A hydrophobic residue in position 15 of the rP2X3 receptor slows desensitization and reveals properties beneficial for pharmacological analysis and high-throughput screening

Hausmann, Ralf; Bahrenberg, Gregor; Kuhlmann, Daniel; Schumacher, Michaela; Braam, U.; Bieler, Dagmar; Schlusche, Ilka; Schmalzing, Günther

doi:10.1016/j.neuropharm.2014.01.010

Cited by 25 publications

(33 citation statements)

References 86 publications

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“…While this article was in preparation, another group identified two amino acids downstream of the P2X2 second transmembrane region that regulate recovery from desensitization (Hausmann et al, 2014). These amino acids are between the P2X2 second transmembrane region pore-forming sequence and the C-terminal of P2X2TR translation; possibly this is a region where P2X2 makes molecular contact with a6b4.…”

Section: Discussionmentioning

confidence: 99%

Subtype-Specific Mechanisms for Functional Interaction between α6β4* Nicotinic Acetylcholine Receptors and P2X Receptors

2014

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P2X receptors and nicotinic acetylcholine receptors (nAChRs) display functional and physical interactions in many cell types and heterologous expression systems, but interactions between a6b4-containing (a6b4*) nAChRs and P2X2 receptors and/or P2X3 receptors have not been fully characterized. We measured several types of crosstalk in oocytes coexpressing a6b4 nAChRs and P2X2, P2X3, or P2X2/3 receptors. A novel form of crosstalk occurs between a6b4 nAChRs and P2X2 receptors. P2X2 receptors were forced into a prolonged desensitized state upon activation by ATP through a mechanism that does not depend on the intracellular C terminus of the P2X2 receptors. Coexpression of a6b4 nAChRs with P2X3 receptors shifts the ATP dose-response relation to the right, even in the absence of acetylcholine (ACh). Moreover, currents become nonadditive when ACh and ATP are coapplied, as previously reported for other Cys-loop receptors interacting with P2X receptors, and this crosstalk is dependent on the presence of the P2X3 C-terminal domain. P2X2 receptors also functionally interact with a6b4b3 but through a different mechanism from a6b4. The interaction with P2X3 receptors is less pronounced for the a6b4b3 nAChR than the a6b4 nAChR. We also measured a functional interaction between the a6b4 nAChRs and the heteromeric P2X2/3 receptor. Experiments with the nAChR channel blocker mecamylamine on P2X2-a6b4 oocytes point to the loss of P2X2 channel activity during the crosstalk, whereas the ion channel pores of the P2X receptors were fully functional and unaltered by the receptor interaction for P2X2-a6b4b3, P2X2/3-a6b4, and P2X2/3-a6b4b3. These results may be relevant to dorsal root ganglion cells and to other neurons that coexpress these receptor subunits.

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Section: Discussionmentioning

confidence: 99%

Subtype-Specific Mechanisms for Functional Interaction between α6β4* Nicotinic Acetylcholine Receptors and P2X Receptors

2014

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“…Other nAChR–P2X receptor interactions increase with the densities of the receptors (21). The details of the contacts, the receptor states involved, and the possible roles of ion flux are not fully known; but modifications to desensitization could play a role (28, 45). Purinergic receptors are important pain processing molecules known to be expressed on nociceptive small diameter neurons in the DRG (46), with important roles having been demonstrated for P2X2/3 (23, 24, 47), P2X4 (48), and P2X7 (49).…”

Section: Discussionmentioning

confidence: 99%

The nicotinic α6 subunit gene determines variability in chronic pain sensitivity via cross-inhibition of P2X2/3 receptors

et al. 2015

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Chronic pain is a highly prevalent and poorly managed human health problem. We used microarray-based expression genomics in 25 inbred mouse strains to identify dorsal root ganglion (DRG)-expressed genetic contributors to mechanical allodynia, a prominent symptom of chronic pain. We identified expression levels of Chrna6, which encodes the α6 subunit of the nicotinic acetylcholine receptor (nAChR), as highly associated with allodynia. We confirmed the importance of α6* (i.e., α6-containing) nAChRs by analyzing both gain- and loss-of-function mutants. We find that mechanical allodynia associated with neuropathic and inflammatory injuries is significantly altered in α6* mutants, and that α6* but not α4* nicotinic receptors are absolutely required for peripheral and/or spinal nicotine analgesia. Furthermore, we show that Chrna6’s role in analgesia is at least partially due to direct interaction and cross-inhibition of α6* nAChRs with P2X2/3 receptors in DRG nociceptors. Finally, we establish relevance of our results to humans by the observation of genetic association in patients suffering from chronic postsurgical pain and temporomandibular pain.

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“…The P2X2R COOH-terminal domain also plays a role in receptor desensitization, as can be deduced from the different properties of its COOH-terminal splice variants (3,(25)(26)(27). The NH 2 -terminal residues also participate in P2XR desensitization; the threonine-18 residue has been reported to be important for P2X2R desensitization (2), and the hydrophobic serine-15 residues have been reported for P2X3R desensitization (17). In the P2X1R, the contribution of both NH 2 and COOH termini to receptor desensitization domains has been found with the use of chimeric receptors and voltage-clamp fluorometry (1,16).…”

Section: Discussionmentioning

confidence: 99%

Role of domain calcium in purinergic P2X2 receptor channel desensitization

Coddou

Yan

Stojilković

2015

American Journal of Physiology-Cell Physiology

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Coddou C, Yan Z, Stojilkovic SS. Role of domain calcium in purinergic P2X2 receptor channel desensitization. Am J Physiol Cell Physiol 308: C729 -C736, 2015. First published February 11, 2015 doi:10.1152/ajpcell.00399.2014.-Activation of P2X2 receptor channels (P2X2Rs) is characterized by a rapid current growth accompanied by a decay of current during sustained ATP application, a phenomenon known as receptor desensitization. Using rat, mouse, and human receptors, we show here that two processes contribute to receptor desensitization: bath calcium-independent desensitization and calcium-dependent desensitization. Calcium-independent desensitization is minor and comparable during repetitive agonist application in cells expressing the full size of the receptor but is pronounced in cells expressing shorter versions of receptors, indicating a role of the COOH terminus in control of receptor desensitization. Calciumdependent desensitization is substantial during initial agonist application and progressively increases during repetitive agonist application in bath ATP and calcium concentration-dependent manners. Experiments with substitution of bath Na ϩ with N-methyl-D-glucamine (NMDG ϩ ), a large organic cation, indicate that receptor pore dilation is a calcium-independent process in contrast to receptor desensitization. A decrease in the driving force for calcium by changing the holding potential from Ϫ60 to ϩ120 mV further indicates that calcium influx through the channel pores at least partially accounts for receptor desensitization. Experiments with various receptor chimeras also indicate that the transmembrane and/or intracellular domains of P2X2R are required for development of calcium-dependent desensitization and that a decrease in the amplitude of current slows receptor desensitization. Simultaneous calcium and current recording shows development of calcium-dependent desensitization without an increase in global intracellular calcium concentrations. Combined with experiments with clamping intrapipette concentrations of calcium at various levels, these experiments indicate that domain calcium is sufficient to establish calcium-dependent receptor desensitization in experiments with whole-cell recordings.ATP; calcium; desensitization; pore dilation; purinergic receptors PURINERGIC P2X RECEPTORS (P2XRs) are ATP-gated nonselective cation channels expressed in various tissues and have multiple physiological roles (4, 30). Some notable examples of their roles in the central and peripheral nervous system include presynaptic P2X2Rs that contribute to an increase of glutamate release in mice interneurons (24), postsynaptic P2X4Rs that contribute to long-term potentiation in CA1 neurons from mice (34), involvement of P2X4Rs and P2X7Rs in neuropathic pain (10,36,37), and P2X3Rs and P2X2R acting through a heteromeric receptor for pain transmission in mammalian sensory neurons (28). On the other hand, the specific properties of each P2XR, such as ATP affinity (7), pore dilation (32), and kinetics of receptor desensitizati...

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A hydrophobic residue in position 15 of the rP2X3 receptor slows desensitization and reveals properties beneficial for pharmacological analysis and high-throughput screening

Cited by 25 publications

References 86 publications

Subtype-Specific Mechanisms for Functional Interaction between α6β4* Nicotinic Acetylcholine Receptors and P2X Receptors

Subtype-Specific Mechanisms for Functional Interaction between α6β4* Nicotinic Acetylcholine Receptors and P2X Receptors

The nicotinic α6 subunit gene determines variability in chronic pain sensitivity via cross-inhibition of P2X2/3 receptors

Role of domain calcium in purinergic P2X2 receptor channel desensitization

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