Pore architecture and ion sites in acid-sensing ion channels and P2X receptors

ASIC3 is an acid-sensing ion channel expressed in sensory neurons, where it participates in acidic and inflammatory pain. In addition to the "classical" transient current, ASIC3 generates a sustained current essential for pain perception. Using chimeras between the ASIC3 and ASIC1a channels we show that the first transmembrane domain (TM1), combined with the N-terminal domain, is the key structural element generating the low pH (<6.5)-evoked sustained current. The TM1 domain also modulates the pH-dependent activation of the fast transient current thus contributing to a constitutive window current, another type of sustained current present near physiological pH. The C-terminal and the TM2 domains negatively regulate both types of sustained current, and the extracellular loop affects its kinetics. These data provide new information to aid understanding the mechanisms of the multifaceted pH gating of ASIC3. Together with the peak current, both components of the sustained current (window and sustained at pH <6.5) allow ASIC3 to adapt its behavior to a wide range of extracellular pH variations by generating transient and/or sustained responses that contribute to nociceptor excitability.Ischemia, inflammation, tumors, or tissue damages are associated with a decrease in extracellular pH (1). Activation of nociceptive sensory nerve endings by tissue acidosis correlates with pain sensations in human and rodents (1-4). The acidsensing ion channel 3 (ASIC3) 2 is predominantly expressed in sensory neurons and has been recently shown to be a sensor of acid-mediated and primary inflammatory pain in rats (4). ASIC3 belongs to the acid-sensing ion channel family, which comprises at least seven isoforms encoded by four different genes (5-7). These isoforms, which comprise cytosolic N and C termini, two transmembrane helices, and a disulfide-rich, multidomain extracellular region, can associate into homo-or heterotrimers (8, 9). ASICs are amiloride-sensitive voltage-independent cationic channels that are activated by a decrease in extracellular pH. Protons trigger a transient inward current that desensitizes rapidly in all of the ASICs except ASIC3, which carries in addition to the transient current a sustained current that does not fully inactivate while the pH remains acidic (10 -12). For moderate extracellular acidification (between pH 7.3 and 6.7 for the rat ASIC3 channel), the sustained current results from the window of overlap between inactivation and activation of the fast transient current (4, 13), whereas the sustained current that is activated by lower pH seems to involve a different, uncharacterized mechanism. Despite the important role proposed for the ASIC3-sustained current in pain perception (4, 13, 14), very little is known about the molecular mechanisms underlying this current. This paper identifies the key structural elements involved in the generation and in the modulation of the window current (I window ) observed at modest acidifications near the physiological pH and of the low pH (Ͻ6.5)-evoked sustained cur...

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Structural Elements for the Generation of Sustained Currents by the Acid Pain Sensor ASIC3

Salinas

Lazdunski

Lingueglia

2009

“…The P2X receptor topology was similar to that of acid-sensing ion channels (42,43), a possibility that had been raised by earlier modeling studies (44). However, it is important to note that the extracellular domains of P2X and acid-sensing ion channels are distinct with little sequence or structural homology, whereas the pore domains share similar architectures (43). The availability of direct structural information for P2X receptors substantiated most of the aforementioned mutational studies and for the first time provided a framework to understand their atomic origins (5).…”

mentioning

confidence: 92%

Gated Access to the Pore of a P2X Receptor

Kracun

Chaptal

Abramson

et al. 2010

P2X receptors (P2X1-P2X7) are cell surface cation channels that transition from closed to open states upon binding ATP. They represent a physiologically and medically important (1), as well as a structurally distinct family of ligand-gated ion channels that are quite different to Cys-loop and ionotropic glutamate receptors (2-5). P2X receptors are found in many species from several phyla, implying important roles in diverse life forms (6, 7). A key goal is to understand how P2X receptors operate at the chemical level.The first P2X receptor genes were cloned in 1994 (8, 9) leading to important progress over the last decade in understanding how P2X receptors function (10, 11) using biophysical and biochemical approaches. P2X subunits are thus known to possess intracellular N and C termini and two transmembrane (TM) 2 segments separated by a large extracellular loop (12-14). The trimeric architecture of P2X receptors is also well established based on subunit concatemers, blue-native PAGE, and atomic force microscopy experiments (15-18). It is also well established that the P2X pore is lined by TM2 (19 -22), with TM1 making little contribution to ion flow (23, 24). Moreover, careful studies of permeation and selectivity have suggested that the same area of TM2 is responsible for both ion selection and channel opening (25-31). Mutational approaches have shed light on the extracellular domain of P2X receptors, including how the ATP binding pocket may form (32), how the 10 conserved cysteines contribute to the fold of the protein (33, 34), and how cations such as Zn 2ϩ profoundly affect receptor function (35)(36)(37)(38). Finally, meticulous analysis of voltage-jump relaxations for ATP-evoked currents combined with mutagenesis has suggested that TM2 may display hinge-like flexibility (39, 40), and recently the P2X4 receptor open pore has been imaged with fast scanning atomic force microscopy (41). Most of these past studies were conducted on rat P2X2 (rP2X2) receptors and have provided a basis to explore P2X receptor pores and the gating process.In 2009, the field was immeasurably advanced by the landmark achievement and report of a crystal structure of the zebrafish (zf) P2X4.1 receptor in the closed state (42). The P2X receptor topology was similar to that of acid-sensing ion channels (42, 43), a possibility that had been raised by earlier modeling studies (44). However, it is important to note that the extracellular domains of P2X and acid-sensing ion channels are distinct with little sequence or structural homology, whereas the pore domains share similar architectures (43). The availability of direct structural information for P2X receptors substantiated most of the aforementioned mutational studies and for the first time provided a framework to understand their atomic origins (5). Of most relevance here, it directly revealed that the P2X pore was indeed formed by TM2 ␣-helices arranged around a 3-fold molecular axis of symmetry. These were arranged ϳ45°to the membrane normal and crossed about halfway along their l...

“…However, the mechanisms of pH-dependent gating are currently not well understood. The high resolution chicken ASIC1 crystal structure has recently been reported from a truncated construct that was crystallized at acidic pH, therefore most likely representing the channel in its inactivated state (25,26).…”

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confidence: 99%

A Combined Computational and Functional Approach Identifies New Residues Involved in pH-dependent Gating of ASIC1a

Liechti

Bernèche

Bargeton

et al. 2010

Acid-sensing ion channels (ASICs) are key receptors for extracellular protons. These neuronal nonvoltage-gated Na ؉ channels are involved in learning, the expression of fear, neurodegeneration after ischemia, and pain sensation. We have applied a systematic approach to identify potential pH sensors in ASIC1a and to elucidate the mechanisms by which pH variations govern ASIC gating. We first calculated the pK a value of all extracellular His, Glu, and Asp residues using a Poisson-Boltzmann continuum approach, based on the ASIC three-dimensional structure, to identify candidate pH-sensing residues. The role of these residues was then assessed by site-directed mutagenesis and chemical modification, combined with functional analysis. The localization of putative pH-sensing residues suggests that pH changes control ASIC gating by protonation/ deprotonation of many residues per subunit in different channel domains. Analysis of the function of residues in the palm domain close to the central vertical axis of the channel allowed for prediction of conformational changes of this region during gating. Our study provides a basis for the intrinsic ASIC pH dependence and describes an approach that can also be applied to the investigation of the mechanisms of the pH dependence of other proteins.Acid-sensing ion channels (ASICs) 3 are neuronal nonvoltage-gated Na ϩ channels that are activated by a rapid drop in extracellular pH (1, 2). They are members of the epithelial Na ϩ channel/Degenerin family of ion channel proteins (3). Expression in nociceptive neurons and activation by protons suggest that ASICs may act as pain receptors (4), and evidence for such a role has been provided in several animal pain models (5-7). ASIC1a in the central nervous system plays a role in memory formation and the expression of fear (8, 9). ASIC1a is also an important mediator of cell injury induced by conditions associated with acidosis in the mammalian nervous system (10). Functional ASICs are formed by homo-or heterotrimeric assembly of ASIC subunits 1a, 1b, 2a, 2b, and 3. Each subunit has short intracellular N and C termini and two transmembrane domains that are separated by a large extracellular domain.Extracellular acidification opens ASICs. The ASIC activity is terminated in the continued presence of the acidic stimulus within hundreds of milliseconds to seconds by open channel inactivation, which is also called desensitization (11). Only in some ASIC isoforms and under certain pH conditions can acidification induce a sustained current, which has in most cases a much smaller amplitude than the peak current (12-14). At pH values slightly below the physiological pH, ASICs inactivate without apparent channel opening in a process that is called steady-state inactivation (SSIN) (15). By these two ways ASICs enter the inactivated state, which is a nonconducting, absorbing state. Experimental protocols have been applied to determine the kinetics of the recovery from the inactivated state, showing that channels need exposure for a certain duration ...