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1.
J Neurochem ; 165(6): 874-891, 2023 06.
Article in English | MEDLINE | ID: mdl-36945903

ABSTRACT

P2X receptors (P2X1-7) are trimeric ion channels activated by extracellular ATP. Each P2X subunit contains two transmembrane helices (TM1 and TM2). We substituted all residues in TM1 of rat P2X7 with alanine or leucine one by one, expressed mutants in HEK293T cells, and examined the pore permeability by recording both membrane currents and fluorescent dye uptake in response to agonist application. Alanine substitution of G27, K30, H34, Y40, F43, L45, M46, and D48 inhibited agonist-stimulated membrane current and dye uptake, and all but one substitution, D48A, prevented surface expression. Mutation V41A partially reduced both membrane current and dye uptake, while W31A and A44L showed reduced dye uptake not accompanied by reduced membrane current. Mutations T28A, I29A, and L33A showed small changes in agonist sensitivity, but they had no or small impact on dye uptake function. Replacing charged residues with residues of the same charge (K30R, H34K, and D48E) rescued receptor function, while replacement with residues of opposite charge inhibited (K30E and H34E) or potentiated (D48K) receptor function. Prolonged stimulation with agonist-induced current facilitation and a leftward shift in the dose-response curve in the P2X7 wild-type and most functional mutants, but sensitization was absent in the W31A, L33A, and A44L. Detailed analysis of the decay of responses revealed two kinetically distinct mechanisms of P2X7 deactivation: fast represents agonist unbinding, and slow might represent resetting of the receptor to the resting closed state. These results indicate that conserved and receptor-specific TM1 residues control surface expression of the P2X7 protein, non-polar residues control receptor sensitization, and D48 regulates intrinsic channel properties.


Subject(s)
Ion Channels , Receptors, Purinergic P2X7 , Rats , Humans , Animals , HEK293 Cells , Biological Transport , Mutation/genetics , Protein Domains , Ion Channels/metabolism , Receptors, Purinergic P2X7/genetics , Receptors, Purinergic P2X7/metabolism , Adenosine Triphosphate/pharmacology , Adenosine Triphosphate/metabolism
2.
Int J Mol Sci ; 21(22)2020 Nov 10.
Article in English | MEDLINE | ID: mdl-33182845

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.


Subject(s)
Receptors, Purinergic P2X7/genetics , Receptors, Purinergic P2X7/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Conserved Sequence , HEK293 Cells , Humans , Ion Channel Gating , Kinetics , Luminescent Proteins/chemistry , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Models, Molecular , Mutagenesis, Site-Directed , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Protein Domains , Protein Interaction Domains and Motifs , Rats , Receptors, Purinergic P2X7/chemistry , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Static Electricity
3.
J Neurochem ; 150(1): 28-43, 2019 07.
Article in English | MEDLINE | ID: mdl-31069814

ABSTRACT

P2X receptors (P2XRs) are ATP-gated cationic channels that are allosterically modulated by numerous compounds, including steroids and neurosteroids. These compounds may both inhibit and potentiate the activity of P2XRs, but sex steroids such as 17ß-estradiol or progesterone are reported to be inactive. Here, we tested a hypothesis that testosterone, another sex hormone, modulates activity of P2XRs. We examined actions of native testosterone and a series of testosterone derivatives on the gating of recombinant P2X2R, P2X4R and P2X7R and native channels expressed in pituitary cells and hypothalamic neurons. The 17ß-ester derivatives of testosterone rapidly and positively modulate the 1 µM ATP-evoked currents in P2X2R- and P2X4R-expressing cells, but not agonist-evoked currents in P2X7R-expressing cells. In general, most of the tested testosterone derivatives are more potent modulators than endogenous testosterone. The comparison of chemical structures and whole-cell recordings revealed that their interactions with P2XRs depend on the lipophilicity and length of the alkyl chain at position C-17. Pre-treatment with testosterone butyrate or valerate increases the sensitivity of P2X2R and P2X4R to ATP by several fold, reduces the rate of P2X4R desensitization, accelerates resensitization, and enhances ethidium uptake by P2X4R. Native channels are also potentiated by testosterone derivatives, while endogenously expressed GABA receptors type A are inhibited. The effect of ivermectin, a P2X4R-specific allosteric modulator, on deactivation is antagonized by testosterone derivatives in a concentration-dependent manner. Together, our results provide evidence for potentiation of particular subtypes of P2XRs by testosterone derivatives and suggest a potential role of ivermectin binding site for steroid-induced modulation. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.


Subject(s)
Ion Channel Gating/drug effects , Ion Channel Gating/physiology , Receptors, Purinergic P2X2/metabolism , Receptors, Purinergic P2X4/metabolism , Testosterone/pharmacology , Animals , HEK293 Cells , Humans , Rats , Rats, Wistar
4.
J Neurochem ; 133(6): 815-27, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25712548

ABSTRACT

In the sustained presence of agonist, the opening of P2X7R channel is followed by pore dilatation, which causes an increase in its permeability to larger organic cations, accompanied by receptor sensitization. To explore the molecular mechanisms by which the conductivity and sensitivity are increased, we analyzed the electrophysiological properties and YO-PRO-1 uptake of selected alanine mutants in the first and second transmembrane domains of the rat P2X7R. Substitution of residues Y40, F43, G338, and D352 with alanine reduced membrane trafficking, and the D352A was practically non-functional. The Y40A and F43A mutants that were expressed in the membrane lacked pore dilation ability. Moreover, the Y40A and Y40F displayed desensitization, whereas the Y40W partially recovered receptor function. The G338A/S mutations favored the open state of the channel and displayed instantaneous permeability to larger organic cations. The G338P was non-functional. The L341A and G345A displayed normal trafficking, current amplitude, and sensitization, but both mutations resulted in a decreased pore formation and dye uptake. These results showed that the increase in P2X7R conductivity and sensitivity is critically dependent on residues Y40 and F43 in the TM1 domain and that the region located at the intersection of TM2 helices controls the rate of large pore opening. We investigated the mechanism of the proapoptotic receptor P2X7R's large pore opening and its sensitization. We found that aromatic residues in the upper part of the first transmembrane domain (TM1) are critical for both the P2X7R channel pore opening and receptor sensitization, and residues located at or below the intersection of the second transmembrane domains (TM2) control the rate of pore opening. These findings identify new residues involved in pore formation of P2X7R.


Subject(s)
Receptors, Purinergic P2X7/chemistry , Receptors, Purinergic P2X7/metabolism , Amino Acid Sequence , Animals , HEK293 Cells , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Patch-Clamp Techniques , Protein Structure, Tertiary , Protein Transport , Rats , Transfection
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