Functional Assessment of Temperature-Gated Ion-Channel Activity Using a Real-Time PCR Machine

Abstract

The functional activity of a number of ion channels is highly sensitive to large changes in temperature. Foremost among these are the thermosensing TRP channels which include cold- (TRPM8, TRPA1), warmth- (TRPV3, TRPV4), and heat-sensing (TRPV1, TRPV2) members. TRPV1, also known as the vanilloid receptor (VR1), is activated by ligands such as capsaicin, acidic pH, and heat (an increase in temperature to approximately 42 degrees C will lead to channel opening). Screening against the thermal gating of TRPV1 is generally performed using perfusion systems or water baths for temperature control, in conjunction with electrophysiology or Ca2 + influx readouts for direct functional assessment. These approaches are very useful, but have limited throughput or minimal thermo-temporal control. A standard real-time PCR machine with standard microplates allowed us to combine fluorescent Ca2 + detection with precise temperature manipulation to develop a homogeneous (Z' = 0.53), cell-based assay that uses temperature as the agonist. A temperature response curve of TRPV1 was obtained, which provided a T50 of 46.1 degrees C, and IC50 values against heat agonism were determined for known TRPV1 antagonists. Furthermore, we expanded this approach to a cold-activated ion channel, TRPM8. We developed and validated an analytical technique with broad applications for the study and screening of temperature-gated ion channels.

The Chimeric Approach Reveals That Differences in the TRPV1 Pore Domain Determine Species-specific Sensitivity to Block of Heat Activation

“…reported (21). We also developed the RT-PCR heat activation assay (data presented by Papakosta et al at FASEB Ion Channel Conference, June, 2009) but we found that our FLIPR heat activation assay was far superior in terms of assay window and reproducibility in a high-throughput format.…”

Elephantid Genomes Reveal the Molecular Bases of Woolly Mammoth Adaptations to the Arctic

“…To functionally characterize the effects of the mammothspecific N647D substitution, we resurrected the AncMammoth and AncGajah TRPV3 genes and measured their temperaturedependent gating in transiently transfected HEK293 cells using Fluo-4 calcium flux assays (Aneiros and Dabrowski, 2009;Reubish et al, 2009). We found that both the AncMammoth and AncGajah TRPV3 proteins were expressed at similar levels (Figure S5I) and that the overall gating dynamics of channels were very similar.…”

“…Temperature-dependent calcium influx was assayed using the Fluo-4 NW Calcium Assay Kit (Molecular Probes) and a high-throughout qPCR-based assay (Aneiros and Dabrowski, 2009;Reubish et al, 2009). After an initial 30-min loading at 25 C, the temperature was raised from 15 C to 57 C in 2 C steps, and fluorescence was measured after 2 min at each temperature using a Bio-Rad CFX-96 real-time PCR machine.…”

Elephantid genomes reveal the molecular bases of Woolly Mammoth adaptations to the arctic

“…To functionally characterize the effects of the mammoth-specific N647D substitution we resurrected the AncMammoth and AncGajah TRPV3 genes and measured their temperature-dependent gating in transiently transfected human embryonic kidney cells (HEK293) using Fluo-4 calcium flux assays Reubish et al, 2009). We found that both the AncMammoth and AncGajah TRPV3 proteins were expressed at similar levels (SI Fig.…”