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1.
Deval E Friend V Thirant C Salinas M Jodar M Lazdunski M Lingueglia E 《The Journal of biological chemistry》2006,281(3):1796-1807
Acid-sensing ion channels (ASICs) are cationic channels activated by extracellular protons. The ASIC3 subunit is largely expressed in the peripheral nervous system, where it contributes to pain perception and to some aspects of mechanosensation. We report here a PDZ-dependent and protein kinase C-modulated association between ASIC3 and the Na+/H+ exchanger regulatory factor-1 (NHERF-1) adaptor protein. We show that NHERF-1 and ASIC3 are co-expressed in dorsal root ganglion neurons. NHERF-1 enhances the ASIC3 peak current in heterologous cells, including F-11 dorsal root ganglion cells, by increasing the amount of channel at the plasma membrane. Perhaps more importantly, we show that the plateau current of ASIC3 can be dramatically increased (10-30-fold) by association with NHERF-1, leading to a significant sustained current at pH 6.6. In the presence of NHERF-1, the ASIC3 subcellular localization is modified, and the channel co-localizes with ezrin, a member of the ezrin-radixin-moesin family of actin-binding proteins, providing the first direct link between ASIC3 and the cortical cytoskeleton. Given the importance of the ASIC3 sustained current in nociceptor excitability, it is likely that NHERF-1 participates in channel functions associated with nociception and mechanosensation. 相似文献
2.
The hypothesis that the intracellularNa+ concentration([Na+]i)is a regulator of the epithelialNa+ channel (ENaC) was tested withthe Xenopus oocyte expression systemby utilizing a dual-electrode voltage clamp.[Na+]iaveraged 48.1 ± 2.2 meq (n = 27)and was estimated from the amiloride-sensitive reversal potential.[Na+]iwas increased by direct injection of 27.6 nl of 0.25 or 0.5 MNa2SO4.Within minutes of injection,[Na+]istabilized and remained elevated at 97.8 ± 6.5 meq(n = 9) and 64.9 ± 4.4 (n = 5) meq 30 min after theinitial injection of 0.5 and 0.25 MNa2SO4,respectively. This increase of[Na+]icaused a biphasic inhibition of ENaC currents. In oocytes injected with0.5 MNa2SO4(n = 9), a rapid decrease of inwardamiloride-sensitive slope conductance(gNa) to 0.681 ± 0.030 of control within the first 3 min and a secondary, slowerdecrease to 0.304 ± 0.043 of control at 30 min were observed.Similar but smaller inhibitions were also observed with the injectionof 0.25 MNa2SO4.Injection of isotonicK2SO4(70 mM) or isotonicK2SO4made hypertonic with sucrose (70 mMK2SO4-1.2M sucrose) was without effect. Injection of a 0.5 M concentration ofeitherK2SO4,N-methyl-D-glucamine (NMDG) sulfate, or 0.75 M NMDG gluconate resulted in a much smaller initial inhibition (<14%) and little or no secondary decrease. Thusincreases of[Na+]ihave multiple specific inhibitory effects on ENaC that can betemporally separated into a rapid phase that was complete within 2-3 min and a delayed slow phase that was observed between 5 and 30 min. 相似文献
3.
Jie Yu Bing Zhang Yixiao Zhang Cong-qiao Xu Wei Zhuo Jingpeng Ge Jun Li Ning Gao Yang Li Maojun Yang 《蛋白质与细胞》2018,9(7):629
Mechanosensitive (MS) channels are extensively studied membrane protein for maintaining intracellular homeostasis through translocating solutes and ions across the membrane, but its mechanisms of channel gating and ion selectivity are largely unknown. Here, we identified the YnaI channel as the Na+/K+ cation-selective MS channel and solved its structure at 3.8 Å by cryo-EM single-particle method. YnaI exhibits low conductance among the family of MS channels in E. coli, and shares a similar overall heptamer structure fold with previously studied MscS channels. By combining structural based mutagenesis, quantum mechanical and electrophysiological characterizations, we revealed that ion selective filter formed by seven hydrophobic methionine (YnaIMet158) in the transmembrane pore determined ion selectivity, and both ion selectivity and gating of YnaI channel were affected by accompanying anions in solution. Further quantum simulation and functional validation support that the distinct binding energies with various anions to YnaIMet158 facilitate Na+/K+ pass through, which was defined as bindingblock mechanism. Our structural and functional studies provided a new perspective for understanding the mechanism of how MS channels select ions driven by mechanical force. 相似文献
4.
Ishikita H 《PloS one》2011,6(2):e16920
Acid-sensing ion channels (ASICs) are proton-gated cation channels that exist throughout the mammalian central and peripheral nervous systems. ASIC1 is the most abundant of all the ASICs and is likely to modulate synaptic transmission. Identifying the proton-binding sites of ASCI1 is required to elucidate its pH-sensing mechanism. By using the crystal structure of ASIC1, the protonation states of each titratable site of ASIC1 were calculated by solving the Poisson-Boltzmann equation under conditions wherein the protonation states of all these sites are simultaneously in equilibrium. Four acidic-acidic residue pairs--Asp238-Asp350, Glu220-Asp408, Glu239-Asp346, and Glu80-Glu417--were found to be highly protonated. In particular, the Glu80-Glu417 pair in the inner pore was completely protonated and possessed 2 H(+), implying its possible importance as a proton-binding site. The pK(a) of Glu239, which forms a pair with a possible pH-sensing site Asp346, differs among each homo-trimer subunit due to the different H-bond pattern of Thr237 in the different protein conformations of the subunits. His74 possessed a pK(a) of ≈6-7. Conservation of His74 in the proton-sensitive ASIC3 that lacks a residue corresponding to Asp346 may suggest its possible pH-sensing role in proton-sensitive ASICs. 相似文献
5.
Tolino LA Okumura S Kashlan OB Carattino MD 《The Journal of biological chemistry》2011,286(18):16297-16307
Acid-sensing ion channels (ASICs) are trimeric cation channels that undergo activation and desensitization in response to extracellular acidification. The underlying mechanism coupling proton binding in the extracellular region to pore gating is unknown. Here we probed the reactivity toward methanethiosulfonate (MTS) reagents of channels with cysteine-substituted residues in the outer vestibule of the pore of ASIC1a. We found that positively-charged MTS reagents trigger pore opening of G428C. Scanning mutagenesis of residues in the region preceding the second transmembrane spanning domain indicated that the MTSET-modified side chain of Cys at position 428 interacts with Tyr-424. This interaction was confirmed by double-mutant cycle analysis. Strikingly, Y424C-G428C monomers were associated by intersubunit disulfide bonds and were insensitive to MTSET. Despite the spatial constraints introduced by these intersubunit disulfide bonds in the outer vestibule of the pore, Y424C-G428C transitions between the resting, open, and desensitized states in response to extracellular acidification. This finding suggests that the opening of the ion conductive pathway involves coordinated rotation of the second transmembrane-spanning domains. 相似文献
6.
Selective regulation of acid-sensing ion channel 1 by serine proteases 总被引:10,自引:0,他引:10
Poirot O Vukicevic M Boesch A Kellenberger S 《The Journal of biological chemistry》2004,279(37):38448-38457
Acid-sensing ion channels (ASICs) are neuronal Na(+) channels that belong to the epithelial Na(+) channel/degenerin family. ASICs are transiently activated by a rapid drop in extracellular pH. Conditions of low extracellular pH, such as ischemia and inflammation in which ASICs are thought to be active, are accompanied by increased protease activity. We show here that serine proteases modulate the function of ASIC1a and ASIC1b but not of ASIC2a and ASIC3. We show that protease exposure shifts the pH dependence of ASIC1a activation and steady-state inactivation to more acidic pH. As a consequence, protease exposure leads to a decrease in current response if ASIC1a is activated by a pH drop from pH 7.4. If, however, acidification occurs from a basal pH of approximately 7, protease-exposed ASIC1a shows higher activity than untreated ASIC1a. We provide evidence that this bi-directional regulation of ASIC1a function also occurs in neurons. Thus, we have identified a mechanism that modulates ASIC function and may allow ASIC1a to adapt its gating to situations of persistent extracellular acidification. 相似文献
7.
The ability of acid-sensing ion channels (ASICs) to discriminate among cations was assessed based on changes in conductance and reversal potential with ion substitution. Human ASIC1a was expressed in Xenopus laevis oocytes, and acid-induced currents were measured using two-electrode voltage clamp. Replacement of extracellular Na+ with Li+, K+, Rb+, or Cs+ altered inward conductance and shifted the reversal potentials consistent with a selectivity sequence of Li ∼ Na > K > Rb > Cs. Permeability decreased more rapidly than conductance as a function of atomic size, with PK/PNa = 0.1 and GK/GNa = 0.7 and PRb/PNa = 0.03 and GRb/GNa = 0.3. Stimulation of Cl− currents when Na+ was replaced with Ca2+, Sr2+, or Ba2+ indicated a finite permeability to divalent cations. Inward conductance increased with extracellular Na+ in a hyperbolic manner, consistent with an apparent affinity (Km) for Na+ conduction of 25 mM. Nitrogen-containing cations, including NH4+, NH3OH+, and guanidinium, were also permeant. In addition to passing through the channels, guanidinium blocked Na+ currents, implying competition for a site within the pore. The role of negative charges in an external vestibule of the pore was evaluated using the point mutation D434N. The mutant channel had a decreased single-channel conductance, measured in excised outside-out patches, and a macroscopic slope conductance that increased with hyperpolarization. It had a weakened interaction with Na+ (Km = 72 mM) and a selectivity that was shifted toward larger atomic sizes. We conclude that the selectivity of ASIC1 is based at least in part on interactions with binding sites both within and internal to the outer vestibule. 相似文献
8.
9.
S Kellenberger N Hoffmann-Pochon I Gautschi E Schneeberger L Schild 《The Journal of general physiology》1999,114(1):13-30
The epithelial Na+ channel (ENaC) is highly selective for Na+ and Li+ over K+ and is blocked by the diuretic amiloride. ENaC is a heterotetramer made of two alpha, one beta, and one gamma homologous subunits, each subunit comprising two transmembrane segments. Amino acid residues involved in binding of the pore blocker amiloride are located in the pre-M2 segment of beta and gamma subunits, which precedes the second putative transmembrane alpha helix (M2). A residue in the alpha subunit (alphaS589) at the NH2 terminus of M2 is critical for the molecular sieving properties of ENaC. ENaC is more permeable to Li+ than Na+ ions. The concentration of half-maximal unitary conductance is 38 mM for Na+ and 118 mM for Li+, a kinetic property that can account for the differences in Li+ and Na+ permeability. We show here that mutation of amino acid residues at homologous positions in the pre-M2 segment of alpha, beta, and gamma subunits (alphaG587, betaG529, gammaS541) decreases the Li+/Na+ selectivity by changing the apparent channel affinity for Li+ and Na+. Fitting single-channel data of the Li+ permeation to a discrete-state model including three barriers and two binding sites revealed that these mutations increased the energy needed for the translocation of Li+ from an outer ion binding site through the selectivity filter. Mutation of betaG529 to Ser, Cys, or Asp made ENaC partially permeable to K+ and larger ions, similar to the previously reported alphaS589 mutations. We conclude that the residues alphaG587 to alphaS589 and homologous residues in the beta and gamma subunits form the selectivity filter, which tightly accommodates Na+ and Li+ ions and excludes larger ions like K+. 相似文献
10.
Ishikawa T Jiang C Stutts MJ Marunaka Y Rotin D 《The Journal of biological chemistry》2003,278(40):38276-38286
The epithelial Na+ channel (ENaC), composed of three subunits (alphabetagamma), is expressed in various Na(+)-absorbing epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. By using patch clamp techniques, we have examined the effect of cytosolic ATP on the activity of the rat alphabetagammaENaC (rENaC) stably expressed in NIH-3T3 cells and in Madin-Darby canine kidney epithelial cells. The inward whole-cell current attributable to rENaC activity ran down when these cells were dialyzed with an ATP-free pipette solution in the conventional whole-cell voltage-clamping technique. This run down was prevented by 2 mM ATP (but not by AMP or ADP) in the pipette solution or by the poorly or non-hydrolyzable analogues of ATP (adenosine 5'-O-(thiotriphosphate) and adenosine 5'-(beta,gamma-imino)triphosphate) in both cell lines, suggesting that protection from run down was mediated through non-hydrolytic nucleotide binding. Accordingly, we demonstrate binding of ATP (but not AMP) to alpharENaC expressed in Madin-Darby canine kidney cells, which was inhibited upon mutation of the two putative nucleotide-binding motifs of alpharENaC. Single channel analyses indicated that the run down of currents observed in the whole-cell recording was attributable to run down of channel activity, defined as NPo (the product of the number of channels and open probability). We propose that this novel ATP regulation of ENaC may be, at least in part, involved in the fine-tuning of ENaC activity under physiologic and pathophysiologic conditions. 相似文献
11.
Indirect activation of the epithelial Na+ channel by trypsin 总被引:1,自引:0,他引:1
We tested the hypothesis that the serine protease trypsin can indirectly activate the epithelial Na(+) channel (ENaC). Experiments were carried out in Xenopus oocytes and examined the effects on the channel formed by all three human ENaC subunits and that formed by Xenopus epsilon and human beta and gamma subunits (epsilonbetagammaENaC). Low levels of trypsin (1-10 ng/ml) were without effects on the oocyte endogenous conductances and were specifically used to test the effects on ENaC. Addition of 1 ng/ml trypsin for 60 min stimulated the amiloride-sensitive human ENaC conductance (g(Na)) by approximately 6-fold. This effect on the g(Na) was [Na(+)]-independent, thereby ruling out an interaction with channel feedback inhibition by Na(+). The indirect nature of this activation was confirmed in cell-attached patch clamp experiments with trypsin added to the outside of the pipette. Trypsin was comparatively ineffective at activating epsilonbetagammaENaC, a channel that exhibited a high spontaneous open probability. These observations, in combination with surface binding experiments, indicated that trypsin indirectly activated membrane-resident channels. Activation by trypsin was also dependent on catalytic activity of this protease but was not accompanied by channel subunit proteolysis. Channel activation was dependent on downstream activation of G-proteins and was blocked by G-protein inhibition by injection of guanyl-5'-yl thiophosphate and by pre-stimulation of phospholipase C. These data indicate a receptor-mediated activation of ENaC by trypsin. This trypsin-activated receptor is distinct from that of protease-activated receptor-2, because the response to trypsin was unaffected by protease-activated receptor-2 overexpression or knockdown. 相似文献
12.
Paukert M Chen X Polleichtner G Schindelin H Gründer S 《The Journal of biological chemistry》2008,283(1):572-581
Acid-sensing ion channels are ligand-gated cation channels, gated by extracellular H(+). H(+) is the simplest ligand possible, and whereas for larger ligands that gate ion channels complex binding sites in the three-dimensional structure of the proteins have to be assumed, H(+) could in principle gate a channel by titration of a single amino acid. Experimental evidence suggests a more complex situation, however. For example, it has been shown that extracellular Ca(2+) ions compete with H(+); probably Ca(2+) ions bound to the extracellular loop of ASICs stabilize the closed state of the channel and have to be displaced before the channel can open. In such a scheme, amino acids contributing to Ca(2+) binding would also be candidates contributing to H(+) gating. In this study we systematically screened more than 40 conserved, charged amino acids in the extracellular region of ASIC1a for a possible contribution to H(+) gating. We identified four amino acids where substitution strongly affects H(+) gating: Glu(63), His(72)/His(73), and Asp(78). These amino acids are highly conserved among H(+)-sensitive ASICs and are candidates for the "H(+) sensor" of ASICs. 相似文献
13.
Cl- interference with the epithelial Na+ channel ENaC 总被引:2,自引:0,他引:2
Bachhuber T König J Voelcker T Mürle B Schreiber R Kunzelmann K 《The Journal of biological chemistry》2005,280(36):31587-31594
The cystic fibrosis transmembrane conductance regulator (CFTR) is a protein kinase A and ATP-regulated Cl- channel that also controls the activity of other membrane transport proteins, such as the epithelial Na+ channel ENaC. Previous studies demonstrated that cytosolic domains of ENaC are critical for down-regulation of ENaC by CFTR, whereas others suggested a role of cytosolic Cl- ions. We therefore examined in detail the anion dependence of ENaC and the role of its cytosolic domains for the inhibition by CFTR and the Cl- channel CLC-0. Coexpression of rat ENaC with human CFTR or the human Cl- channel CLC-0 caused inhibition of amiloride-sensitive Na+ currents after cAMP-dependent stimulation and in the presence of a 100 mM bath Cl- concentration. After activation of CFTR by 3-isobutyl-1-methylxanthine and forskolin or expression of CLC-0, the intracellular Cl- concentration was increased in Xenopus oocytes in the presence of a high bath Cl- concentration, which inhibited ENaC without changing surface expression of alpha beta gammaENaC. In contrast, a 5 mM bath Cl- concentration reduced the cytosolic Cl- concentration and enhanced ENaC activity. ENaC was also inhibited by injection of Cl- into oocytes and in inside/out macropatches by exposure to high cytosolic Cl- concentrations. The effect of Cl- was mimicked by Br-, Br-, NO3(-), and I-. Inhibition by Cl- was reduced in trimeric channels with a truncated COOH terminus of betaENaC and gammaENaC, and it was no longer detected in dimeric alpha deltaCbeta ENaC channels. Deletion of the NH2 terminus of alpha-, beta-, or gammaENaC, mutations in the NH2-terminal phosphatidylinositol bisphosphate-binding domain of betaENaC and gammaEnaC, and activation of phospholipase C, all reduced ENaC activity but allowed for Cl(-)-dependent inhibition of the remaining ENaC current. The results confirm a role of the carboxyl terminus of betaENaC for Cl(-)-dependent inhibition of the Na+ channel, which, however, may only be part of a complex regulation of ENaC by CFTR. 相似文献
14.
Actin filaments regulate epithelial Na+ channel activity 总被引:14,自引:0,他引:14
Cantiello H. F.; Stow J. L.; Prat A. G.; Ausiello D. A. 《American journal of physiology. Cell physiology》1991,261(5):C882
15.
Na+ activation of the muscarinic K+ channel by a G-protein-independent mechanism 总被引:6,自引:0,他引:6
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《The Journal of general physiology》1996,108(5):381-391
Muscarinic potassium channels (KACh) are composed of two subunits, GIRK1 and GIRK4 (or CIR), and are directly gated by G proteins. We have identified a novel gating mechanism of KACh, independent of G-protein activation. This mechanism involved functional modification of KACh which required hydrolysis of physiological levels of intracellular ATP and was manifested by an increase in the channel mean open time. The ATP-modified channels could in turn be gated by intracellular Na+, starting at approximately 3 mM with an EC50 of approximately 40 mM. The Na(+)-gating of KACh was operative both in native atrial cells and in a heterologous system expressing recombinant channel subunits. Block of the Na+/K+ pump (e.g., by cardiac glycosides) caused significant activation of KACh in atrial cells, with a time course similar to that of Na+ accumulation and in a manner indistinguishable from that of Na(+)-mediated activation of the channel, suggesting that cardiac glycosides activated KACh by increasing intracellular Na+ levels. These results demonstrate for the first time a direct effect of cardiac glycosides on atrial myocytes involving ion channels which are critical in the regulation of cardiac rhythm. 相似文献
16.
The epithelial Na(+) channel (ENaC) is implicated in the pathogenesis of salt-sensitive hypertension. Recent evidence from animal models suggests that the vasoactive peptide, endothelin (ET-1), may be an important negative regulator of ENaC in vivo. We investigated the signaling pathway involved in endothelin-mediated ENaC inhibition. Experiments were performed in NIH 3T3 cells stably expressing genes for the three (alpha, beta, and gamma) ENaC subunits. In whole cell patch clamp experiments, we found that ET-1 treatment induced a dose-dependent decrease in amiloride-sensitive currents. Using receptor-specific antagonists, we determined that the effects of ET-1 were attributed to activation of the ET(B) receptor. Moreover, the inhibitory effect of ET-1 on ENaC could be completely blocked when cells were pretreated with the selective Src family kinase inhibitor, PP2. Further studies revealed that basal Src family kinase activity strongly regulates ENaC whole cell currents and single channel gating. These results suggest that Src family kinases lie in a signaling pathway activated by ET-1 and are components of a novel negative regulatory cascade resulting in ENaC inhibition. 相似文献
17.
Identification of the Ca2+ blocking site of acid-sensing ion channel (ASIC) 1: implications for channel gating 总被引:5,自引:0,他引:5
Acid-sensing ion channels ASIC1a and ASIC1b are ligand-gated ion channels that are activated by H+ in the physiological range of pH. The apparent affinity for H+ of ASIC1a and 1b is modulated by extracellular Ca2+ through a competition between Ca2+ and H+. Here we show that, in addition to modulating the apparent H+ affinity, Ca2+ blocks ASIC1a in the open state (IC50 approximately 3.9 mM at pH 5.5), whereas ASIC1b is blocked with reduced affinity (IC50 > 10 mM at pH 4.7). Moreover, we report the identification of the site that mediates this open channel block by Ca2+. ASICs have two transmembrane domains. The second transmembrane domain M2 has been shown to form the ion pore of the related epithelial Na+ channel. Conserved topology and high homology in M2 suggests that M2 forms the ion pore also of ASICs. Combined substitution of an aspartate and a glutamate residue at the beginning of M2 completely abolished block by Ca2+ of ASIC1a, showing that these two amino acids (E425 and D432) are crucial for Ca2+ block. It has previously been suggested that relief of Ca2+ block opens ASIC3 channels. However, substitutions of E425 or D432 individually or in combination did not open channels constitutively and did not abolish gating by H+ and modulation of H+ affinity by Ca2+. These results show that channel block by Ca2+ and H+ gating are not intrinsically linked. 相似文献
18.
The tarantula toxin psalmotoxin 1 inhibits acid-sensing ion channel (ASIC) 1a by increasing its apparent H+ affinity 总被引:3,自引:0,他引:3
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Acid-sensing ion channels (ASICs) are ion channels activated by extracellular protons. They are involved in higher brain functions and perception of pain, taste, and mechanical stimuli. Homomeric ASIC1a is potently inhibited by the tarantula toxin psalmotoxin 1. The mechanism of this inhibition is unknown. Here we show that psalmotoxin 1 inhibits ASIC1a by a unique mechanism: the toxin increases the apparent affinity for H(+) of ASIC1a. Since ASIC1a is activated by H(+) concentrations that are only slightly larger than the resting H(+) concentration, this increase in H(+) affinity is sufficient to shift ASIC1a channels into the desensitized state. As activation of ASIC1a has recently been linked to neurodegeneration associated with stroke, our results suggest chronic desensitization of ASIC1a by a slight increase of its H(+) affinity as a possible way of therapeutic intervention in stroke. 相似文献
19.
Bachhuber T Almaça J Aldehni F Mehta A Amaral MD Schreiber R Kunzelmann K 《The Journal of biological chemistry》2008,283(19):13225-13232
CK2 is a ubiquitous, pleiotropic, and constitutively active Ser/Thr protein kinase that controls protein expression, cell signaling, and ion channel activity. Phosphorylation sites for CK2 are located in the C terminus of both beta- and gamma-subunits of the epithelial Na(+) channel (ENaC). We examined the role of CK2 on the regulation of both endogenous ENaC in native murine epithelia and in Xenopus oocytes expressing rENaC. In Ussing chamber experiments with mouse airways, colon, and cultured M1-collecting duct cells, amiloride-sensitive Na(+) transport was inhibited dose-dependently by the selective CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB). In oocytes, ENaC currents were also inhibited by TBB and by the structurally unrelated inhibitors heparin and poly(E:Y). Expression of a trimeric channel lacking both CK2 sites (alphabeta(S631A)gamma(T599A)) produced a largely attenuated amiloride-sensitive whole cell conductance and rendered the mutant channel insensitive to CK2. In Xenopus oocytes, CK2 was translocated to the cell membrane upon expression of wt-ENaC but not of alphabeta(S631A)gamma(T599A)-ENaC. Phosphorylation by CK2 is essential for ENaC activation, and to a lesser degree, it also controls membrane expression of alphabetagamma-ENaC. Channels lacking the Nedd4-2 binding motif in beta-ENaC (R561X, Y618A) no longer required the CK2 site for channel activity and siRNA-knockdown of Nedd4-2 eliminated the effects of TBB. This implies a role for CK2 in inhibiting the Nedd4-2 pathway. We propose that the C terminus of beta-ENaC is targeted by this essential, conserved pleiotropic kinase that directs its constitutive activity toward many cellular protein complexes. 相似文献
20.
Acid-sensing ion channels (ASICs) have long been considered as extracellular proton (H(+))-gated cation channels, and peripheral ASIC3 channels seem to be a natural sensor of acidic pain. Here, we report the identification of a nonproton sensor on ASIC3. We show first that 2-guanidine-4-methylquinazoline (GMQ) causes persistent ASIC3 channel activation at the normal pH. Using GMQ as a probe and combining mutagenesis and covalent modification analysis, we then uncovered a ligand sensor lined by residues around E423 and E79 of the extracellular "palm" domain of the ASIC3 channel that is crucial for activation by nonproton activators. Furthermore, we show that GMQ activates sensory neurons and causes pain-related behaviors in an ASIC3-dependent manner, indicating the functional significance of ASIC activation by nonproton ligands. Thus, natural ligands beyond protons may activate ASICs under physiological and pathological conditions through the nonproton ligand sensor, serving for channel activation independent of abrupt and marked acidosis. 相似文献