Measurement of Rapid Amiloride-Dependent pH Changes at the Cell Surface Using a Proton-Sensitive Field-Effect TransistorReportar como inadecuado




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1

Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Bldg 21-4-403B, 2-3-10 Kanda-Surugadai, Chiyoda, Tokyo 101-0062, Japan

2

Department of Physiology, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390, USA





*

Author to whom correspondence should be addressed.



Academic Editors: Mark A. Reed and Mathias Wipf

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-isopropylamiloride 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. View Full-Text

Keywords: ISFET; pH; proton; CHO; MSF; ammonia; amiloride; EIPA; NHE ISFET; pH; proton; CHO; MSF; ammonia; amiloride; EIPA; NHE





Autor: Daniel Schaffhauser 1, Michael Fine 2, Miyuki Tabata 1, Tatsuro Goda 1 and Yuji Miyahara 1,*

Fuente: http://mdpi.com/



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