AcidAcid-sensing ion channels (ASICs) 3 are H ϩ -gated members of the DEG/ENaC ion channel family. In mammals, ASICs include four genes (ASIC1, -2, -3, and -4) that encode for six subunits (ASIC1 and -2 both have alternative splice transcripts as follows: ASIC1a, -1b, -2a, and -2b) (1, 2). Functional ASIC channels consist of a complex of three subunits (3), and they are principally expressed in neurons in the central nervous system and in peripheral sensory nerves. In the brain, the isoform ASIC1a is the best studied, and evidence suggests it plays a role in learning and memory. Genetic or pharmacological perturbation of ASIC1a affects spatial memory, eye-blink conditioning, and it seems to be particularly important for fear-related learning and behaviors (4 -6). ASIC1a also has important functions during pathological conditions, including stroke, seizures, depression, and brain tumors (7-10).For several reasons, ASIC channels are ideally positioned to sense changes in brain interstitium. First, the structure of ASICs is unique for ion channels in that ϳ70% of the entire protein consists of a single large extracellular loop. Second, ASIC1a homomeric channels are profoundly sensitive to subtle pH changes; the threshold of activation is ϳ7.0, and half-maximal activation occurs at pH ϳ6.8 (11), which is well within the range that occurs in the brain interstitium during ischemia, seizures, or spreading depression (12, 13). In fact, loss of ASIC1a abolishes currents in central nervous system neurons evoked by extracellular pH changes in the range between 7.2 and 6.0 (6). Third, multiple other chemicals that are released by metabolically stressed brain cells can potentiate ASICs. For example, ASIC currents are increased by physiological concentrations of lactate, ATP, or arachidonic acid (14 -16), all of which are released into the interstitium during brain ischemia (17)(18)(19).The recently resolved ASIC1a crystal structure revealed the surprising finding that three Cl Ϫ ions were bound to the channel complex in the extracellular domain (3). These sites are coordinated by two nearby residues (Arg-310 and Glu-314) on an ␣-helix of one subunit, a residue (Lys-212) from an adjacent subunit, and are almost completely conserved between all H ϩ -gated ASIC isoforms. Extracellular anions are known to modulate a wide variety of ion channels, including the ASICrelated epithelial sodium channel ENaC (20 -23). However, the significance of Cl Ϫ binding to ASIC channels is unknown. Here, we investigated the effect of extracellular Cl Ϫ and other anions on heterologously expressed ASIC1a as well as native ASICs in mammalian central nervous system neurons. EXPERIMENTAL PROCEDURESHeterologous Expression of cDNA in CHO Cells-Mouse ASIC1a was cloned as described previously (11). The mutations ASIC1a K211A , ASIC1a R309A , and ASIC1a E313A (the residues are numbered per the mouse ASIC1a sequence) were generated by site-directed mutagenesis using the QuikChange kit (Stratagene, La Jolla, CA) and sequenced at the University of Iowa DNA c...

1 This study was undertaken to determine whether long-term in vivo administration of nitroglycerine (NTG) downregulates the hyperpolarization induced by acetylcholine (ACh) in aortic valve endothelial cells (AVECs) of the rabbit and, if so, whether antioxidant agents can normalize this downregulated hyperpolarization. 2 ACh (0.03-3 mM) induced a hyperpolarization through activations of both apamin-and charybdotoxin-sensitive Ca 2 þ -activated K þ channels (K Ca ) in rabbit AVECs. The intermediateconductance K Ca channel (IK Ca ) activator 1-ethyl-2-benzimidazolinone (1-EBIO, 0.3 mM) induced a hyperpolarization of the same magnitude as ACh (3 mM).3 The ACh-induced hyperpolarization was significantly weaker, although the ACh-induced [Ca 2 þ ] i increase was unchanged, in NTG-treated rabbits (versus NTG-untreated control rabbits). The hyperpolarization induced by 1-EBIO was also weaker in NTG-treated rabbits. 4 The reduced ACh-induced hyperpolarization seen in NTG-treated rabbits was not modified by in vitro application of the superoxide scavengers Mn-TBAP, tiron or ascorbate, but it was normalized when ascorbate was coadministered with NTG in vivo. 5 Superoxide production within the endothelial cell (estimated by ethidium fluorescence) was increased in NTG-treated rabbits and this increased production was normalized by in vivo coadministration of ascorbate with the NTG. 6 It is suggested that long-term in vivo administration of NTG downregulates the ACh-induced hyperpolarization in rabbit AVECs, possibly through chronic actions mediated by superoxide.

Acid-sensing ion channels (ASICs) are sodium channels gated by extracellular protons. ASIC1a channels possess intersubunit Cl(-)-binding sites in the extracellular domain, which are highly conserved between ASIC subunits. We previously found that anions modulate ASIC1a gating via these sites. Here we investigated the effect of anion substitution on native ASICs in rat sensory neurons and heterologously expressed ASIC2a and ASIC3 channels by whole cell patch clamp. Similar to ASIC1a, anions modulated the kinetics of desensitization of other ASIC channels. However, unlike ASIC1a, anions also modulated the pH dependence of activation. Moreover, the order of efficacy of different anions to modulate ASIC2a and -3 was very different from that of ASIC1a. More surprising, mutations of conserved residues that form an intersubunit Cl(-)-binding site in ASIC1a only partially abrogated the effects of anion modulation of ASIC2a and had no effect on anion modulation of ASIC3. The effects of anions on native ASICs in rat dorsal root ganglion neurons mimicked those in heterologously expressed ASIC1a/3 heteromeric channels. Our data show that anions modulate a variety of ASIC properties and are dependent on the subunit composition, and the mechanism of modulation for ASIC2a and -3 is distinct from that of ASIC1a. We speculate that modulation of ASIC gating by Cl(-) is a novel mechanism to sense shifts in extracellular fluid composition.

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 þ -activated K þ -channel blockers (charybdotoxin (CTX) þ apamin) and the greater part of the response was inhibited by the nitric-oxide-synthase inhibitor3 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.

Acid-sensing ion channels (ASICs) are Na+ channels activated by changes in pH within the peripheral and central nervous systems. Several different isoforms of ASICs combine to form trimeric channels, and their properties are determined by their subunit composition. ASIC2 subunits are widely expressed throughout the brain, where they heteromultimerize with their partnering subunit, ASIC1a. However, ASIC2 contributes little to the pH sensitivity of the channels, and so its function is not well understood. We found that ASIC2 increased cell surface levels of the channel when it is coexpressed with ASIC1a, and genetic deletion of ASIC2 reduced acid-evoked current amplitude in mouse hippocampal neurons. Additionally, ASIC2a interacted with the neuronal synaptic scaffolding protein PSD-95, and PSD-95 reduced cell surface expression and current amplitude in ASICs that contain ASIC2a. Overexpression of PSD-95 also reduced acid-evoked current amplitude in hippocampal neurons. This result was dependent upon ASIC2 since the effect of PSD-95 was abolished in ASIC2−/− neurons. These results lend support to an emerging role of ASIC2 in the targeting of ASICs to surface membranes, and allows for interaction with PSD-95 to regulate these processes.