Abstract
Activation of the P2X7 receptor results in the opening of a large pore that plays a role in immune responses, apoptosis, and many other physiological and pathological processes. Here, we investigated the role of conserved and unique residues in the extracellular vestibule connecting the agonist-binding domain with the transmembrane domain of rat P2X7 receptor. We found that all residues that are conserved among the P2X receptor subtypes respond to alanine mutagenesis with an inhibition (Y51, Q52, and G323) or a significant decrease (K49, G326, K327, and F328) of 2′,3′-O-(benzoyl-4-benzoyl)-ATP (BzATP)-induced current and permeability to ethidium bromide, while the nonconserved residue (F322), which is also present in P2X4 receptor, responds with a 10-fold higher sensitivity to BzATP, much slower deactivation kinetics, and a higher propensity to form the large dye-permeable pore. We examined the membrane expression of conserved mutants and found that Y51, Q52, G323, and F328 play a role in the trafficking of the receptor to the plasma membrane, while K49 controls receptor responsiveness to agonists. Finally, we studied the importance of the physicochemical properties of these residues and observed that the K49R, F322Y, F322W, and F322L mutants significantly reversed the receptor function, indicating that positively charged and large hydrophobic residues are important at positions 49 and 322, respectively. These results show that clusters of conserved residues above the transmembrane domain 1 (K49–Y51–Q52) and transmembrane domain 2 (G326–K327–F328) are important for receptor structure, membrane expression, and channel gating and that the nonconserved residue (F322) at the top of the extracellular vestibule is involved in hydrophobic inter-subunit interaction which stabilizes the closed state of the P2X7 receptor channel.
Highlights
The purinergic P2X receptors (P2X1–7) are ATP-gated cation channels that have a widespread distribution in neuronal and non-neuronal cells and are involved in many physiological and pathophysiological processes [1,2,3,4,5,6]
We report here the functional characterization of selected extracellular vestibule mutants of rat P2X7 (rP2X7) that were analyzed for their capacities to induce membrane current, control receptor expression in the membrane, and open a large membrane pore permeable to ethidium bromide (EtBr)
Our results revealed that clusters of conserved residues above the TM1 (K49–Y51–Q52) and TM2 (G326–K327–F328) domains are important for membrane expression, receptor structure, and gating properties and that the nonconserved residue (F322), which is unique for P2X7 and P2X4, dramatically controls the affinity of the receptor for its ligands, deactivation kinetics, and dye uptake ability
Summary
The purinergic P2X receptors (P2X1–7) are ATP-gated cation channels that have a widespread distribution in neuronal and non-neuronal cells and are involved in many physiological and pathophysiological processes [1,2,3,4,5,6]. The main focus in these studies was on charged residues (E51, E56, and D58, P2X4 numbering) proximal to TM1 that line the lateral portals of the closed human P2X4 receptor These studies indicate that ions access and enter the pore through lateral fenestrations of the extracellular vestibule, with negatively charged side chains of residues E56 and D58 being important for forming an access pathway for cations [37], and residue E51 contributes to the high Ca2+ permeability of P2X4 [35,36,38]. We used alanine- and cysteine-scanning mutagenesis to show that a majority (53%) of the individual substitutions in the extracellular vestibule of rat P2X4 (regions V47-V61 and F324-N338) do not significantly affect receptor function and that five substitutions, namely V49, Y54, Q55, F324, and G325, are critical for P2X4 function [40] These residues are present in P2X7 (except V49), and Y51, Q52, and G323 are fully conserved among the mammalian P2X family. The results show that clusters of conserved residues proximal to TM1 and TM2 are important for receptor trafficking, structure, and gating and that P2X7 function is critically dependent on the properties of the nonconserved residue F322 at the top of the extracellular vestibule and between the extracellular and central vestibules
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