Summary Adenosine-to-inosine RNA editing is crucial for generating molecular diversity, and serves to regulate protein function through recoding of genomic information. Here, we discover editing within CaV1.3 Ca2+ channels, renown for low-voltage Ca2+-influx and neuronal pacemaking. Significantly, editing occurs within the channel’s IQ domain, a calmodulin-binding site mediating inhibitory Ca2+-feedback (CDI) on channels. The editing turns out to require RNA adenosine deaminase ADAR2, whose variable activity could underlie a spatially diverse pattern of CaV1.3 editing seen across the brain. Edited CaV1.3 protein is detected both in brain tissue and within the surface membrane of primary neurons. Functionally, edited CaV1.3 channels exhibit strong reduction of CDI; in particular, neurons within the suprachiasmatic nucleus show diminished CDI, with higher frequencies of repetitive action-potential and calcium-spike activity, in wildtype versus ADAR2 knockout mice. Our study reveals a mechanism for fine-tuning CaV1.3 channel properties in CNS, which likely impacts a broad spectrum of neurobiological functions.

Insulin-like growth factor 1 (IGF-1) is implicated in the nociceptive (pain) sensitivity of primary afferent neurons. We found that the IGF-1 receptor (IGF-1R) functionally stimulated voltage-gated T-type Ca(2+) (CaV3) channels in mouse dorsal root ganglia (DRG) neurons through a mechanism dependent on heterotrimeric G protein (heterotrimeric guanine nucleotide-binding protein) signaling. IGF-1 increased T-type channel currents in small-diameter DRG neurons in a manner dependent on IGF-1 concentration and IGF-1R but independent of phosphatidylinositol 3-kinase (PI3K). The intracellular subunit of IGF-1R coimmunoprecipitated with Gαo. Blocking G protein signaling by the intracellular application of guanosine diphosphate (GDP)-β-S or with pertussis toxin abolished the stimulatory effects of IGF-1. Antagonists of protein kinase Cα (PKCα), but not of PKCβ, abolished the IGF-1-induced T-type channel current increase. Application of IGF-1 increased membrane abundance of PKCα, and PKCα inhibition (either pharmacologically or genetically) abolished the increase in T-type channel currents stimulated by IGF-1. IGF-1 increased action potential firing in DRG neurons and increased the sensitivity of mice to both thermal and mechanical stimuli applied to the hindpaw, both of which were attenuated by intraplantar injection of a T-type channel inhibitor. Furthermore, inhibiting IGF-1R signaling or knocking down CaV3.2 or PKCα in DRG neurons abolished the increased mechanical and thermal sensitivity that mice exhibited under conditions modeling chronic hindpaw inflammation. Together, our results showed that IGF-1 enhances T-type channel currents through the activation of IGF-1R that is coupled to a G protein-dependent PKCα pathway, thereby increasing the excitability of DRG neurons and the sensitivity to pain.

Hydrogen sulfide (H(2)S), an endogenous gas molecule synthesized by cystathionine-β-synthetase (CBS), is involved in inflammation and nociceptive signaling. However, the molecular and epigenetic mechanisms of CBS-H(2)S signaling in peripheral nociceptive processing remain unknown. We demonstrated that peripheral inflammation induced by intraplantar injection of complete Freund adjuvant significantly up-regulated expression of CBS at both protein and mRNA levels in rat dorsal root ganglia (DRG). The CBS inhibitors hydroxylamine and aminooxyacetic acid attenuated mechanical hyperalgesia in a dose-dependent manner and reversed hyperexcitability of DRG neurons in inflamed rats. Intraplantar administration of NaHS (its addition mimics CBS production of H(2)S) or l-cysteine in healthy rats elicited mechanical hyperalgesia. Application of NaHS in vitro enhanced excitability and tetrodotoxin (TTX)-resistant sodium current of DRG neurons from healthy rats, which was attenuated by pretreatment of protein kinase A inhibitor H89. Methylation-specific PCR and bisulfite sequencing demonstrated that promoter region of cbs gene was less methylated in DRG samples from inflamed rats than that from controls. Peripheral inflammation did not alter expression of DNA methyltransferase 3a and 3b, the 2 major enzymes for DNA methylation, but led to a significant up-regulation of methyl-binding domain protein 4 and growth arrest and DNA damage inducible protein 45α, the enzymes involved in active DNA demethylation. Our findings suggest that epigenetic regulation of CBS expression may contribute to inflammatory hyperalgesia. H(2)S seems to increase TTX-resistant sodium channel current, which may be mediated by protein kinase A pathway, thus identifying a potential therapeutic target for the treatment of chronic pain.

BACKGROUND AND PURPOSEEndostatin (ES) is a c-terminal proteolytic fragment of collagen XVIII with promising antitumour properties in several tumour models, including human glioblastoma. We hypothesized that this peptide could interact with plasma membrane ion channels and modulate their functions. EXPERIMENTAL APPROACHUsing cell proliferation and migration assays, patch clamp and Western blot analysis, we studied the effects of ES on the proliferation and migration of human glioblastoma U87 cells, mediated by T-type Ca 2+ channels. KEY RESULTSExtracellular application of ES reversibly inhibited T-type Ca 2+ channel currents (T-currents) in U87 cells, whereas L-type Ca 2+ currents were not affected. This inhibitory effect was associated with a hyperpolarizing shift in the voltage-dependence of inactivation but was independent of G-protein and protein tyrosine kinase-mediated pathways. All three a1 subunits of T-type Ca 2+ channels (CaV3), a1G (CaV3.1), a1H (CaV3.2) and a1I (CaV3.3), were endogenously expressed in U87 cells. Using transfected HEK293 or CHO cells, we showed that only CaV3.1 and CaV3.2, but not CaV3.3 or CaV1.2 (L-type), channel currents were significantly inhibited. More interestingly, ES inhibited the proliferation and migration of U87 cells in a dose-dependent manner. Pretreatment of the cells with the specific T-type Ca 2+ channel blocker mibefradil occluded these inhibitory effects of ES. CONCLUSION AND IMPLICATIONSThis study provides the first evidence that the antitumour effects of ES on glioblastoma cells is through direct inhibition of T-type Ca 2+ channels and gives new insights into the future development of a new class of antiglioblastoma agents that target the proliferation and migration of these cells. LINKED ARTICLEThis article is commented on by Santoni et al., pp. 1244Santoni et al., pp. -1246

Low-voltage-activated T-type Ca²⁺ channels contribute to a wide variety of physiological functions, most predominantly in the nervous, cardiovascular and endocrine systems. Studies have documented the roles of T-type channels in sleep, neuropathic pain, absence epilepsy, cell proliferation and cardiovascular function. Importantly, novel aspects of the modulation of T-type channels have been identified over the last few years, providing new insights into their physiological and pathophysiological roles. Although there is substantial literature regarding modulation of native T-type channels, the underlying molecular mechanisms have only recently begun to be addressed. This review focuses on recent evidence that the Ca(v)3 subunits of T-type channels, Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3, are differentially modulated by a multitude of endogenous ligands including anandamide, monocyte chemoattractant protein-1, endostatin, and redox and oxidizing agents. The review also provides an overview of recent knowledge gained concerning downstream pathways involving G-protein-coupled receptors. This article is part of a Special Issue entitled: Calcium channels.

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Mitochondria are the double membrane organelles providing most of the energy for cells. In addition, mitochondria also play essential roles in various cellular biological processes such as calcium signaling, apoptosis, ROS generation, cell growth, and cell cycle. Mitochondrial dysfunction is observed in various neurological disorders which harbor acute and chronic neural injury such as neurodegenerative diseases and ischemia, hypoxia-induced brain injury. In this review, we describe how mitochondrial dysfunction contributes to the pathogenesis of neurological disorders which manifest chronic or acute neural injury.

The corticotropin-releasing factor (CRF) peptides CRF and urocortins 1 to 3 are crucial regulators of mammalian stress and inflammatory responses, and they are also implicated in disorders such as anxiety, depression, and drug addiction. There is considerable interest in the physiological mechanisms by which CRF receptors mediate their widespread effects, and here we report that the native CRF receptor 1 (CRFR1) endogenous to the human embryonic kidney 293 cells can functionally couple to mammalian Ca V 3.2 T-type calcium channels. Activation of CRFR1 by either CRF or urocortin (UCN) 1 reversibly inhibits Ca V 3.2 currents (IC 50 of ϳ30 nM), but it does not affect Ca V 3.1 or Ca V 3.3 channels. Blockade of CRFR1 by the antagonist astressin abolished the inhibition of Ca V 3.2 channels. The CRFR1-dependent inhibition of Ca V 3.2 channels was independent of the activities of phospholipase C, tyrosine kinases, Ca 2ϩ /calmodulin-dependent protein kinase II, protein kinase C, and other kinase pathways, but it was dependent upon a cholera toxin-sensitive G protein-mediated mechanism relying upon G protein ␤␥ subunits (G␤␥). The inhibition of Ca V 3.2 channels via the activation of CRFR1 was due to a hyperpolarized shift in their steady-state inactivation, and it was reversible upon washout of the agonists. Given that UCN affect multiple aspects of cardiac and neuronal physiology and that Ca V 3.2 channels are widespread throughout the cardiovascular and nervous systems, the results point to a novel and functionally relevant CRFR1-Ca V 3.2 T-type calcium channel signaling pathway.The corticotropin-releasing factor (CRF) family, consisting of CRF, urocortin 1 (UCN), UCN2, and UCN3, are critical regulators of stress and inflammatory responses, and they have been variously associated with being cardioprotective and contributing toward alcohol and drug dependencies (Reul and Holsboer, 2002;Bale and Vale, 2004;Bruijnzeel and Gold, 2005;Gravanis and Margioris, 2005). The two major receptors for CRF and UCNs, CRF receptor (CRFR)1 and CRFR2, have been identified as G protein-coupled receptors (GPCRs) that can mediate responses via activation of the protein kinase signaling pathways (Bruijnzeel and Gold, 2005;Gravanis and Margioris, 2005). CRF has a higher affinity for CRFR1 than for CRFR2, UCN shows high affinity for both CRFR1 and CRFR2, whereas UCN2 and UCN3 are selective for CRFR2 (Bale and Vale, 2004). The CRFR1 is expressed primarily in the brain and pituitary, and activation of CRFR1 exerts numerous central and peripheral effects associated with pathological diseases (Dautzenberg and Hauger, 2002). Within the hypothalamus-pituitary axis, CRF and CRF-related peptides such as UCN activate CRFR1 receptors to regulate pituitary function in response to stress T.W