84 research outputs found
The selectivity, voltage-dependence and acid sensitivity of the tandem pore potassium channel TASK-1 : contributions of the pore domains
We have investigated the contribution to ionic
selectivity of residues in the selectivity filter and pore
helices of the P1 and P2 domains in the acid sensitive
potassium channel TASK-1. We used site directed mutagenesis
and electrophysiological studies, assisted by structural
models built through computational methods. We have
measured selectivity in channels expressed in Xenopus
oocytes, using voltage clamp to measure shifts in reversal
potential and current amplitudes when Rb+ or Na+ replaced
extracellular K+. Both P1 and P2 contribute to selectivity,
and most mutations, including mutation of residues in the
triplets GYG and GFG in P1 and P2, made channels nonselective.
We interpret the effects of theseβand of other
mutationsβin terms of the way the pore is likely to be
stabilised structurally. We show also that residues in the
outer pore mouth contribute to selectivity in TASK-1.
Mutations resulting in loss of selectivity (e.g. I94S, G95A)
were associated with slowing of the response of channels to
depolarisation. More important physiologically, pH sensitivity
is also lost or altered by such mutations. Mutations
that retained selectivity (e.g. I94L, I94V) also retained their
response to acidification. It is likely that responses both to
voltage and pH changes involve gating at the selectivity filter
The response of the tandem pore potassium channel TASK-3 (K2P9.1) to voltage : gating at the cytoplasmic mouth
Although the tandem pore potassium channel TASK-3 is thought to open and shut at its
selectivity filter in response to changes of extracellular pH, it is currently unknown whether the
channel also shows gating at its inner, cytoplasmic mouth through movements of membrane
helices M2 and M4.We used two electrode voltage clamp and single channel recording to show
that TASK-3 responds to voltage in a way that reveals such gating. In wild-type channels, Popen
was very low at negative voltages, but increased with depolarisation. The effect of voltage was
relatively weak and the gating charge small, βΌ0.17.Mutants A237T (in M4) and N133A (in M2)
increased Popen at a given voltage, increasing mean open time and the number of openings per
burst. In addition, the relationship between Popen andvoltagewas shifted to lesspositive voltages.
Mutation of putative hinge glycines (G117A, G231A), residues that are conserved throughout
the tandem pore channel family, reduced Popen at a given voltage, shifting the relationship
with voltage to a more positive potential range. None of these mutants substantially affected
the response of the channel to extracellular acidification. We have used the results from single
channel recording to develop a simple kinetic model to show how gating occurs through two
classes of conformation change, with two routes out of the open state, as expected if gating
occurs both at the selectivity filter and at its cytoplasmic mouth
Ξ±-Syntrophin Modulates Myogenin Expression in Differentiating Myoblasts
Ξ±-Syntrophin is a scaffolding protein linking signaling proteins to the sarcolemmal dystrophin complex in mature muscle. However, Ξ±-syntrophin is also expressed in differentiating myoblasts during the early stages of muscle differentiation. In this study, we examined the relationship between the expression of Ξ±-syntrophin and myogenin, a key muscle regulatory factor.The absence of Ξ±-syntrophin leads to reduced and delayed myogenin expression. This conclusion is based on experiments using muscle cells isolated from Ξ±-syntrophin null mice, muscle regeneration studies in Ξ±-syntrophin null mice, experiments in Sol8 cells (a cell line that expresses only low levels of Ξ±-syntrophin) and siRNA studies in differentiating C2 cells. In primary cultured myocytes isolated from Ξ±-syntrophin null mice, the level of myogenin was less than 50% that from wild type myocytes (p<0.005) 40 h after differentiation induction. In regenerating muscle, the expression of myogenin in the Ξ±-syntrophin null muscle was reduced to approximately 25% that of wild type muscle (p<0.005). Conversely, myogenin expression is enhanced in primary cultures of myoblasts isolated from a transgenic mouse over-expressing Ξ±-syntrophin and in Sol8 cells transfected with a vector to over-express Ξ±-syntrophin. Moreover, we find that myogenin mRNA is reduced in the absence of Ξ±-syntrophin and increased by Ξ±-syntrophin over-expression. Immunofluorescence microscopy shows that Ξ±-syntrophin is localized to the nuclei of differentiating myoblasts. Finally, immunoprecipitation experiments demonstrate that Ξ±-syntrophin associates with Mixed-Lineage Leukemia 5, a regulator of myogenin expression.We conclude that Ξ±-syntrophin plays an important role in regulating myogenesis by modulating myogenin expression
Retrograde trafficking of Ξ²-dystroglycan from the plasma membrane to the nucleus
Ξ²-Dystroglycan (Ξ²-DG) is a transmembrane protein with critical roles in cell adhesion, cytoskeleton remodeling and nuclear architecture. This functional diversity is attributed to the ability of Ξ²-DG to target to, and conform specific protein assemblies at the plasma membrane (PM) and nuclear envelope (NE). Although a classical NLS and importin Ξ±/Ξ² mediated nuclear import pathway has already been described for Ξ²-DG, the intracellular trafficking route by which Ξ²-DG reaches the nucleus is unknown. In this study, we demonstrated that Ξ²-DG undergoes retrograde intracellular trafficking from the PM to the nucleus via the endosome-ER network. Furthermore, we provided evidence indicating that the translocon complex Sec61 mediates the release of Ξ²-DG from the ER membrane, making it accessible for importins and nuclear import. Finally, we show that phosphorylation of Ξ²-DG at Tyr890 is a key stimulus for Ξ²-DG nuclear translocation. Collectively our data describe the retrograde intracellular trafficking route that Ξ²-DG follows from PM to the nucleus. This dual role for a cell adhesion receptor permits the cell to functionally connect the PM with the nucleus and represents to our knowledge the first example of a cell adhesion receptor exhibiting retrograde nuclear trafficking and having dual roles in PM and NE
Ξ²1-Syntrophin Modulation by miR-222 in mdx Mice
Background: In mdx mice, the absence of dystrophin leads to the deficiency of other components of the dystrophin-glycoprotein complex (DAPC), making skeletal muscle fibers more susceptible to necrosis. The mechanisms involved in the disappearance of the DAPC are not completely understood. The muscles of mdx mice express normal amounts of mRNA for the DAPC components, thus suggesting post-transcriptional regulation. Methodology/Principal Findings: We investigated the hypothesis that DAPC reduction could be associated with the microRNA system. Among the possible microRNAs (miRs) found to be upregulated in the skeletal muscle tissue of mdx compared to wt mice, we demonstrated that miR-222 specifically binds to the 3β²-UTR of Ξ²1-syntrophin and participates in the downregulation of Ξ²1-syntrophin. In addition, we documented an altered regulation of the 3β²-UTR of Ξ²1-syntrophin in muscle tissue from dystrophic mice. Conclusion/Significance: These results show the importance of the microRNA system in the regulation of DAPC components in dystrophic muscle, and suggest a potential role of miRs in the pathophysiology of dystrophy. Β© 2010 De Arcangelis et al
Glial Tumor Necrosis Factor Alpha (TNFΞ±) Generates Metaplastic Inhibition of Spinal Learning
Injury-induced overexpression of tumor necrosis factor alpha (TNFΞ±) in the spinal cord can induce chronic neuroinflammation and excitotoxicity that ultimately undermines functional recovery. Here we investigate how TNFΞ± might also act to upset spinal function by modulating spinal plasticity. Using a model of instrumental learning in the injured spinal cord, we have previously shown that peripheral intermittent stimulation can produce a plastic change in spinal plasticity (metaplasticity), resulting in the prolonged inhibition of spinal learning. We hypothesized that spinal metaplasticity may be mediated by TNFΞ±. We found that intermittent stimulation increased protein levels in the spinal cord. Using intrathecal pharmacological manipulations, we showed TNFΞ± to be both necessary and sufficient for the long-term inhibition of a spinal instrumental learning task. These effects were found to be dependent on glial production of TNFΞ± and involved downstream alterations in calcium-permeable AMPA receptors. These findings suggest a crucial role for glial TNFΞ± in undermining spinal learning, and demonstrate the therapeutic potential of inhibiting TNFΞ± activity to rescue and restore adaptive spinal plasticity to the injured spinal cord. TNFΞ± modulation represents a novel therapeutic target for improving rehabilitation after spinal cord injury
A Common Carcinogen Benzo[a]pyrene Causes Neuronal Death in Mouse via Microglial Activation
BACKGROUND: Benzo[a]pyrene (B[a]P) belongs to a class of polycyclic aromatic hydrocarbons that serve as micropollutants in the environment. B[a]P has been reported as a probable carcinogen in humans. Exposure to B[a]P can take place by ingestion of contaminated (especially grilled, roasted or smoked) food or water, or inhalation of polluted air. There are reports available that also suggests neurotoxicity as a result of B[a]P exposure, but the exact mechanism of action is unknown. METHODOLOGY/PRINCIPAL FINDINGS: Using neuroblastoma cell line and primary cortical neuron culture, we demonstrated that B[a]P has no direct neurotoxic effect. We utilized both in vivo and in vitro systems to demonstrate that B[a]P causes microglial activation. Using microglial cell line and primary microglial culture, we showed for the first time that B[a]P administration results in elevation of reactive oxygen species within the microglia thereby causing depression of antioxidant protein levels; enhanced expression of inducible nitric oxide synthase, that results in increased production of NO from the cells. Synthesis and secretion of proinflammatory cytokines were also elevated within the microglia, possibly via the p38MAP kinase pathway. All these factors contributed to bystander death of neurons, in vitro. When administered to animals, B[a]P was found to cause microglial activation and astrogliosis in the brain with subsequent increase in proinflammatory cytokine levels. CONCLUSIONS/SIGNIFICANCE: Contrary to earlier published reports we found that B[a]P has no direct neurotoxic activity. However, it kills neurons in a bystander mechanism by activating the immune cells of the brain viz the microglia. For the first time, we have provided conclusive evidence regarding the mechanism by which the micropollutant B[a]P may actually cause damage to the central nervous system. In today's perspective, where rising pollution levels globally are a matter of grave concern, our study throws light on other health hazards that such pollutants may exert
Synaptic Neurotransmission Depression in Ventral Tegmental Dopamine Neurons and Cannabinoid-Associated Addictive Learning
Drug addiction is an association of compulsive drug use with long-term associative learning/memory. Multiple forms of learning/memory are primarily subserved by activity- or experience-dependent synaptic long-term potentiation (LTP) and long-term depression (LTD). Recent studies suggest LTP expression in locally activated glutamate synapses onto dopamine neurons (local Glu-DA synapses) of the midbrain ventral tegmental area (VTA) following a single or chronic exposure to many drugs of abuse, whereas a single exposure to cannabinoid did not significantly affect synaptic plasticity at these synapses. It is unknown whether chronic exposure of cannabis (marijuana or cannabinoids), the most commonly used illicit drug worldwide, induce LTP or LTD at these synapses. More importantly, whether such alterations in VTA synaptic plasticity causatively contribute to drug addictive behavior has not previously been addressed. Here we show in rats that chronic cannabinoid exposure activates VTA cannabinoid CB1 receptors to induce transient neurotransmission depression at VTA local Glu-DA synapses through activation of NMDA receptors and subsequent endocytosis of AMPA receptor GluR2 subunits. A GluR2-derived peptide blocks cannabinoid-induced VTA synaptic depression and conditioned place preference, i.e., learning to associate drug exposure with environmental cues. These data not only provide the first evidence, to our knowledge, that NMDA receptor-dependent synaptic depression at VTA dopamine circuitry requires GluR2 endocytosis, but also suggest an essential contribution of such synaptic depression to cannabinoid-associated addictive learning, in addition to pointing to novel pharmacological strategies for the treatment of cannabis addiction
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