Organization of ATP-gated P2X1 receptor intracellular termini in apo and desensitized states.

The human P2X1 receptor (hP2X1R) is a trimeric ligand-gated ion channel opened by extracellular ATP. The intracellular amino and carboxyl termini play significant roles in determining the time-course and regulation of channel gating-for example, the C terminus regulates recovery from the desensitized state following agonist washout. This suggests that the intracellular regions of the channel have distinct structural features. Studies on the hP2X3R have shown that the intracellular regions associate to form a cytoplasmic cap in the open state of the channel. However, intracellular features could not be resolved in the agonist-free apo and ATP-bound desensitized structures. Here we investigate the organization of the intracellular regions of hP2X1R in the apo and ATP-bound desensitized states following expression in HEK293 cells. We couple cysteine scanning mutagenesis of residues R25-G30 and H355-R360 with the use of bi-functional cysteine reactive cross-linking compounds of different lengths (MTS-2-MTS, BMB, and BM(PEG)2), which we use as molecular calipers. If two cysteine residues come into close proximity, we predict they will be cross-linked and result in ∼66% of the receptor subunits running on a Western blot as dimers. In the control construct (C349A) that removed the free cysteine C349, and some cysteine-containing mutants, cross-linker treatment does not result in dimerization. However, we detect efficient dimerization for R25C, G30C, P358C, K359C, and R360C. This selective pattern indicates that there is structural organization to these regions in the apo and desensitized states in a native membrane environment. The existence of such precap (apo) and postcap (desensitized) organization of the intracellular domains would facilitate efficient gating of the channel.

Mapping the binding site of the P2X receptor antagonist PPADS reveals the importance of orthosteric site charge and the cysteine-rich head region.

ATP is the native agonist for cell-surface ligand-gated P2X receptor (P2XR) cation channels. The seven mammalian subunits (P2X1-7) form homo- and heterotrimeric P2XRs having significant physiological and pathophysiological roles. Pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) is an effective antagonist at most mammalian P2XRs. Lys-249 in the extracellular domain of P2XR has previously been shown to contribute to PPADS action. To map this antagonist site, we generated human P2X1R cysteine substitutions within a circle centered at Lys-249 (with a radius of 13 Å equal to the length of PPADS). We hypothesized that cysteine substitutions of residues involved in PPADS binding would (i) reduce cysteine accessibility (measured by MTSEA-biotinylation), (ii) exhibit altered PPADS affinity, and (iii) quench the fluorescence of cysteine residues modified with MTS-TAMRA. Of the 26 residues tested, these criteria were met by only four (Lys-70, Asp-170, Lys-190, and Lys-249), defining the antagonist site, validating molecular docking results, and thereby providing the first experimentally supported model of PPADS binding. This binding site overlapped with the ATP-binding site, indicating that PPADS sterically blocks agonist access. Moreover, PPADS induced a conformational change at the cysteine-rich head (CRH) region adjacent to the orthosteric ATP-binding pocket. The importance of this movement was confirmed by demonstrating that substitution introducing positive charge present in the CRH of the hP2X1R causes PPADS sensitivity at the normally insensitive rat P2X4R. This study provides a template for developing P2XR subtype selectivity based on the differences among the mammalian subunits around the orthosteric P2XR-binding site and the CRH.

Mechanistic insights from resolving ligand-dependent kinetics of conformational changes at ATP-gated P2X1R ion channels.

Structural studies of P2X receptors show a novel U shaped ATP orientation following binding. We used voltage clamp fluorometry (VCF) and molecular dynamics (MD) simulations to investigate agonist action. For VCF the P2X1 receptor (P2X1R) K190C mutant (adjacent to the agonist binding pocket) was labelled with the fluorophore MTS-TAMRA and changes in fluorescence on agonist treatment provided a real time measure of conformational changes. Studies with heteromeric channels incorporating a key lysine mutation (K68A) in the ATP binding site demonstrate that normally three molecules of ATP activate the receptor. The time-course of VCF responses to ATP, 2'-deoxy ATP, 3'-deoxy ATP, Ap5A and αßmeATP were agonist dependent. Comparing the properties of the deoxy forms of ATP demonstrated the importance of the 2' hydroxyl group on the ribose ring in determining agonist efficacy consistent with MD simulations showing that it forms a hydrogen bond with the γ-phosphate oxygen stabilizing the U-shaped conformation. Comparison of the recovery of fluorescence on agonist washout, with channel activation to a second agonist application for the partial agonists Ap5A and αßmeATP, showed a complex relationship between conformational change and desensitization. These results highlight that different agonists induce distinct conformational changes, kinetics and recovery from desensitization at P2X1Rs.

Kinetics of conformational changes revealed by voltage-clamp fluorometry give insight to desensitization at ATP-gated human P2X1 receptors.

ATP acts as an extracellular signaling molecule at cell-surface P2X receptors, mediating a variety of important physiologic and pathophysiologic roles. Homomeric P2X1 receptors open on binding ATP and then transition to an ATP-bound closed, desensitized state that requires an agonist-free washout period to recover. Voltage-clamp fluorometry was used to record ion channel activity and conformational changes simultaneously at defined positions in the extracellular loop of the human P2X1 receptor during not only agonist binding and desensitization but also during recovery. ATP evoked distinct conformational changes adjacent to the agonist binding pocket in response to channel activation and desensitization. The speed of recovery of the conformational change on agonist washout was state-dependent, with a faster time constant from the open (5 seconds) compared with the desensitized (75 seconds) form of the channel. The ability of ATP to evoke channel activity on washout after desensitization was not dependent on the degree of conformational rearrangement in the extracellular loop, and desensitization was faster from the partially recovered state. An intracellular mutation in the carboxyl terminus that slowed recovery of P2X1 receptor currents (7-fold less recovery at 30 seconds) had no effect on the time course of the extracellular conformational rearrangements. This study highlights that the intracellular portion of the receptor can regulate recovery and shows for the first time that this is by a mechanism independent of changes in the extracellular domain, suggesting the existence of a distinct desensitization gate in this novel class of ligand gated ion channels.

P2X receptor chimeras highlight roles of the amino terminus to partial agonist efficacy, the carboxyl terminus to recovery from desensitization, and independent regulation of channel transitions.

P2X receptor subtypes can be distinguished by their sensitivity to ATP analogues and selective antagonists. We have used chimeras between human P2X1 and P2X2 receptors to address the contribution of the extracellular ligand binding loop, transmembrane segments (TM1 and TM2), and intracellular amino and carboxyl termini to the action of partial agonists (higher potency and efficacy of BzATP and Ap5A at P2X1 receptors) and antagonists. Sensitivity to the antagonists NF449, suramin, and PPADS was conferred by the nature of the extracellular loop (e.g. nanomolar for NF449 at P2X1 and P2X2-1EXT and micromolar at P2X2 and P2X1-2EXT). In contrast, the effectiveness of partial agonists was similar to P2X1 levels for both of the loop transfers, suggesting that interactions with the rest of the receptor played an important role. Swapping TM2 had reciprocal effects on partial agonist efficacy. However, TM1 swaps increased partial agonist efficacy at both chimeras, and this was similar for swaps of both TM1 and 2. Changing the amino terminus had no effect on agonist potency but increased partial agonist efficacy at P2X2-1N and decreased it at P2X1-2N chimeras, demonstrating that potency and efficacy can be independently regulated. Chimeras and point mutations also identified residues in the carboxyl terminus that regulated recovery from channel desensitization. These results show that interactions among the intracellular, transmembrane, and extracellular portions of the receptor regulate channel properties and suggest that transitions to channel opening, the behavior of the open channel, and recovery from the desensitized state can be controlled independently.

Ototrauma induces sodium channel plasticity in auditory afferent neurons.

Exposure to intense sound can cause damage to the delicate sensory and neuronal components of the cochlea leading to hearing loss. Such damage often causes the dendrites of the spiral ganglion neurons (SGN), the neurons that provide the afferent innervation of the hair cells, to swell and degenerate thus damaging the synapse. In models of neuropathic pain, axotomy, another form of afferent nerve damage, is accompanied by altered voltage-gated sodium channel (VGSC) expression, leading to neuronal hyperactivity. In this study, adult Wistar rats were exposed to noise which produced a mild, 20 dB hearing threshold elevation and their VGSC expression was investigated. Quantitative PCR showed decreased Na(V)1.1 and Na(V)1.6 mRNA expression in the SGN following noise exposure (29% and 56% decrease respectively) while Na(V)1.7 mRNA expression increased by approximately 20% when compared to control rats. Immunohistochemistry extended these findings, revealing increased staining for Na(V)1.1 along the SGN dendrites and Na(V)1.7 in the cell bodies after noise. These results provide the first evidence for selective changes in VGSC expression following moderate noise-induced hearing loss and could contribute to elevated hearing thresholds and to the generation of perceptual anomalies commonly associated with cochlear damage, such as tinnitus and hyperacusis.