7 results on '"Cavanaugh, Eric J."'
Search Results
2. Heteromeric TASK-1/TASK-3 is the major oxygen-sensitive background K+ channel in rat carotid body glomus cells.
- Author
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Kim, Donghee, Cavanaugh, Eric J., Kim, Insook, and Carroll, John L.
- Abstract
Carotid body (CB) glomus cells from rat express a TASK-like background K
+ channel that is believed to play a critical role in the regulation of excitability and hypoxia-induced increase in respiration. Here we studied the kinetic behaviour of single channel openings from rat CB cells to determine the molecular identity of the ‘TASK-like’ K+ channels. In outside-out patches, the TASK-like background K+ channel in CB cells was inhibited >90% by a reduction of pHo from 7.3 to 5.8. In cell-attached patches with 140 mm KCl and 1 mm Mg2+ in the bath and pipette solutions, two main open levels with conductance levels of ∼14 pS and ∼32 pS were recorded at a membrane potential of −60 mV. The K+ channels showed kinetic properties similar to TASK-1 (∼14 pS), TASK-3 (∼32 pS) and TASK-1/3 heteromer (∼32 pS). The presence of three TASK isoforms was tested by reducing [Mg2+ ]o to ∼0 mm, which had no effect on the conductance of TASK-1, but increased those of TASK-1/3 and TASK-3 to 42 pS and 74 pS, respectively. In CB cells, the reduction of [Mg2+ ]o to ∼0 mm also caused the appearance of ∼42 pS (TASK-1/3-like) and ∼74 pS (TASK-3-like) channels, in addition to the ∼14 pS (TASK-1-like) channel. The 42 pS channel was the most abundant, contributing ∼75% of the current produced by TASK-like channels. Ruthenium red (5 μm) had no effect on TASK-1 and TASK-1/3, but inhibited TASK-3 by 87%. In CB cells, ruthenium red caused ∼12% inhibition of TASK-like activity. Methanandamide reduced the activity of all three TASKs by 80–90%, and that of TASK-like channels in CB cell also by ∼80%. In CB cells, hypoxia caused inhibition of TASK-like channels, including TASK-1/3-like channels. These results show that TASK-1, TASK-1/3 and TASK-3 are all functionally expressed in isolated CB cells, and that the TASK-1/3 heteromer provides the major part of the oxygen-sensitive TASK-like background K+ conductance. [ABSTRACT FROM AUTHOR]- Published
- 2009
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3. Pore dilation occurs in TRPA1 but not in TRPM8 channels.
- Author
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Jun Chen, Donghee Kim, Bianchi, Bruce R., Cavanaugh, Eric J., Faltynek, Connie R., Kym, Philip R., and Reilly, Regina M.
- Abstract
Abundantly expressed in pain-sensing neurons, TRPV1, TRPA1 and TRPM8 are major cellular sensors of thermal, chemical and mechanical stimuli. The function of these ion channels has been attributed to their selective permeation of small cations (e.g., Ca
2+ , Na+ and K+ ), and the ion selectivity has been assumed to be an invariant fingerprint to a given channel. However, for TRPV1, the notion of invariant ion selectivity has been revised recently. When activated, TRPV1 undergoes time and agonist-dependent pore dilation, allowing permeation of large organic cations such as Yo-Pro and NMDG+ . The pore dilation is of physiological importance, and has been exploited to specifically silence TRPV1-positive sensory neurons. It is unknown whether TRPA1 and TRPM8 undergo pore dilation. Here we show that TRPA1 activation by reactive or non-reactive agonists induces Yo-Pro uptake, which can be blocked by TRPA1 antagonists. In outside-out patch recordings using NMDG+ as the sole external cation and Na+ as the internal cation, TRPA1 activation results in dynamic changes in permeability to NMDG+ . In contrast, TRPM8 activation does not produce either Yo-Pro uptake or significant change in ion selectivity. Hence, pore dilation occurs in TRPA1, but not in TRPM8 channels. [ABSTRACT FROM AUTHOR]- Published
- 2009
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4. Control of the single channel conductance of K2P10.1 (TREK-2) by the amino-terminus: role of alternative translation initiation.
- Author
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Simkin, Dina, Cavanaugh, Eric J., and Kim, Donghee
- Abstract
TREK-2 expressed in mammalian cells exhibits small (∼52 pS) and large (∼220 pS) unitary conductance levels. Here we tested the role of the N-terminus (69 amino acids long) in the control of the unitary conductance, and role of the alternative translation initiation as a mechanism that produces isoforms of TREK-2 that show different conductance levels. Deletion of the first half (Δ1–36) of the N-terminus had no effect. However, deletion of most of the N-terminus (Δ1–66) resulted in the appearance of only the large-conductance channel (∼220 pS). In support of the critical function of the distal half of the N-terminus, the deletion mutants Δ1–44 and Δ1–54 produced ∼90 pS and 188 pS channels, respectively. In Western blot analysis, TREK-2 antibody detected two immunoreactive bands at ∼54 kDa and ∼60 kDa from cells expressing wild-type TREK-2 that has three potential translation initiation sites (designated M
1 M2 M3 ) within the N-terminus. Mutation of the second and third initiation sites from Met to Leu (M1 L2 L3 ) produced only the ∼60 kDa isoform and the small-conductance channel (∼52 pS). Mutants designed to produce translation from the second (M2 L3 ) or third (M3 ) initiation site produced the ∼54 kDa isoform, and the large conductance channel (∼185–224 pS). M1 L2 L3 , M2 L3 and M3 were relatively selectively permeable to K+ , as judged by the 51–55 mV shifts in reversal potential following a 10-fold change in [K+ ]o . PNa / PK values were also similar for M1 L2 L3 (∼0.02), M2 L3 (∼0.02) and M3 (∼0.03). Arachidonic acid, proton and membrane stretch activated, whereas dibutyryl-cAMP inhibited all three isoforms of TREK-2, indicating that deletion of the N-terminus does not abolish modulation. These results show that the small and large conductance TREK-2 channels are produced as a result of alternative translation initiation, producing isoforms with long and short N-termini, and that the distal half of the N-terminus controls the unitary conductance. [ABSTRACT FROM AUTHOR]- Published
- 2008
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5. Inhibition of transient receptor potential A1 channel by phosphatidylinositol-4,5-bisphosphate.
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Kim, Donghee, Cavanaugh, Eric J., and Simkin, Dina
- Abstract
Membrane phosphatidylinositol- 4,5-bisphosphate (PIP2) is critical for the function of many transient receptor potential (TRP) ion channels. The role of PIP2 in TRPA1 function is not well known. The effect of PIP2 on TRPA1 was investigated by direct application of PIP2 and by using polylysine and PIP2 antibody that sequester PIP2. In inside-out patches from HeLa cells expressing mouse TRPA1, polytriphosphate (PPPi) was added to the bath solution to keep TRPA1 sensitive to allyl isothiocyanate (AITC; mustard oil). Direct application of PIP2 (10 µM) to inside-out patches did not activate TRPA1, but AITC and Δ
9 -tetrahydrocannabinol (THC) produced strong activation. In inside-out patches in which TRPA1 was first activated with AITC (in the presence of PPPi), further addition of PIP2 produced a concentration-dependent inhibition of TRPA1 [agonist concentration producing half-maximal activity (K1/2 ), 2.8 µM]. Consistent with the inhibition of TRPA1 by PIP2, AITC activated a large whole cell current when polylysine or PIP2 antibody was added to the pipette but a markedly diminished current when PIP2 was added to the pipette. In inside-out patches with PPPi in the bath solution, application of PIP2 antibody or polylysine caused activation of TRPA1, and this was blocked by PIP2. However, TRPA1 was not activated by polylysine and PIP2 antibody under whole cell conditions, suggesting a more complex regulation of TRPA1 by PIP2 in intact cells. These results show that PIP2 inhibits TRPA1 and reduces the sensitivity of TRPA1 to AITC. [ABSTRACT FROM AUTHOR]- Published
- 2008
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6. Cutaneous nociception evoked by 15-delta PGJ2 via activation of ion channel TRPA1.
- Author
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Cruz-Orengo, Lillian, Dhaka, Ajay, Heuermann, Robert J., Young, Timothy J., Montana, Michael C., Cavanaugh, Eric J., Kim, Donghee, and Story, Gina M.
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PROSTAGLANDINS ,NEURONS ,G proteins ,HYPERALGESIA ,ION channels ,CINNAMON ,ELECTROPHILES - Abstract
Background: A number of prostaglandins (PGs) sensitize dorsal root ganglion (DRG) neurons and contribute to inflammatory hyperalgesia by signaling through specific G protein-coupled receptors (GPCRs). One mechanism whereby PGs sensitize these neurons is through modulation of "thermoTRPs," a subset of ion channels activated by temperature belonging to the Transient Receptor Potential ion channel superfamily. Acrid, electrophilic chemicals including cinnamaldehyde (CA) and allyl isothiocyanate (AITC), derivatives of cinnamon and mustard oil respectively, activate thermoTRP member TRPA1 via direct modification of channel cysteine residues. Results: Our search for endogenous chemical activators utilizing a bioactive lipid library screen identified a cyclopentane PGD
2 metabolite, 15-deoxy-δ12,14 -prostaglandin J2 (15d-PGJ2 ), as a TRPA1 agonist. Similar to CA and AITC, this electrophilic molecule is known to modify cysteines of cellular target proteins. Electophysiological recordings verified that 15d-PGJ2 specifically activates TRPA1 and not TRPV1 or TRPM8 (thermoTRPs also enriched in DRG). Accordingly, we identified a population of mouse DRG neurons responsive to 15d-PGJ2 and AITC that is absent in cultures derived from TRPA1 knockout mice. The irritant molecules that activate TRPA1 evoke nociceptive responses. However, 15d-PGJ2 has not been correlated with painful sensations; rather, it is considered to mediate anti-inflammatory processes via binding to the nuclear peroxisome proliferator-activated receptor gamma (PPARγ). Our in vivo studies revealed that 15d-PGJ2 induced acute nociceptive responses when administered cutaneously. Moreover, mice deficient in the TRPA1 channel failed to exhibit such behaviors. Conclusion: In conclusion, we show that 15d-PGJ2 induces acute nociception when administered cutaneously and does so via a TRPA1-specific mechanism. [ABSTRACT FROM AUTHOR]- Published
- 2008
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7. Requirement of a Soluble Intracellular Factor for Activation of Transient Receptor Potential A1 by Pungent Chemicals: Role of Inorganic Polyphosphates.
- Author
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Kim, Donghee and Cavanaugh, Eric J.
- Subjects
PROTEINS ,CHEMICALS ,NEURONS ,TRP channels ,POLYPHOSPHATES ,PAIN ,PATCH-clamp techniques (Electrophysiology) - Abstract
Pungent chemicals such as allyl isothiocyanate (AITC), cinnamaldehyde, and allicin, produce nociceptive sensation by directly activating transient receptor potential A1 (TRPA1) expressed in sensory afferent neurons. In this study, we found that pungent chemicals added to the pipette or bath solution easily activated TRPA1 in cell-attached patches but failed to do so in inside-out or outside-out patches. Thus, a soluble cytosolic factor was required to activate TRPA1. N-Ethylmaleimide, (2-aminoethyl)-methane thiosulfonate, 2-aminoethoxydiphneyl borate, and trinitrophenol, compounds that are known to activate TRPA1, also failed to activate it in inside-out patches. To identify a factor that supports activation of TRPA1 by pungent chemicals, we screened ∼ 30 intracellular molecules known to modulate ion channels. Among them, pyrophosphate (PPi) and polytriphosphate (PPPi) were found to support activation of TRPA1 by pungent chemicals. Structure--function studies showed that inorganic polyphosphates (polyP
n , where n = number of phosphates) with at least four phosphate groups were highly effective (polyP4 ≈ polyP65 ≈ polyP45 ≈ polyP25 > PPPi > PPi), with K½ values ranging from 0.2 to 2.8 mM. Inositol-trisphosphate and inositol-hexaphosphate also partially supported activation of TRPA1 by AITC. ATP, GTP, and phosphatidylinositol-4,5-bisphosphate that have three phosphate groups did not support TRPA1 activation. TRPA1 recorded from cell bodies of trigeminal ganglion neurons showed similar behavior with respect to sensitivity to pungent chemicals; no activation was observed in inside-out patches unless a polyphosphate was present. These results show that TRPA1 requires an intracellular factor to adopt a functional conformation that is sensitive to pungent chemicals and suggest that polyphosphates may partly act as such a factor. [ABSTRACT FROM AUTHOR]- Published
- 2007
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