Functional assessment of temperature-gated ion-channel activity using a real-time PCR machine
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 rec...
Saved in:
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Taylor & Francis Group
2009-09-01
|
| Series: | BioTechniques |
| Subjects: | |
| Online Access: | https://www.future-science.com/doi/10.2144/000113198 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850152722295160832 |
|---|---|
| author | Derek S. Reubish Daniel E. Emerling Jeff DeFalco Daniel Steiger Cheryl L. Victoria Fabien Vincent |
| author_facet | Derek S. Reubish Daniel E. Emerling Jeff DeFalco Daniel Steiger Cheryl L. Victoria Fabien Vincent |
| author_sort | Derek S. Reubish |
| collection | DOAJ |
| description | 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 ∼42°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°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. |
| format | Article |
| id | doaj-art-667eecac654c40aea4be4a0cc286b2f3 |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2009-09-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-667eecac654c40aea4be4a0cc286b2f32025-08-20T02:25:54ZengTaylor & Francis GroupBioTechniques0736-62051940-98182009-09-01473Siiiix10.2144/000113198Functional assessment of temperature-gated ion-channel activity using a real-time PCR machineDerek S. Reubish0Daniel E. Emerling1Jeff DeFalco2Daniel Steiger3Cheryl L. Victoria4Fabien Vincent51In Vitro Pharmacology Department, Evotec, South San Francisco, CA, USA1In Vitro Pharmacology Department, Evotec, South San Francisco, CA, USA1In Vitro Pharmacology Department, Evotec, South San Francisco, CA, USA1In Vitro Pharmacology Department, Evotec, South San Francisco, CA, USA1In Vitro Pharmacology Department, Evotec, South San Francisco, CA, USA1In Vitro Pharmacology Department, Evotec, South San Francisco, CA, USAThe 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 ∼42°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°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.https://www.future-science.com/doi/10.2144/000113198Real-time PCRtemperatureheatcoldion channelassay |
| spellingShingle | Derek S. Reubish Daniel E. Emerling Jeff DeFalco Daniel Steiger Cheryl L. Victoria Fabien Vincent Functional assessment of temperature-gated ion-channel activity using a real-time PCR machine BioTechniques Real-time PCR temperature heat cold ion channel assay |
| title | Functional assessment of temperature-gated ion-channel activity using a real-time PCR machine |
| title_full | Functional assessment of temperature-gated ion-channel activity using a real-time PCR machine |
| title_fullStr | Functional assessment of temperature-gated ion-channel activity using a real-time PCR machine |
| title_full_unstemmed | Functional assessment of temperature-gated ion-channel activity using a real-time PCR machine |
| title_short | Functional assessment of temperature-gated ion-channel activity using a real-time PCR machine |
| title_sort | functional assessment of temperature gated ion channel activity using a real time pcr machine |
| topic | Real-time PCR temperature heat cold ion channel assay |
| url | https://www.future-science.com/doi/10.2144/000113198 |
| work_keys_str_mv | AT dereksreubish functionalassessmentoftemperaturegatedionchannelactivityusingarealtimepcrmachine AT danieleemerling functionalassessmentoftemperaturegatedionchannelactivityusingarealtimepcrmachine AT jeffdefalco functionalassessmentoftemperaturegatedionchannelactivityusingarealtimepcrmachine AT danielsteiger functionalassessmentoftemperaturegatedionchannelactivityusingarealtimepcrmachine AT cheryllvictoria functionalassessmentoftemperaturegatedionchannelactivityusingarealtimepcrmachine AT fabienvincent functionalassessmentoftemperaturegatedionchannelactivityusingarealtimepcrmachine |