MEASURING FASTER PHYSIOLOGICAL EVENTS WITH ELECTROCHEMICAL PROBES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cells maintain a dynamic interaction with their environment by acquiring and expelling inorganic ions, gases and organic compounds ranging from metabolic wastes to chemical messengers. Release of a compound results in a surface high concentration that produces a gradient as the compound diffuses away from the cell; uptake results in an inverse gradient. These gradients are measured using modulation of electrochemical probes that enhance the signal to noise ratio but to date have been restricted to relatively steady state applications. Studies conducted at the BRC reviewed the expected response time for electrochemical sensors and noted that several of the potentiometric design can achieve 90% response in less than 20msec. This speed brings us to within the scope of measuring channel activities. We have used K+-selective microelectrodes to monitor changes in external [K+] after efflux through artificial channels in a planar lipid bilayer and after efflux through Ca2+-activated K+ channels expressed in Xenopus oocytes and Chinese Hamster Ovary cells. A combination of modeling and data analysis schemes has been used to confirm single channel detection and identify the strengths and weaknesses of the system. Combining these non-invasive sensors and analysis approaches with a scanning technique described elsewhere will provide a unique insight into cellular organization, revealing finer details of spatial and temporal regulation of cellular processes from chemical gradients surrounding cells. Self-referencing with ion-selective electrodes (ISEs) has been used noninvasively, to measure relatively steady ionic gradients near cells and tissues. However, these relatively steady gradients are the average of many discrete events including transport through channels or transporters. The data collection and averaging scheme used for measuring the steady gradients blurs the individual events, leading to the loss of useful information regarding the nature of the ionic gradient. By using fast responding electrodes with signal analysis methods we hope to characterize ion channels and transporters under normal and pathogenic conditions in order to study the diseased state with a non-invasive approach.