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

Abstract

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...

Inter- and intrasubunit interactions between transmembrane helices in the open state of P2X receptor channels

“…For example, ATP binding to the cleft and subsequent cleft closure were predicted to occur based on proximity tethering (11), normal mode analysis (12), and both metal bridging and spectroscopic studies (13,14). In addition, the accessibility of both methanethiolsulfonate compounds and metals (Ag + and Cd 2+ ) to cysteine residues introduced into TM1 and TM2 (6)(7)(8) is consistent with the expansion of the external pore predicted from the zfP2X4 structures (Fig.…”

Relative motions between left flipper and dorsal fin domains favour P2X4 receptor activation

“…Those conformational changes are distinct from previously identified allosteric events essential for channel activations, such as the downward motion of cysteine-rich head domain, tightening of the ATP-binding site jaw and the upper vestibule expansions 5,6,8,9 . As revealed by the spontaneous closure of the ATP-binding site jaw (Fig.…”

“…Numerous studies have demonstrated that a downward motion of the cysteine-rich head domain, tightening of the ATP-binding site jaw and upper vestibule expansion are essential for activation, desensitization and sustained activation of P2X receptors [5][6][7][8][9][10] , with data obtained from the application of multiple approaches, including electron microscopy 10 , voltage-clamp fluorometry 9 , engineering EC metal bridges 8,11 , normal mode analysis (NMA) 7 , molecular dynamics (MD) simulations 7 and fast-scanning atomic force microscopy 6 . Those studies provide new insights into the dynamics of EC domain as well as the movements of the whole receptor during channel gating.…”

A Highly Conserved Salt Bridge Stabilizes the Kinked Conformation of β2,3-Sheet Essential for Channel Function of P2X4 Receptors

“…3), the decreased current density in those mutations might be attributed to altered channel gating. Previous studies demonstrated that the head, the DF, and the LF domains have participated in nearly every essential or constituent element of P2X channel gating (20 -22, 24, 39, 42), including channel desensitization (24). Given that the changed salt bridge possibly resulted in the conformational changes in ␤2,3 and its physically coupled head and LF domains, the decreased current density in those mutants may be attributed to the combination of the affecting factors mentioned above.…”

“…1F). The increased EC 50 may originate from the fact that the motion of this domain was associated with the downward movement of the head domain, which is coupled with the ATP binding and channel gating activity (20,24,42). However, compared with the mutations at positively charged residues, which interact directly with the negative triphosphate of ATP, such as K71A and K69R (the EC 50 of ATP Ͼ20 mM) (43,44), the rightward shift of the concentrationresponse curve for D85A was nearly negligible.…”