Background:Ca 2ϩ /calcineurin-dependent transcription factor NFAT regulates many neuronal functions. Results: Depolarization/Ca 2ϩ influx-induced activation and nuclear translocation are markedly faster and stronger for NFATc3 than NFATc4 in neurons, which is determined in part by SP-containing regions of NFATc3 and NFATc4. Conclusion: Activation of NFATc3 and NFATc4 is distinctly regulated in neurons. Significance: The distinct regulation of NFATc3 and NFATc4 may underlie isoform-specific NFAT functions in neurons.

Key points• The first sensory synapse formed between the central processes of primary afferents and dorsal horn neurons plays an important role in controlling the flow of nociceptive information from the periphery to the CNS, and plasticity at this synapse contributes to centrally mediated pain hypersensitivity.• Although exocytosis and synaptic plasticity are regulated by presynaptic Ca 2+ , the mechanisms underlying presynaptic Ca 2+ signalling at the first sensory synapse are not well understood.• In this study we show that the plasma membrane Ca 2+ -ATPase and mitochondria are the major regulators of presynaptic Ca 2+ signalling in capsaicin-sensitive dorsal root ganglion neurons accounting for ∼47 and ∼40% of presynaptic Ca 2+ clearance, respectively.• Quantitative analysis of changes in cytosolic and mitochondrial Ca 2+ concentrations demonstrates that the mitochondrial Ca 2+ uniporter is highly sensitive to cytosolic Ca 2+ at this synapse.• These results help us understand presynaptic mechanisms at the first sensory synapse. AbstractThe central processes of primary nociceptors form synaptic connections with the second-order nociceptive neurons located in the dorsal horn of the spinal cord. These synapses gate the flow of nociceptive information from the periphery to the CNS, and plasticity at these synapses contributes to centrally mediated hyperalgesia and allodynia. Although exocytosis and synaptic plasticity are controlled by Ca 2+ at the release sites, the mechanisms underlying presynaptic Ca 2+ signalling at the nociceptive synapses are not well characterized. We examined the presynaptic mechanisms regulating Ca 2+ clearance following electrical stimulation in capsaicin-sensitive nociceptors using a dorsal root ganglion (DRG)/spinal cord neuron co-culture system.

Background Leucine-rich repeat containing 8A (LRRC8A) is an ubiquitously expressed transmembrane protein with 17 leucine-rich repeats (LRRs) at its C-terminal end and is an essential component of the volume-regulated anion channel (VRAC), which controls cellular volume. A heterozygous mutation in LRRC8A that truncates the 2 terminal LRRs was reported in a patient with agammaglobulinemia and absent B cells and was demonstrated to exert a dominant negative effect on T- and B-cell development in mice. Lrrc8a−/− mice have severely defective T-cell development and function. It is not known whether the T- and B-cell defects caused by LRRC8A deficiency are caused by loss of VRAC activity. Objective We sought to determine whether VRAC activity is required for normal T-cell development and function. Methods VRAC activity was examined by using patch-clamp analysis. Flow cytometry was used to examine T-cell development. T-cell proliferation, cytokine secretion, and antibody titers were measured by using standard techniques. Results We demonstrate that the spontaneous mouse mutant ébouriffé (ebo/ebo) harbors a homozygous 2-bp frameshift mutation in Lrrc8a that truncates the 15 terminal LRRs of LRRC8A. The Lrrc8aebo mutation does not affect protein expression but drastically diminishes VRAC activity in T cells. ebo/ebo mice share features with Lrrc8a−/− mice that include curly hair, infertility, reduced longevity, and kidney abnormalities. However, in contrast to Lrrc8a−/− mice, ebo/ebo mice have normal T-cell development and function and intact antibody response to T-dependent antigen. Conclusion LRRC8A-dependent VRAC activity is dispensable for T-cell development and function.

Calcium (Ca2+) influx into the mitochondrial matrix stimulates ATP synthesis. Here, we investigate whether mitochondrial Ca2+ transport pathways are altered in the setting of deficient mitochondrial energy synthesis, as increased matrix Ca2+ may provide a stimulatory boost. We focused on mitochondrial cardiomyopathies, which feature such dysfunction of oxidative phosphorylation. We study a mouse model where the main transcription factor for mitochondrial DNA (transcription factor A, mitochondrial, Tfam) has been disrupted selectively in cardiomyocytes. By the second postnatal week (10–15 day old mice), these mice have developed a dilated cardiomyopathy associated with impaired oxidative phosphorylation. We find evidence of increased mitochondrial Ca2+ during this period using imaging, electrophysiology, and biochemistry. The mitochondrial Ca2+ uniporter, the main portal for Ca2+ entry, displays enhanced activity, whereas the mitochondrial sodium-calcium (Na+-Ca2+) exchanger, the main portal for Ca2+ efflux, is inhibited. These changes in activity reflect changes in protein expression of the corresponding transporter subunits. While decreased transcription of Nclx, the gene encoding the Na+-Ca2+ exchanger, explains diminished Na+-Ca2+ exchange, the mechanism for enhanced uniporter expression appears to be post-transcriptional. Notably, such changes allow cardiac mitochondria from Tfam knockout animals to be far more sensitive to Ca2+-induced increases in respiration. In the absence of Ca2+, oxygen consumption declines to less than half of control values in these animals, but rebounds to control levels when incubated with Ca2+. Thus, we demonstrate a phenotype of enhanced mitochondrial Ca2+ in a mitochondrial cardiomyopathy model, and show that such Ca2+ accumulation is capable of rescuing deficits in energy synthesis capacity in vitro.

NaChBac, the first bacterial voltage-gated Na+(Nav) channel to be characterized, has been the prokaryotic prototype for studying the structure–function relationship of Navchannels. Discovered nearly two decades ago, the structure of NaChBac has not been determined. Here we present the single particle electron cryomicroscopy (cryo-EM) analysis of NaChBac in both detergent micelles and nanodiscs. Under both conditions, the conformation of NaChBac is nearly identical to that of the potentially inactivated NavAb. Determining the structure of NaChBac in nanodiscs enabled us to examine gating modifier toxins (GMTs) of Navchannels in lipid bilayers. To study GMTs in mammalian Navchannels, we generated a chimera in which the extracellular fragment of the S3 and S4 segments in the second voltage-sensing domain from Nav1.7 replaced the corresponding sequence in NaChBac. Cryo-EM structures of the nanodisc-embedded chimera alone and in complex with HuwenToxin IV (HWTX-IV) were determined to 3.5 and 3.2 Å resolutions, respectively. Compared to the structure of HWTX-IV–bound human Nav1.7, which was obtained at an overall resolution of 3.2 Å, the local resolution of the toxin has been improved from ∼6 to ∼4 Å. This resolution enabled visualization of toxin docking. NaChBac can thus serve as a convenient surrogate for structural studies of the interactions between GMTs and Navchannels in a membrane environment.