Abstract
We present a novel method for the rapid measurement of pH fluxes at close proximity to the surface of the plasma membrane in mammalian cells using an ion-sensitive field-effect transistor (ISFET). In conjuction with an efficient continuous superfusion system, the ISFET sensor was capable of recording rapid changes in pH at the cells’ surface induced by intervals of ammonia loading and unloading, even when using highly buffered solutions. Furthermore, the system was able to isolate physiologically relevant signals by not only detecting the transients caused by ammonia loading and unloading, but display steady-state signals as would be expected by a proton transport-mediated influence on the extracellular proton-gradient. Proof of concept was demonstrated through the use of 5-(N-ethyl-N-isopropyl)amiloride (EIPA), a small molecule inhibitor of sodium/hydrogen exchangers (NHE). As the primary transporter responsible for proton balance during cellular regulation of pH, non-electrogenic NHE transport is notoriously difficult to detect with traditional methods. Using the NHE positive cell lines, Chinese hamster ovary (CHO) cells and NHE3-reconstituted mouse skin fibroblasts (MSF), the sensor exhibited a significant response to EIPA inhibition, whereas NHE-deficient MSF cells were unaffected by application of the inhibitor.
Highlights
Proton-dependent membrane transport plays a significant role in many physiological processes, such as pH regulation in blood and tissue, maintenance of cellular volume, glycolysis or as a co-requisite for the transport and absorption of amino acids, peptides and iron [1,2]
Based on our previous experimental findings and success with the ion-sensitive field-effect transistor (ISFET) sensor, we developed a system for the measurement of pH at the surface of immobilized mammalian cells
Based on our previous experimental findings and success with the ISFET sensor, we developed a previously been demonstrated on tumor cells that it is possible to measure their extracellular proton system for the measurement of pH at using the surface immobilized mammalian cells
Summary
Proton-dependent membrane transport plays a significant role in many physiological processes, such as pH regulation in blood and tissue, maintenance of cellular volume, glycolysis or as a co-requisite for the transport and absorption of amino acids, peptides and iron [1,2]. These processes generate changes in extracellular proton gradients that are difficult to detect as the length of proton flux is largely determined by the buffering capacity of the extracellular bulk solution [3]. One study on the purple membrane of H. salinarium involved the use of Pyranine as a fluorescent pH indicator where the time constant for transfer of a proton along the membrane surface was shown to be almost six times faster than the one to the bulk solution [4]
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