Modulation of Acid-Sensing Ion Channels by Tannic Acid and Green Tea via a Membrane-Mediated Mechanism.

Acid-sensing ion channels (ASICs) are proton-gated ion channels that contribute to pain perception and neurotransmission. Being involved in sensing inflammation and ischemia, ASIC1a and ASIC3 are promising drug targets. Polyphenol tannic acid (TA) as well as green tea can interact with a variety of ion channels, but their effect on ASICs remains unknown. In addition, it is unknown whether they interact with ion channels via a common mechanism. Here, we show that TA is a potent modulator of ASICs. TA inhibited the transient current of rat ASIC3 expressed in HEK cells with an apparent IC50 of 2.2 ± 0.6 µM; it potentiated the sustained current and induced a slowly declining decay current. In addition, it produced an acidic shift of the pH-dependent activation of ASIC3 and inhibited the window current at pH 7.0. Moreover, TA inhibited the transient current of ASIC1a, ASIC1b, and ASIC2a. Pentagalloylglucose that is chemically identical to the central part of TA and a green tea extract both had effects on ASIC3 comparable to TA. TA and green tea inhibited inward currents generated by gramicidin channels, indicating interaction with the membrane. These results show that TA, pentagalloylglucose, and green tea modulate ASICs and identify alteration of the membrane as the potential common mechanism of this modulation. These properties will limit clinical application of these molecules.

Personalized Medicine Approach in a DCM Patient with LMNA Mutation Reveals Dysregulation of mTOR Signaling.

BACKGROUND: Mutations in the Lamin A/C (LMNA) gene are responsible for about 6% of all familial dilated cardiomyopathy (DCM) cases which tend to present at a young age and follow a fulminant course. METHODS: We report a 47-year-old DCM patient with severely impaired left ventricular ejection fraction and NYHA functional class IV despite optimal heart failure treatment. Whole-exome sequencing revealed an LMNA E161K missense mutation as the pathogenetic cause for DCM in this patient. We generated a patient-specific LMNA-knock in (LMNA-KI) in vitro model using mES cells. RESULTS: Beta adrenergic stimulation of cardiomyocytes derived from LMNA-KI mES cells resulted in augmented mTOR signaling and increased dysregulation of action potentials, which could be effectively prevented by the mTOR-inhibitor rapamycin. A cardiac biopsy confirmed strong activation of the mTOR-signaling pathway in the patient. An off-label treatment with oral rapamycin was initiated and resulted in an improvement in left ventricular ejection fraction (27.8% to 44.5%), NT-BNP (8120 ng/L to 2210 ng/L) and NYHA functional class. CONCLUSION: We have successfully generated the first in vitro model to recapitulate a patient-specific LMNA E161K mutation which leads to a severe form of DCM. The model may serve as a template for individualized and specific treatment of heart failure.

Dynorphin Neuropeptides Decrease Apparent Proton Affinity of ASIC1a by Occluding the Acidic Pocket.

Prolonged acidosis, as it occurs during ischemic stroke, induces neuronal death via acid-sensing ion channel 1a (ASIC1a). Concomitantly, it desensitizes ASIC1a, highlighting the pathophysiological significance of modulators of ASIC1a acid sensitivity. One such modulator is the opioid neuropeptide big dynorphin (Big Dyn) which binds to ASIC1a and enhances its activity during prolonged acidosis. The molecular determinants and dynamics of this interaction remain unclear, however. Here, we present a molecular interaction model showing a dynorphin peptide inserting deep into the acidic pocket of ASIC1a. We confirmed experimentally that the interaction is predominantly driven by electrostatic forces, and using noncanonical amino acids as photo-cross-linkers, we identified 16 residues in ASIC1a contributing to Big Dyn binding. Covalently tethering Big Dyn to its ASIC1a binding site dramatically decreased the proton sensitivity of channel activation, suggesting that Big Dyn stabilizes a resting conformation of ASIC1a and dissociates from its binding site during channel opening.

Efficient expression of a cnidarian peptide-gated ion channel in mammalian cells.

Hydra Na+ channels (HyNaCs) are peptide-gated ion channels of the DEG/ENaC gene family that are directly activated by neuropeptides of the Hydra nervous system. They have previously been successfully characterized in Xenopus oocytes. To establish their expression in mammalian cells, we transiently expressed heteromeric HyNaC2/3/5 in human HEK 293 and monkey COS-7 cells. We found that the expression of HyNaC2/3/5 using native cDNAs was inefficient and that codon optimization strongly increased protein expression and current amplitude in patch-clamp experiments. We used the improved expression of codon-optimized channel subunits to perform Ca2+ imaging and to demonstrate their glycosylation pattern. In summary, we established efficient expression of a cnidarian ion channel in mammalian cell lines.

Protons as Messengers of Intercellular Communication in the Nervous System.

In this review, evidence demonstrating that protons (H+) constitute a complex, regulated intercellular signaling mechanisms are presented. Given that pH is a strictly regulated variable in multicellular organisms, localized extracellular pH changes may constitute significant signals of cellular processes that occur in a cell or a group of cells. Several studies have demonstrated that the low pH of synaptic vesicles implies that neurotransmitter release is always accompanied by the co-release of H+ into the synaptic cleft, leading to transient extracellular pH shifts. Also, evidence has accumulated indicating that extracellular H+ concentration regulation is complex and implies a source of protons in a network of transporters, ion exchangers, and buffer capacity of the media that may finally establish the extracellular proton concentration. The activation of membrane transporters, increased production of CO2 and of metabolites, such as lactate, produce significant extracellular pH shifts in nano- and micro-domains in the central nervous system (CNS), constituting a reliable signal for intercellular communication. The acid sensing ion channels (ASIC) function as specific signal sensors of proton signaling mechanism, detecting subtle variations of extracellular H+ in a range varying from pH 5 to 8. The main question in relation to this signaling system is whether it is only synaptically restricted, or a volume modulator of neuron excitability. This signaling system may have evolved from a metabolic activity detection mechanism to a highly localized extracellular proton dependent communication mechanism. In this study, evidence showing the mechanisms of regulation of extracellular pH shifts and of the ASICs and its function in modulating the excitability in various systems is reviewed, including data and its role in synaptic neurotransmission, volume transmission and even segregated neurotransmission, leading to a reliable extracellular signaling mechanism.

Acid-Sensing Ion Channels as Potential Therapeutic Targets in Neurodegeneration and Neuroinflammation.

Acid-sensing ion channels (ASICs) are a family of proton-sensing channels that are voltage insensitive, cation selective (mostly permeable to Na+), and nonspecifically blocked by amiloride. Derived from 5 genes (ACCN1-5), 7 subunits have been identified, 1a, 1b, 2a, 2b, 3, 4, and 5, that are widely expressed in the peripheral and central nervous system as well as other tissues. Over the years, different studies have shown that activation of these channels is linked to various physiological and pathological processes, such as memory, learning, fear, anxiety, ischemia, and multiple sclerosis to name a few, so their potential as therapeutic targets is increasing. This review focuses on recent advances that have helped us to better understand the role played by ASICs in different pathologies related to neurodegenerative diseases, inflammatory processes, and pain.