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 lowest point in the gradient is near the cell.

We measure these gradients using modulation of electrochemical detection which enhances the signal to noise ratio of the measuring system. 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. The standard self-referencing approach collects a differential measurement each 3.3 s and therefore cannot accurately resolve a rapid transient shorter than about 10 s. The prolonged data collection and averaging scheme used for measuring the steady gradients blur the individual events, leading to the loss of useful information regarding the specific characteristics of the gradient. By using fast responding electrodes with signal analysis methods we hope to characterize rapid physiological and metabolic events under normal and pathogenic conditions in order to study the diseased state with a non-invasive approach.

Studies conducted at the BRC reviewed the response times for ion-selective (H+, Ca2+ and K+) and O2 microelectrodes and noted that several of the potentiometric designs can achieve 90% response in less than 20 msec (Smith, Sanger and Messerli, 2007). This rapid response brings us to within the scope of measuring ion gradients from single channels with brief (~10 ms) open times and opens up a realm of possibilities for capturing other rapid biological events.

In order to confirm the measurement of brief ion channel gradients with ion-selective microelectrodes we explored mapping large conductance Ca2+-activated K+ channels in heterologous expression systems, Xenopus oocytes (Messerli et al., 2007) and Chinese Hamster Ovary (CHO) cells (Messerli et al., 2009). Predictive modeling of the single channel gradients and capture of relatively long channel open times is reported in Messerli et al. (2007). Better control over the cytosolic Ca2+ concentration and membrane potential by using a mammalian cell line as well as improvement of the response time of the detection system enabled generation and capture of very brief events that more closely resemble single channel events under native conditions (Messerli et al., 2009).

The figure below shows a representative recording of rapid K+ gradients from single channels in CHO cells. For these controlled experiments we chose to activate the channels through depolarizing the plasma membrane with the whole cell voltage clamp (VC) method while measuring extracellular K+ gradients with an ion-selective (K+) microelectrode (ISM), both illustrated as used next to the cell in the left inset to the figure. The main figure shows the K+ activity dependent voltage recorded by the ISM in a position near the cell and a position 20um further away. The recording in the away position reports the background K+ activity while the recording in the near position shows spiking superimposed on a higher background K+ activity. The spiking is due to rapid generation of extracellular K+ gradients from efflux through the overexpressed K+ channels. The high density of spiking was not captured in parental cell lines or after the application of the K+ channel blocker, iberiotoxin. Single channel K+ gradients were capture from these channels under many different circumstances (Messerli et al., 2009).

These results helped support the future capabilities of rapid monitoring of biological events.  The technical advances that have been achieved also provide avenues for expanding more complex use of extracellular electrochemical sensors. Combining these non-invasive sensors 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.