Acid‐sensing ion channels (ASICs) have Cl − ‐binding sites in the extracellular domain that are highly conserved between ASIC isoforms. In this experiment, we investigated the effect of Cl − ‐substitution on heterologously expressed ASIC1a current and ASIC current in hippocampal neurons by whole‐cell patch‐clamp method. Substitution of Cl − with the impermeable and inert anion, methansulfonate (MeS) did not alter the maximal current amplitude, pH sensitivity or steady‐state desensitization. However, the rate of desensitization was faster in MeS than in Cl − , and the tachyphylaxis of ASIC current was attenuated in MeS. Other anions (thiocyanate, Br − and I − ) also modified the kinetics of desensitization and tachyphylaxis. Next, we studied the effect of mutation of Cl − ‐binding sites. The pH sensitivity of mutants (K211A, R309A, E313A) was similar to that of wild type channel. However, these mutants showed a faster desensitization and an attenuated rundown. These mutations abolished the effect of anion substitution. Finally, we investigated the effect of Cl − substitution in hippocampal neuron. The modulation of the ASIC1a current in hippocampal neuron was similar to that in heterologously expressed ASIC1a. In addition to sensing pH changes, modulation of ASICs by anion fluxes associated with pathological conditions further positions the channels to sense changes in the extracellular environment.
Acid-Sensing Ion Channels (ASICs) are proton-gated sodium-selective cation channels that have emerged as metabolic and pain sensors in peripheral sensory neurons and contribute to neurotransmission in the CNS. These channels and their related degenerin/epithelial sodium channel (DEG/ENaC) family are often characterized by their sensitivity to amiloride inhibition. However, amiloride can also cause paradoxical potentiation of ASIC currents under certain conditions. Here we characterized and investigated the determinants of paradoxical potentiation by amiloride on ASIC3 channels. While inhibiting currents induced by acidic pH, amiloride potentiated sustained currents at neutral pH activation. These effects were accompanied by alterations in gating properties including (1) an alkaline shift of pH-dependent activation, (2) inhibition of pH-dependent steady-state desensitization (SSD), (3) prolongation of desensitization kinetics, and (4) speeding of recovery from desensitization. Interestingly, extracellular Ca2+ was required for paradoxical potentiation and it diminishes the amiloride-induced inhibition of SSD. Site-directed mutagenesis within the extracellular non-proton ligand-sensing domain (E79A, E423A) demonstrated that these residues were critical in mediating the amiloride-induced inhibition of SSD. However, disruption of the purported amiloride binding site (G445C) within the channel pore blunted both the inhibition and potentiation of amiloride. Together, our results suggest that the myriad of modulatory and blocking effects of amiloride are the result of a complex competitive interaction between amiloride, Ca2+, and protons at probably more than one site in the channel.
Abstract Thyroglossal duct on the dorsum of the tongue in the pediatric patient can cause a difficult airway due to the large mass and risk of airway obstruction associated with a swollen tongue after surgery.
1 This study was undertaken to determine whether long-term in vivo administration of nitroglycerine (NTG) downregulates the endothelium-dependent relaxation induced by acetylcholine (ACh) in the rabbit intrapulmonary vein and, if so, whether the type 1 angiotensin II receptor (AT(1)R) blocker valsartan normalizes this downregulated relaxation. 2 In strips treated with the cyclooxygenase inhibitor diclofenac, ACh induced a relaxation only when the endothelium was intact. A small part of this ACh-induced relaxation was inhibited by coapplication of two Ca(2+)-activated K(+)-channel blockers (charybdotoxin (CTX)+apamin) and the greater part of the response was inhibited by the nitric-oxide-synthase inhibitor N(omega)-nitro-L-arginine (L-NNA). 3 The endothelium-dependent relaxation induced by ACh, but not the endothelium-independent relaxation induced by the nitric oxide donor NOC-7, was significantly reduced in NTG-treated rabbits (versus those in NTG-nontreated control rabbits). The attenuated relaxation was normalized by coapplication of valsartan with the NTG. 4 In the vascular wall, both the amount of localized angiotensin II and the production of superoxide anion were increased by in vivo NTG treatment. These variables were normalized by coapplication of valsartan with the NTG. 5 It is suggested that long-term in vivo administration of NTG downregulates the ACh-induced endothelium-dependent relaxation, mainly through an inhibition of endothelial nitric oxide production in the rabbit intrapulmonary vein. A possible role for AT(1)R is proposed in the mechanism underlying this effect.
In the setting of nitrate tolerance, endothelium-dependent relaxation is reduced in several types of peripheral vessels. However, it is unknown whether chronic in vivo administration of nitroglycerine modulates such relaxation in cerebral arteries.Isometric force and smooth muscle cell membrane potential were measured in endothelium-intact strips from rabbit middle cerebral artery (MCA) and posterior cerebral artery (PCA).ACh (0.1-10 microM) concentration-dependently induced endothelium-dependent relaxation during the contraction induced by histamine in both MCA and PCA. Chronic (10 days) in vivo administration of nitroglycerine reduced the ACh-induced relaxation in PCA but not in MCA, in the presence of the cyclooxygenase inhibitor diclofenac (3 microM). In the presence of the NO-synthase inhibitor N (omega)-nitro-L-arginine (L-NNA, 0.1 mM) plus diclofenac, in MCA from both nitroglycerine-untreated control and -treated rabbits, ACh (0.1-10 microM) induced a smooth muscle cell hyperpolarization and relaxation, and these were blocked by the small-conductance Ca(2+)-activated K(+)-channel inhibitor apamin (0.1 microM), but not by the large- and intermediate-conductance Ca(2+)-activated K(+)-channel inhibitor charybdotoxin (0.1 microM). In contrast, in PCA, ACh (<3 microM) induced neither hyperpolarization nor relaxation under these conditions, suggesting that the endothelium-derived relaxing factor is NO in PCA, whereas endothelium-derived hyperpolarizing factor (EDHF) plays a significant role in MCA.It is suggested that in rabbit cerebral arteries, the function of the endothelium-derived relaxing factor NO and that of EDHF may be modulated differently by chronic in vivo administration of nitroglycerine.
