Description

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 an extracellular gradient as the compound diffuses away from the cell; uptake results in an extracellular gradient where the low point in the gradient is near the cell. These gradients are measured using modulation of electrochemical probes that enhance the signal to noise ratio. To date these methods have been restricted to measuring relatively steady state applications. 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.

Studies conducted at the BRC reviewed the expected response time for electrochemical sensors and noted that several of the potentiometric designs can achieve 90% response in less than 20msec (Smith, Sanger and Messerli, 2007). This speed brings us to within the scope of measuring ion gradients from single channels. 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 and reveal finer details of spatial and temporal regulation of cellular processes from chemical gradients surrounding cells.

Recent Update

Measurement of ion gradients from single channels has been achieved near Xenopus oocytes overexpressing Ca²⁺ activated K⁺ channels (Messerli, Corson and Smith, 2007).  The measured gradients fit to a simple diffusion model when the response time of the measuring system was taken into account. Best-fit alignment of the measured gradients enabled determination of the distance from the ion-selective electrode and channels as well as the open time of the channels. Future effort will be to improve the response time of the K⁺-selective electrodes as well as to implement feedback positioning of the K⁺-selective microelectrode.