Abstract:Chloride homeostasis has a pivotal role in controlling neuronal excitability in the adult brain and during development. The intracellular concentration of chloride is regulated by the dynamic equilibrium between passive fluxes through membrane conductances and the active transport mediated by importers and exporters. In cortical neurons, chloride fluxes are coupled to network activity by the opening of the ionotropic GABAA receptors that provides a direct link between the activity of interneurons and chloride … Show more
“…Both human and murine lysosomes in normal macrophages showed chloride concentrations close to ~118 mM, revealing that lysosomes have the highest chloride levels compared to any other endocytic organelle (Saha et al, 2015; Sonawane et al, 2002). This is nearly 10–15% higher than even extracellular chloride concentrations, which reaches only up to 105–110 mM (Arosio and Ratto, 2014). 10.7554/eLife.28862.014Figure 4.Lysosomal chloride is substantially depleted in mammalian cell culture models of lysosomal storage diseases.( a ) Calibration profile of Clensor in cells (red) and in vitro (grey) showing normalized Alexa 647 (R) and BAC (G) intensity (R/G) ratios versus [Cl - ].…”
Lysosomes are organelles responsible for the breakdown and recycling of cellular machinery. Dysfunctional lysosomes give rise to lysosomal storage disorders as well as common neurodegenerative diseases. Here, we use a DNA-based, fluorescent chloride reporter to measure lysosomal chloride in Caenorhabditis elegans as well as murine and human cell culture models of lysosomal diseases. We find that the lysosome is highly enriched in chloride, and that chloride reduction correlates directly with a loss in the degradative function of the lysosome. In nematodes and mammalian cell culture models of diverse lysosomal disorders, where previously only lysosomal pH dysregulation has been described, massive reduction of lumenal chloride is observed that is ~103 fold greater than the accompanying pH change. Reducing chloride within the lysosome impacts Ca2+ release from the lysosome and impedes the activity of specific lysosomal enzymes indicating a broader role for chloride in lysosomal function.DOI:
http://dx.doi.org/10.7554/eLife.28862.001
“…Both human and murine lysosomes in normal macrophages showed chloride concentrations close to ~118 mM, revealing that lysosomes have the highest chloride levels compared to any other endocytic organelle (Saha et al, 2015; Sonawane et al, 2002). This is nearly 10–15% higher than even extracellular chloride concentrations, which reaches only up to 105–110 mM (Arosio and Ratto, 2014). 10.7554/eLife.28862.014Figure 4.Lysosomal chloride is substantially depleted in mammalian cell culture models of lysosomal storage diseases.( a ) Calibration profile of Clensor in cells (red) and in vitro (grey) showing normalized Alexa 647 (R) and BAC (G) intensity (R/G) ratios versus [Cl - ].…”
Lysosomes are organelles responsible for the breakdown and recycling of cellular machinery. Dysfunctional lysosomes give rise to lysosomal storage disorders as well as common neurodegenerative diseases. Here, we use a DNA-based, fluorescent chloride reporter to measure lysosomal chloride in Caenorhabditis elegans as well as murine and human cell culture models of lysosomal diseases. We find that the lysosome is highly enriched in chloride, and that chloride reduction correlates directly with a loss in the degradative function of the lysosome. In nematodes and mammalian cell culture models of diverse lysosomal disorders, where previously only lysosomal pH dysregulation has been described, massive reduction of lumenal chloride is observed that is ~103 fold greater than the accompanying pH change. Reducing chloride within the lysosome impacts Ca2+ release from the lysosome and impedes the activity of specific lysosomal enzymes indicating a broader role for chloride in lysosomal function.DOI:
http://dx.doi.org/10.7554/eLife.28862.001
“…Several ion sensors based on GFP have been developed (20), starting from Clomeleon (44). Clomeleon consists of the fusion of CFP and Topaz, a YFP mutant, with fluorescence that is reduced on binding with Cl − ions.…”
Section: Discussion Properties and Advantages Of Lssmclophensor In Nementioning
confidence: 99%
“…Thus, a technique for monitoring [Cl − ] i in vivo would substantially add to our understanding of Cl − -regulatory mechanisms and their roles in brain development, plasticity, and disease. The need for an imaging-based method has long been recognized, and several fluorescent sensors have been designed for this purpose (20); however, no reliable data have been obtained so far.…”
Intracellular chloride ([Cl−] i ) and pH (pH i ) are fundamental regulators of neuronal excitability. They exert wide-ranging effects on synaptic signaling and plasticity and on development and disorders of the brain. The ideal technique to elucidate the underlying ionic mechanisms is quantitative and combined two-photon imaging of [Cl − ] i and pH i , but this has never been performed at the cellular level in vivo. Here, by using a genetically encoded fluorescent sensor that includes a spectroscopic reference (an element insensitive to Cl − and pH), we show that ratiometric imaging is strongly affected by the optical properties of the brain. We have designed a method that fully corrects for this source of error. Parallel measurements of [Cl − ] i and pH i at the single-cell level in the mouse cortex showed the in vivo presence of the widely discussed developmental fall in [Cl − ] i and the role of the K-Cl cotransporter KCC2 in this process. Then, we introduce a dynamic twophoton excitation protocol to simultaneously determine the changes of pH i and [Cl − ] i in response to hypercapnia and seizure activity.I ntracellular ion concentrations are controlled by plasmalemmal transporters and channels, which generate and dissipate ionic electrochemical gradients, respectively (1). In recent years, regulation of the intracellular Cl − concentration ([Cl − ] i ) in neurons has attracted lots of attention, because it is the main ion that carries current across GABA A (and also, glycine) receptors. Changes in [Cl − ] i exert an immediate effect on the reversal potential of GABAergic currents (E GABA ) and, thereby, on the properties of GABA A receptor-mediated transmission (2-4). The "ionic plasticity" of GABAergic signaling involves not only the passive flux of Cl − ions through membrane channels but also, a number of ion transporters that regulate [Cl − ] i . Furthermore, this mechanism is under the control of intracellular signaling cascades that regulate the expression patterns as well as functional properties of ion transporters and channels (5, 6). With regard to long-term ionic modulation of GABAergic transmission, a case in point is the decrease in [Cl − ] i that is generally thought to take place during maturation of most central neurons. According to this widely accepted scenario, the Na-K-2Cl cotransporter NKCC1 accumulates Cl − in immature neurons, thereby promoting depolarizing GABA responses (3, 7-9), which is followed by developmental upregulation of the neuron-specific K-Cl cotransporter KCC2 that is required for the generation of classical hyperpolarizing inhibitory postsynaptic potentials (IPSPs) (10).A wealth of electrophysiological evidence dating back to the work in vivo by Eccles and coworkers (11) has provided evidence for active regulation of [Cl − ] i in mammalian central neurons and its crucial effect on the driving force of Cl − in inhibitory synapses (1). However, thus far, there are no direct data on neuronal [Cl − ] i measured in vivo at the single-cell level in the living brain, and for in...
“…41 or due to differences in chloride between somatic and dendritic compartments. 1 The relatively low access resistance of perforated patch recordings mitigates this concern. Also mitigating the concern are findings that neurons in the medullary dorsal horn form GABAergic synapses on their somata and proximal dendrites, 46 suggesting that space clamp limitations were modest.…”
Changes in chloride reversal potential in rat spinal cord neurons have previously been associated with persistent pain in nerve injury and inflammation models. These changes correlate with a decrease in the expression of the potassium chloride transporter, KCC2, and with increases in neuronal excitability. Here, we test the hypothesis that similar changes occur in mice with neuropathic pain induced by chronic constriction injury of the trigeminal infraorbital nerve (CCI-ION). This model allows us to distinguish an acute pain phase (3-5 days after injury) from a persistent pain phase (12-14 days after CCI-ION). Chronic constriction injury of the trigeminal infraorbital nerve induced significant decreases in mechanical pain thresholds in both the acute and persistent phases. To estimate GABAA reversal potentials in neurons from trigeminal nucleus caudalis, we obtained perforated patch recordings in vitro. GABAA reversal potential decreased by 8% during the acute phase in unidentified neurons, but not in GABAergic interneurons. However, at 12 to 14 days after CCI-ION, GABAA reversal potential recovered to normal values. Quantitative real-time polymerase chain reaction analysis revealed no significant changes, at either 3 to 5 days or 12 to 14 days after CCI-ION, in either KCC2 or NKCC1. These findings suggest that CCI-ION in mice results in transient and modest changes in chloride reversal potentials, and that these changes may not persist during the late phase. This suggests that, in the mouse model of CCI-ION, chloride dysregulation may not have a prominent role in the central mechanisms leading to the maintenance of chronic pain.
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