Inter-subunit disulfide cross-linking in homomeric and heteromeric P2X receptors

Marquez-Klaka, Benjamin; Rettinger, Jürgen; Nicke, Annette

doi:10.1007/s00249-008-0325-9

Cited by 29 publications

(31 citation statements)

References 34 publications

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“…Thus, our disulfide trapping experiments are best compatible with the view that inter-subunit disulfide locking of the head and dorsal fin domains or at the level of the lower body domains prevents the gatinginduced conformational rearrangement of the subunits with respect to each other. These results are consistent with findings of former studies on P2X1R, P2X1/2R, P2X2R, and P2X4R, which were elaborated by using a similar approach [12,[14][15][16][17][18][19][20], reviewed in [9].…”

Section: Discussionsupporting

confidence: 92%

“…In accordance with this gating mechanism, several mutational studies on P2X1 or P2X2 subunit-containing receptors have shown that the restriction of the relative movements of adjacent subunits by disulfide locking inhibits the channel function [12][13][14][15][16][17][18][19][20]. In the hP2X3R, we have recently shown by disulfide trapping analysis that the conformational mobility of domains constituting the inter-subunit ATP-binding site is essential for ATP-induced channel opening [21].…”

Section: Electronic Supplementary Materialsmentioning

confidence: 72%

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Inter-subunit disulfide locking of the human P2X3 receptor elucidates ectodomain movements associated with channel gating

Stephan

Kowalski-Jahn

Zens

et al. 2016

Purinergic Signalling

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P2X3 receptors (P2X3R) are trimeric ATP-gated cation channels involved in sensory neurotransmission and inflammatory pain. We used homology modeling and molecular dynamic simulations of the hP2X3R to identify intersubunit interactions of residues that are instrumental to elucidate conformational changes associated with gating of the hPX3R. We identified an ionic interaction between E112 and R198 of the head domain and dorsal fin domain, respectively, and E57 and T263 of the lower body domains of adjacent subunits and detected a marked rearrangement of these domains during gating of the hP3X3R. Double-mutant cycle analysis of the inter-subunit residue pairs E112/R198 and E57/ T263 revealed significant interaction-free energies. Disulfide locking of the hP2X3R E112C/R198C or the E57C/T263C double cysteine mutants markedly reduced the ATP-induced current responses. The decreased current amplitude following inter-subunit disulfide cross-linking indicates that disulfide locking of the head and dorsal fin domains or at the level of the lower body domains of the hP2X3R prevents the gatinginduced conformational rearrangement of the subunits with respect to each other. The distinct reorganization of the subunit interfaces during gating of the hP2X3R is generally consistent with the gating mechanism of other P2XRs. Chargereversal mutagenesis and methanethiosulfonate (MTS)-modification of substituted cysteines demonstrated that E112 and R198 interact electrostatically. Both disulfide locking and salt bridge breaking of the E112/R198 interaction reduced the hP2X3R function. We conclude that the inter-subunit salt bridge between E112 and R198 of the head and dorsal fin domains, respectively, serves to control the mobility of these domains during agonist-activation of the hP2X3R.

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Section: Discussionsupporting

confidence: 92%

Section: Electronic Supplementary Materialsmentioning

confidence: 72%

Inter-subunit disulfide locking of the human P2X3 receptor elucidates ectodomain movements associated with channel gating

Stephan

Kowalski-Jahn

Zens

et al. 2016

Purinergic Signalling

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“…We have previously shown that replacement of two of these residues, K68 and F291, by cysteine residues allows disulfide cross-linking between neighboring P2X1 subunits and that this reaction is prevented in the presence of ATP. Based on these data, we concluded that the ATP binding sites are located at the subunit interfaces (7,8). This conclusion is in good agreement with the positions of the relevant amino acids in the crystal structure of the unliganded P2X4R from zebrafish (2).…”

mentioning

confidence: 84%

Involvement of the cysteine-rich head domain in activation and desensitization of the P2X1 receptor

Lörinczi

Bhargava

Marino

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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P2X receptors (P2XRs) are ligand-gated ion channels activated by extracellular ATP. Although the crystal structure of the zebrafish P2X4R has been solved, the exact mode of ATP binding and the conformational changes governing channel opening and desensitization remain unknown. Here, we used voltage clamp fluorometry to investigate movements in the cysteine-rich head domain of the rat P2X1R (A118-I125) that projects over the proposed ATP binding site. On substitution with cysteine residues, six of these residues (N120-I125) were specifically labeled by tetramethyl-rhodaminemaleimide and showed significant changes in the emission of the fluorescence probe on application of the agonists ATP and benzoylbenzoyl-ATP. Mutants N120C and G123C showed fast fluorescence decreases with similar kinetics as the current increases. In contrast, mutants P121C and I125C showed slow fluorescence increases that seemed to correlate with the current decline during desensitization. Mutant E122C showed a slow fluorescence increase and fast decrease with ATP and benzoyl-benzoyl-ATP, respectively. Application of the competitive antagonist 2′,3′-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) resulted in large fluorescence changes with the N120C, E122C, and G123C mutants and minor or no changes with the other mutants. Likewise, TNP-ATP-induced changes in control mutants distant from the proposed ATP binding site were comparably small or absent. Combined with molecular modeling studies, our data confirm the proposed ATP binding site and provide evidence that ATP orients in its binding site with the ribose moiety facing the solution. We also conclude that P2XR activation and desensitization involve movements of the cysteine-rich head domain. P 2X receptors (P2XRs) represent a family of nonselective cation channels gated by extracellular ATP. They are widely distributed in mammalian tissues and have been shown to be involved in diverse physiological functions (1). The seven known subunits all contain two transmembrane domains linked by a large extracellular loop. Functional receptors are homo-or heteromeric trimers (2, 3).Based on mutagenesis studies, it has been suggested that conserved positively charged and aromatic residues are crucial for ATP binding, presumably by interacting with the negatively charged phosphate chain of ATP (4-6) and its adenine ring (6), respectively. We have previously shown that replacement of two of these residues, K68 and F291, by cysteine residues allows disulfide cross-linking between neighboring P2X1 subunits and that this reaction is prevented in the presence of ATP. Based on these data, we concluded that the ATP binding sites are located at the subunit interfaces (7,8). This conclusion is in good agreement with the positions of the relevant amino acids in the crystal structure of the unliganded P2X4R from zebrafish (2). This zP2X4 structure revealed an ion channel architecture that resembles a dolphin, with the transmembrane helices and the extracellular region forming the fluke and the upper body, respectively. Att...

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“…In the zfP2X4R crystal structure, these conserved residues line an open-jaw-shaped pocket, strongly suggesting the pocket as the ATP binding site (9). This putative binding site, located at the trimeric interface and approximately 45 Å from the transmembrane domain (TMD), is supported by intersubunit cross-linking between substituted cysteine residues (15,21). Recently Jiang et al (22) presented additional evidence by cross-linking 8-thiocyano-ATP (NCS-ATP) with single cysteine substitutions in the putative binding site of a P2X2R.…”

mentioning

confidence: 97%

Gating mechanism of a P2X4 receptor developed from normal mode analysis and molecular dynamics simulations

Dong

Zhou

2012

Proc. Natl. Acad. Sci. U.S.A.

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P2X receptors are trimeric ATP-gated cation channels participating in diverse physiological processes. How ATP binding triggers channel opening remains unclear. Here the gating mechanism of a P2X receptor was studied by normal mode analysis and molecular dynamics (MD) simulations. Based on the resting-state crystal structure, a normal mode involving coupled motions of three β-strands (β1, β13, and β14) at the trimeric interface of the ligandbinding ectodomain and the pore-lining helix (TM2) in the transmembrane domain (TMD) was identified. The resulting widening of the fenestrations above the TMD and opening of the transmembrane pore produce known signatures of channel activation. In MD simulations, ATP was initially placed in the putative binding pocket (defined by four charged residues located in β1, β13 and β14) in two opposite orientations, with the adenine either proximal or distal to the TMD. In the proximal orientation, the triphosphate group extends outward to draw in the four charged residues, leading to closure of β13/β14 toward β1. The adenine ring, wedged between β1 and β13, acts as a fulcrum for the β14 lever, turning a modest closure around the triphosphate group into significant opening of the pre-TM2 loop. The motions of these β-strands are similar to those in the putative channel-activation normal mode. In the distal orientation, the ATP stabilizes the trimeric interface and the closure of the pre-TM2 loop, possibly representing desensitization. Our computational studies produced the first complete model, supported by experimental data, for how ATP binding triggers activation of a P2X receptor.antagonist design | ATP binding mode | channel-activation mechanism | ligand-gated ion channels P 2X receptors (P2XRs) are a family of ligand-gated nonselective cation channels activated by extracelluar ATP (1). Distributed throughout the human body, they participate in diverse physiological processes such as synaptic transmission, response to inflammation, and pain sensation, and thus are potential drug targets (2-7). Seven subtypes of P2XRs, P2XR1 to P2X7R, have been identified in mammals. They assemble to form functional homo-or hetero-trimeric channels (8), distinguishing them from the better-studied tetrameric and pentameric ligand-gated ionchannel families. The recent crystal structure of zebrafish P2X4 receptor (zfP2X4R) in the resting state represents a major advance in understanding the structure and function of P2XR ion channels (9). Each subunit of zfP2X4R contains two transmembrane helices (TM1 and TM2), which link the intracellular amino and carboxyl termini to the large ligand-binding ectodomain (Fig. 1). In the ectodomain, each subunit consists of 4 α-helices and 14 β-strands (β1-β14). β1 and β14 are connected to TM1 and TM2, respectively.Studies based on mutagenesis have identified several highly conserved ectodomain residues as involved in ATP binding (10-18). The conserved residues include four cationic ones: K70 and K72 on β1, R298 on β13, and K316 on β14 (10,19,20). Aromatic and polar r...

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Inter-subunit disulfide cross-linking in homomeric and heteromeric P2X receptors

Cited by 29 publications

References 34 publications

Inter-subunit disulfide locking of the human P2X3 receptor elucidates ectodomain movements associated with channel gating

Inter-subunit disulfide locking of the human P2X3 receptor elucidates ectodomain movements associated with channel gating

Involvement of the cysteine-rich head domain in activation and desensitization of the P2X1 receptor

Gating mechanism of a P2X4 receptor developed from normal mode analysis and molecular dynamics simulations

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