Novel Temperature Activation Cell-Based Assay on Thermo-TRP Ion Channels

Aneiros, Eduardo; Dabrowski, Michael

doi:10.1177/1087057109336595

Cited by 13 publications

(13 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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.…”

Section: Substitutions In Temperature-sensitive Transient Receptor Pomentioning

confidence: 94%

“…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.…”

Section: Trpv3 Function Assaysmentioning

confidence: 99%

See 1 more Smart Citation

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

et al. 2015

View full text Add to dashboard Cite

Woolly mammoths and living elephants are characterized by major phenotypic differences that have allowed them to live in very different environments. To identify the genetic changes that underlie the suite of woolly mammoth adaptations to extreme cold, we sequenced the nuclear genome from three Asian elephants and two woolly mammoths, and we identified and functionally annotated genetic changes unique to woolly mammoths. We found that genes with mammoth-specific amino acid changes are enriched in functions related to circadian biology, skin and hair development and physiology, lipid metabolism, adipose development and physiology, and temperature sensation. Finally, we resurrected and functionally tested the mammoth and ancestral elephant TRPV3 gene, which encodes a temperature-sensitive transient receptor potential (thermoTRP) channel involved in thermal sensation and hair growth, and we show that a single mammoth-specific amino acid substitution in an otherwise highly conserved region of the TRPV3 channel strongly affects its temperature sensitivity.

show abstract

Section: Substitutions In Temperature-sensitive Transient Receptor Pomentioning

confidence: 94%

Section: Trpv3 Function Assaysmentioning

confidence: 99%

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

et al. 2015

View full text Add to dashboard Cite

show abstract

“…To functionally characterize the mammoth-specific amino acid substitutions in TRPM8, we resurrected the AncMammoth and AncGajah TRPM8 genes (Fig. 3A) and measured their temperature-dependent activity in transiently transfected HEK293 cells using a Fluo-4 calcium influx assay (Aneiros and Dabrowski, 2009;Reubish et al, 2009). We found that the AncGajah TRPM8 had a similar activation profile as other mammalian TRPM8 proteins, for example, it was activated at ~22°C, had half maximum activities (T50) at ~17°C, and maximal activities (Tmax) at 2°C (Fig.…”

Section: Thermal Desensitization Of Trpm8 In Mammothsmentioning

confidence: 99%

“…48 hr after transfection, cells were harvested by trypsinization, centrifuged, resuspended in Hank's balanced salt solution (HBSS) and HEPES assay buffer containing 2.5 mM probenecid, and transferred to a 96-well plate at 150,000 cells/well in 50 μl assay buffer. Temperature-dependent calcium influx was assayed using the Fluo-4 NW Calcium Assay Kit (Molecular Probes) and a high-throughout qPCR-based assay (Aneiros andDabrowski, 2009, Reubish et al, 2009). After an initial 30-min loading at 37°C, the temperature was lowered from 37°C to 2°C in 5°C steps, and fluorescence was measured after 30 s at each temperature using a Bio-Rad CFX-96 real-time PCR machine.…”

Section: Trpm8 Function Assaysmentioning

confidence: 99%

Relaxed constraint and thermal desensitization of the cold-sensing ion channel TRPM8 in mammoths

Chigurapati

Sulak

Miller

et al. 2018

Preprint

View full text Add to dashboard Cite

Unlike the living elephants, which live in warm tropical and subtropical habitats, mammoths lived in extreme cold environments where average winter temperatures ranged from −30° to −50°C.Like other cold adapted and artic species, mammoths evolved of suite of morphological and molecular adaptations that facilitated life in the cold. Here we reanalyze mammoth genomes and find that genes with mammoth-specific amino acid substitutions are enriched in functions related to temperature sensation. Among the genes with mammoth-specific amino acid substitutions is TRPM8, which mediates sensitivity to nonnoxious cool temperatures from 25-28°C and cooling sensations induced by the chemical agonists menthol and icilin. We find that TRPM8 evolved rapidly in the mammoth stem-lineage, likely because of an episode of relaxed purifying selection. Functional characterization of resurrected mammoth and ancestral TRPM8 indicates that the mammoth TRPM8 is desensitized to cold but maintains sensitivity to menthol and icilin. These data suggest that as mammoths evolved into a cold tolerant species they lost the need for a cold-sensitive TRPM8.

show abstract

“…A versatile temperature-dependent assay was reported that relied on the rapid TaqMan temperature control to trigger thermoTRPs activation; results were shown to reliably translate to native rat dorsal root ganglia neurons in either 384 or 96 well formats [197].…”

Section: Targeting the Temperature Sensitivity Of Trpa1 With In Vitromentioning

confidence: 99%

Roles of TRPA1 in Pain Pathophysiology and Implications for the Development of a New Class of Analgesic Drugs

Radresa¹,

Dahllöf²,

Nyman³

et al. 2013

TOPAINJ

View full text Add to dashboard Cite

Abstract:The Transient Receptor Potential A1 (TRPA1) ion channel has evolved in animals to respond to signals from a variety of sensory stimuli. Many structural determinants of its multimodal activation have been identified to date. TRPA1 activities include responses to exogenous chemical irritants, responses to endogenous inflammatory mediators, zinc, voltage, temperature or stretch and subtle yet critical modulation by calcium ions. TRPA1 has emerged as an important target for several types of pain and inflammatory conditions because of its limited expression profile and its demonstrated roles in mediating different types of pain and sensitization of peripheral sensory afferents. Despite observed species differences in channel pharmacology, recent genetic evidence in human brings some hope that preclinical efficacy in disease models will translate to patient condition. During the past decade, various groups have investigated the development of a new class of analgesic drugs or anti-tussive agents aimed at blocking TRPA1 activity in primary sensory afferents. Several companies are advancing toward clinical proof of concept studies. This review aims to summarize key advances in the understanding of TRPA1 with regard to its roles and implications for patient conditions.

show abstract

Novel Temperature Activation Cell-Based Assay on Thermo-TRP Ion Channels

Cited by 13 publications

References 15 publications

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

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

Relaxed constraint and thermal desensitization of the cold-sensing ion channel TRPM8 in mammoths

Roles of TRPA1 in Pain Pathophysiology and Implications for the Development of a New Class of Analgesic Drugs

Contact Info

Product

Resources

About