distinguish between different ionic activities simultaneously, has limited the utility of this technique for HTS. Other ion channel HTS techniques, fluorescent/potentiometric probes, radiolabelled ion flux assays, and automated spectrometric assays have drawbacks related to cytotoxicity and limited information content. Currently there is no technology available which can provide non invasive, dynamic, and autonomous recordings of specific ion channel activities as an in vitro ion channel characterization assay in a miniaturized HTS format. The long term goal of this work is to develop an easy-to-use technology for HTS cell electrophysiology, and screening of ion channel modulators as potential drug targets in an in vitro format. The overall objective of this proposal is to enable simultaneous, quantitative and temporally resolved, real time measurement of extracellular Na+, K+, Cl- and Ca2+ from single cells integrated with whole cell patch clamp. This will enable complete dynamic analysis of ion channel electrophysiology in response to various ion channel therapeutic candidates. We will do this by developing the """"""""Goldman chip"""""""";a microfabricated platform which will enable non- invasive sensing of key extracellular ionic activities in a high resolution, high throughput system by maximizing the information gained from a single experiment. Based on solid state ion- selective sensor technology and planar patch, the Goldman chip will provide simultaneous extracellular Na+, K+, Cl- and Ca2+ measurement capabilities with whole cell electrophysiological recordings. Whole cell current measurement and control will be possible based on integration of planar patch clamp technology, signal processing hardware/software and automated control for high throughput experimentation. By integrating a standard patch clamp electrophysiological interface with Na+, K+, Cl- and Ca2+ ISEs on a single platform, it will be possible to directly identify the ionic component of a whole cell current recording. This technology is highly innovative because the focus is not entirely on increasing raw electrophysiological experimental throughput, but instead offers to qualitatively change the value and scope of the information obtained from a single experiment by incorporating ion-specificity into the approach. Because this is a more holistic approach to electrophysiology experimentation, the overall throughput is expected to increase as the requirement for multiple rounds of ion-replacement, and channel blocker controls will be eliminated.
(provided by the applicant): Narrative Ion channels are a large and very diverse group of integral membrane proteins, which are the source of many diseases including: cardiovascular dysfunction (Long QT syndrome), neurological disorders (multiple sclerosis, epilepsy), deafness and blindness. Now basic biomedical researchers and pharmacologists are increasingly looking at ion channels and approximately 15% of all new drugs being screened are generally classified as """"""""ion channel modifiers."""""""" New technologies are now needed for High Throughput Screening (HTS) for ion channel modifiers, and the proposed work seeks to advance this area by using a bioMEMS approach to integrate patch-clamp electrophysiology with ion-specific microelectrodes.