A cell-based impedance assay for monitoring transient receptor potential (TRP) ion channel activity.
Transient receptor potential (TRP) channels are non-selective ion channels permeable to cations including Na(+), Ca(2+) and Mg(2+). They play a unique role as cellular sensors and are involved in many Ca(2+)-mediated cell functions. Failure in channel gating can contribute to complex pathophysiological mechanisms. Dysfunctions of TRP channels cause diseases but are also involved in the progress of diseases. We present a novel method to analyse chemical compounds as potential activators or inhibitors of TRP channels to provide pharmaceutical tools to regulate channel activity for disease control. Compared to common methods such as patch clamp or Ca(2+) imaging, the presented impedance assay is automatable, experimental less demanding and not restricted to Ca(2+) flow. We have chosen TRPA1 from the TRPA ('ankyrin') family as a model channel which was stimulated by allyl isothiocyanate (AITC). HEK293 cells stably transfected with human TRPA1 cDNA were grown on microelectrode arrays. Confluent cell layers of high density were analysed. Impedance spectra of cell-covered and non-covered electrodes yielded a cell-specific signal at frequencies between 70 and 120 kHz. Therefore, 100 kHz was chosen to monitor TRPA1 activity thereupon. An average impedance decrease to about 70% of its original value was observed after application of 10 μM AITC indicating an increased conductance of the cell layer mediated by TRPA1. Transfected cells pretreated with 10 μM of inhibitor ruthenium red to prevent channel conductance, as well as control cells lacking TRPA1, showed no impedance changes upon AITC stimuli demonstrating the specificity of the novel impedance assay.
Centre for Biotechnology and Biomedicine (BBZ), University of Leipzig, Division of Molecular Biological-Biochemical Processing Technology, Deutscher Platz 5, D-04103 Leipzig, Germany. oliver.paenke@bbz.uni-leipzig.de
Oliver
O
Pänke
Winnie
W
Weigel
Sabine
S
Schmidt
Anja
A
Steude
Andrea A
AA
Robitzki
D001681:Biological Assay / Q000295:instrumentation*; D015374:Biosensing Techniques / Q000295:instrumentation*; D015220:Calcium Channels / Q000502:physiology*; D003217:Conductometry / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D015336:Molecular Probe Techniques / Q000295:instrumentation*; D009419:Nerve Tissue Proteins / Q000502:physiology*; D000074025:TRPA1 Cation Channel; D050051:Transient Receptor Potential Channels / Q000502:physiology*
D015220:Calcium Channels; D009419:Nerve Tissue Proteins; D000074025:TRPA1 Cation Channel; C473350:TRPA1 protein, human; D050051:Transient Receptor Potential Channels