Eric J. Cavanaugh

Heteromeric TASK-1/TASK-3 is the major oxygen-sensitive background K+ channel in rat carotid body glomus cells

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.

Pore dilation occurs in TRPA1 but not in TRPM8 channels

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., Ca2+, 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.

Inhibition of transient receptor potential A1 channel by phosphatidylinositol-4,5-bisphosphate.

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 microM) to inside-out patches did not activate TRPA1, but AITC and Delta(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 (K(1/2)), 2.8 microM]. 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.

Activation of transient receptor potential A1 channels by mustard oil, tetrahydrocannabinol and Ca2+ reveals different functional channel states

Our recent study has shown that activation of transient receptor potential A1 channel (TRPA1) by pungent chemicals such as allyl-isothiocyanate (AITC) requires an unidentified cytosolic factor whose action can be mimicked by inorganic polyphosphates. Thus, AITC and other pungent chemicals fail to activate TRPA1 in excised patches. It is unclear whether TRPA1 switches to a conformation that is insensitive to the pungent chemicals, or whether TRPA1 simply becomes completely non-functional and insensitive to all activators when the cytosolic factor is absent. To help distinguish between these possibilities, the effects of Delta(9)-tetrahydrocannabinol (THC) and Ca(2+) that are structurally different from pungent chemicals were tested on AITC-sensitive and AITC-insensitive states of TRPA1. In HeLa cells transiently expressing mouse TRPA1, activation of TRPA1 by THC was slow and weak from the extracellular side (cell-attached; K(1/2) >20 microM), but was faster and more potent from the intracellular side (inside-out; K(1/2), approximately 0.7 microM), and this did not require the presence of a polyphosphate. Similar results were observed in rat trigeminal neurons. Increasing the extracellular [Ca(2+)] from approximately 0-1-3 mM activated TRPA1 in cell-attached patches. Elevation of cytosolic [Ca(2+)] using thapsigargin (inhibitor of Ca(2+)-ATPAse) and histamine (that elevates IP(3)) also activated TRPA1 in cell-attached patches. Similar to pungent chemicals, Ca(2+) (1-5 microM) failed to activate TRPA1 in inside-out patches, unless polyphosphates were present. These results show that TRPA1 can exist in different functional states: a native state (cell-attached patch) and a non-native state (excised patch). THC can activate TRPA1 even in the absence of polyphosphates, whereas pungent chemicals and Ca(2+) require it for activation.

Control of the single channel conductance of K2P10.1 (TREK‐2) by the amino‐terminus: role of alternative translation initiation

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 M1M2M3) within the N‐terminus. Mutation of the second and third initiation sites from Met to Leu (M1L2L3) produced only the ∼60 kDa isoform and the small‐conductance channel (∼52 pS). Mutants designed to produce translation from the second (M2L3) or third (M3) initiation site produced the ∼54 kDa isoform, and the large conductance channel (∼185–224 pS). M1L2L3, M2L3 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 M1L2L3 (∼0.02), M2L3 (∼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.

Properties of single two-pore domain TREK-2 channels expressed in mammalian cells

TREK‐2 (K2P10.1), a member of the two‐pore domain K+ (K2P) channel family, provides the background K+ conductance in many cell types, and is a target of neurotransmitters that act on receptors coupled to Gs and Gq. We report here that TREK‐2 exhibits small (TREK‐2S) and large (TREK‐2L) conductance phenotypes when expressed in mammalian cell lines (COS‐7, HEK293, HeLa) and in Xenopus oocytes. TREK‐2S phenotype shows a noisy open state with a mean conductance of 54 pS (+40 mV). TREK‐2L phenotype shows a full open state (202 pS) with several short‐lived sub‐conductance levels. Both phenotypes were strongly activated by arachidonic acid, membrane stretch (−40 mmHg) and intracellular acidification (pH 6.4). Phosphorylation of TREK‐2 produced by treatment of cells with activators of protein kinases A and C, and okadaic acid (a serine/threonine phosphatase inhibitor) decreased the current contributed by TREK‐2S and TREK‐2L, and caused partial switching of conductance levels from those of TREK‐2S and TREK‐2L to more intermediate values. Under this condition, TREK‐2 exhibited six conducting levels and one closed level. TREK‐2 mutants in which putative protein kinases A and C phosphorylation sites were mutated to alanines (S326A, S359A, S326A/S359A) displayed mostly TREK‐2S and TREK‐2L phenotypes. However, S326D and S359D mutants (as well as the double mutants) that mimic the phosphorylated state showed all six conducting levels and low channel activity. The S326A and S359A mutants did not significantly affect the intrinsic voltage dependence of TREK‐2 in Mg2+‐free solution. Phenotypes resembling TREK‐2S and TREK‐2L were also observed in cerebellar granule neurons that express TREK‐2 mRNA. These results show that TREK‐2 exhibits two primary modes of gating that give rise to two channel phenotypes under dephosphorylated conditions, and that its phosphorylation shifts the gating mode to include intermediate conducting levels. This represents a novel mechanism by which receptor agonists modulate the function of a K+ channel to alter cell excitability.

Sp3 controls fibroblast growth factor receptor 4 gene activity during myogenic differentiation

Fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) signaling is a critical component in the regulation of myoblast proliferation and differentiation. The transient FGFR4 gene expression during the transition from proliferating myoblasts to differentiated myotubes indicates that FGFR4 regulates this critical phase of myogenesis. The Specificity Protein (SP) family of transcription factors controls FGFR family member gene activity. We sought to determine if members of the Sp family regulate mouse FGFR4 gene activity during myogenic differentiation. RT-PCR and western blot analysis of FGFR4 mRNA and protein revealed transient expression over 72h, with peak expression between 24 and 36h after addition of differentiation medium to C2C12 myogenic cultures. Sp3 also displayed a transient expression pattern with peak expression occurring after 6h of differentiation. We cloned a 1527bp fragment of the mouse FGFR4 promoter into a luciferase reporter. This FGFR4 promoter contains eight putative Sp binding sites and directed luciferase gene activity comparable to native FGFR4 expression. Overexpression of Sp1 and Sp3 showed that Sp1 repressed FGFR4 gene activity, and Sp3 activated FGFR4 gene activity during myogenic differentiation. Mutational analyses of multiple Sp binding sites within the FGFR4 promoter revealed that three of these sites were transcriptionally active. Electromobility shift assays and chromatin immunoprecipitation of the area containing the activator sites showed that Sp3 bound to this promoter location.

C/EBPα represses slow myosin heavy chain 2 gene expression in developing avian myotubes.

BACKGROUND The CCAAT/enhancer binding proteins (C/EBP) comprise a family of transcription factors that regulate many cellular processes. Little is known of their function during embryonic and fetal myogenesis. Slow myosin heavy chain 2 (MyHC2) is a marker of the slow avian skeletal muscle fiber type, and slow MyHC2 gene regulation involves molecular pathways that lead to muscle fiber type diversification. METHODS The biological effects of C/EBPα and C/EBPβ expression were analyzed by use of a general C/EBP activity reporter and by slow MyHC2 promoter-reporter constructs transfected into specific myogenic cell lineages. The effects of C/EBPα and C/EBPβ expression were also analyzed by immunocytochemical detection of slow MyHC2. C/EBPα interaction with the slow MyHC2 promoter was assessed by electromobility shift assays. RESULTS C/EBPα and C/EBPβ are present in embryonic fast and fast/slow avian myogenic lineages. Overexpression of C/EBPα cDNA repressed slow MyHC2 promoter activity in embryonic myotubes and in both electrically stimulated fetal myotubes. Deletion analysis of the slow MyHC2 promoter-luciferase reporter demonstrated that the transcriptional repression mediated by C/EBPα occurs within the first 222bp upstream from exon 1 of the slow MyHC2 gene. Electromobility shift assays determined that C/EBPα can bind to a non-canonical C/EBP site within the slow MyHC2 gene, and mutation of this site reduced transcriptional repression of the slow MyHC2 gene. CONCLUSION C/EBPα, but not C/EBPβ, represses slow MyHC2 promoter activity via a non-canonical C/EBP binding element. GENERAL SIGNIFICANCE Members of the C/EBP family of transcription factors differentially regulate genes indicative of distinct muscle fiber types.