Coupling of proton binding in extracellular domain to channel gating in acid-sensing ion channel.

Protonation of several amino acid residues in the extracellular domain (ECD) of acid-sensing ion channel (ASIC) causes conformational changes that lead to opening of the channel. It is not clear how conformational changes in ECD are coupled to channel gating. Here, we show that the loop connecting ß9 and α4 at the base of the thumb region of ECD interacts with post-TM1 to stabilize the channel in the closed state. Flexibility of these two regions is important for optimum gating of the channel. In ASIC1a, when Y71 (post-TM1) and W287 (ß9-α4 loop) were mutated to cysteine, they formed disulfide bond in the closed state. Breaking of the disulfide bond by reducing agent dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) potentiated the current significantly. Engineered cysteine G288C reacted with sulfhydryl-specific methanethiosulfonate ethyltrimethylammonium (MTSET) in the open state but not in closed/steady desensitized state, suggesting gating-associated conformational movement of this loop. We also identified a salt bridge between highly conserved R64 at TM1 and D432 at TM2 that is important for optimum gating. Based on our results and other published work, we propose that proton binding in ECD is followed by the displacement of the ß9-α4 loop of the thumb, leading to the rotation of TM1. Conformational movement propagates to TM2 and the channel gate opens by the concomitant movement of TM2 and breaking of the salt bridge between R64 and D432.

Proton-gated ion channels in mouse bone marrow stromal cells.

A variety of ion channels like acid sensing ion channels (ASICs) and several members of the transient receptor potential (TRP) cation channel family are known to be activated by protons. The present study describes proton-gated current in mouse bone marrow stromal cells (BMSCs), by using whole cell patch clamp. Rapid application of extracellular solution of pH ≤ 6.5, evoked slow inactivating current with mean peak value of 328 ± 31pA, (n = 25) at pH 5.0. The reversal potential was close to the theoretical Na(+) equilibrium potential, indicating that majority of the current is mediated by Na(+) and partially carried by Ca(2+) as revealed by ion substitution experiments and Ca(2+) imaging. ASICs blocker amiloride (1mM) and nonselective cation channel blocker flufenamic acid (0.3mM) reduced the current amplitudes by 36 ± 5% (n = 10) and 39 ± 7% (n = 14) respectively. Co-application of flufenamic acid and amiloride further decreased the current by 70 ± 7% (n = 7). However, capsazepine, SKF 96365 and ruthenium red had no effect. 10mM of Ca(2+) and 2mM of La(3+) inhibited the current by 39 ± 6% (n = 5) and 46 ± 6% (n = 4) respectively. Zn(2+) (300 µM) and Gd(3+) (500 µM) had no effect on the current amplitude. Low pH mediated cell death was completely inhibited by co-application of La(3+) and amiloride. Reverse Transcriptase-PCR detected expression of mRNAs of ASICs and TRP family. In summary, our results demonstrate the functional expression of low pH-activated ion channels in mouse BMSCs.

Evaluation of the role of nitric oxide in acid sensing ion channel mediated cell death.

Acid sensing ion channels (ASICs) are widely expressed in central and peripheral nervous system. They are involved in a variety of physiological and pathophysiological processes: synaptic transmission, learning and memory, pain perception, ischemia, etc. During ischemia, metabolic acidosis causes the drop of extracellular pH (pHe) which in turn activates ASICs. Activation of calcium permeable ASIC1a has been implicated in neuronal death. ASICs are modulated by several redox reagents, divalent cations and nitric oxide (NO). Although NO potentiates ASIC mediated currents, the physiological significance of such modulation has not been studied in detail. We have evaluated the role of endogenous NO in cell death at different pH, mediated by the activation of ASICs. At pH 6.1, death rates of ASIC1 expressing Neuro2A (N2A) cells are significantly higher in comparison to the cells that do not express ASICs. Amiloride, a blocker of ASICs protects the cell from acid-injury. Sodium nitroprusside, a potent NO donor not only increases the ASIC mediated currents but also increases cell death at low pH. L-Arg, the precursor of NO also potentiates ASICs in a pH dependent manner. L-Arg-induced NO production and potentiation of ASICs were observed at pHs 7.4, 7.2, 7.0 and 6.8. Lowering the pH below 6.8 did not result in significant production of NO or potentiation of ASICs upon L-Arg stimulation. Our results suggest that potentiation of ASICs by NO and subsequent cell death in vivo depends on the severity of acidosis. During mild and moderate acidosis, NO promotes cell death by potentiating ASICs, whereas this potentiation subsides in severe acidosis due to inhibition of NO synthase.