The family of ATP-gated purinergic P2X receptors comprises seven bunits (P2X1-7) that are unevenly distributed in the central and peripheral nervous systems as well as other organs. Endogenous modulators of P2X receptors are phospholipids, steroids and neurosteroids. Here, we analyzed whether bile acids, which are natural products derived from cholesterol, affect P2X receptor activity. We examined the effects of primary and secondary bile acids and newly synthesized derivatives of lithocholic acid on agonist-induced responses in HEK293T cells expressing rat P2X2, P2X4 and P2X7 receptors. Electrophysiology revealed that low micromolar concentrations of lithocholic acid and its structural analog 4-dafachronic acid strongly inhibit ATP-stimulated P2X2 but potentiate P2X4 responses, whereas primary bile acids and other secondary bile acids exhibit no or reduced effects only at higher concentrations. Agonist-stimulated P2X7 responses are significantly potentiated by lithocholic acid at moderate concentrations. Structural modifications of lithocholic acid at positions C-3, C-5 or C-17 abolish both inhibitory and potentiation effects to varying degrees, and the 3α-hydroxy group contributes to the ability of the molecule to switch between potentiation and inhibition. Lithocholic acid allosterically modulates P2X2 and P2X4 receptor sensitivity to ATP, reduces the rate of P2X4 receptor desensitization and antagonizes the effect of ivermectin on P2X4 receptor deactivation. Alanine-scanning mutagenesis of the upper halve of P2X4 transmembrane domain-1 revealed that residues Phe48, Val43 and Tyr42 are important for potentiating effect of lithocholic acid, indicating that modulatory sites for lithocholic acid and ivermectin partly overlap. Lithocholic acid also inhibits ATP-evoked currents in pituitary gonadotrophs expressing native P2X2, and potentiates ATP currents in nonidentified pituitary cells expressing P2X4 receptors. These results indicate that lithocholic acid is a bioactive steroid that may help to further unveil the importance of the P2X2, and P2X4 receptors in many physiological processes.

• MeSH
• Pituitary Gland, Anterior cytology   drug effects   physiology   MeSH
• Purinergic P2X Receptor Agonists pharmacology   MeSH
• Purinergic P2X Receptor Antagonists pharmacology   MeSH
• Ion Channel Gating drug effects   MeSH
• HEK293 Cells MeSH
• Hypothalamus cytology   drug effects   physiology   MeSH
• Lithocholic Acid analogs & derivatives   pharmacology   MeSH
• Humans MeSH
• Neurons drug effects   physiology   MeSH
• Rats, Wistar MeSH
• Receptors, Purinergic P2X2 physiology   MeSH
• Receptors, Purinergic P2X4 physiology   MeSH
• Receptors, Purinergic P2X7 physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Crystallization of the zebrafish P2X4 receptor in both open and closed states revealed conformational differences in the ectodomain structures, including the dorsal fin and left flipper domains. Here, we focused on the role of these domains in receptor activation, responsiveness to orthosteric ATP analogue agonists, and desensitization. Alanine scanning mutagenesis of the R203-L214 (dorsal fin) and the D280-N293 (left flipper) sequences of the rat P2X4 receptor showed that ATP potency/efficacy was reduced in 15 out of 26 alanine mutants. The R203A, N204A, and N293A mutants were essentially non-functional, but receptor function was restored by ivermectin, an allosteric modulator. The I205A, T210A, L214A, P290A, G291A, and Y292A mutants exhibited significant changes in the responsiveness to orthosteric analog agonists 2-(methylthio)adenosine 5'-triphosphate, adenosine 5'-(γ-thio)triphosphate, 2'(3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate, and α,β-methyleneadenosine 5'-triphosphate. In contrast, the responsiveness of L206A, N208A, D280A, T281A, R282A, and H286A mutants to analog agonists was comparable to that of the wild type receptor. Among these mutants, D280A, T281A, R282A, H286A, G291A, and Y292A also exhibited increased time-constant of the desensitizing current response. These experiments, together with homology modeling, indicate that residues located in the upper part of the dorsal fin and left flipper domains, relative to distance from the channel pore, contribute to the organization of the ATP binding pocket and to the initiation of signal transmission towards residues in the lower part of both domains. The R203 and N204 residues, deeply buried in the protein, may integrate the output signal from these two domains towards the gate. In addition, the left flipper residues predominantly account for the control of transition of channels from an open to a desensitized state.

• MeSH
• Adenosine Triphosphate metabolism   MeSH
• Purinergic P2X Receptor Agonists pharmacology   MeSH
• Alanine genetics   metabolism   MeSH
• Ion Channel Gating drug effects   MeSH
• HEK293 Cells MeSH
• Ivermectin pharmacology   MeSH
• Rats MeSH
• Humans MeSH
• Patch-Clamp Techniques MeSH
• Molecular Sequence Data MeSH
• Mutagenesis MeSH
• Receptors, Purinergic P2X4 chemistry   genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Sequence Alignment MeSH
• Molecular Dynamics Simulation MeSH
• Protein Structure, Tertiary MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Purinergic P2X receptors (P2XR), activated by extracellular adenosine 5‘-triphosphate (ATP), represent a specific type of li- gand-gated ion channels. They form functional trimeric homomers or heteromers which are nonselectively cation-perme- able after receptor activation. P2X receptors are widely expressed in excitable and nonexcitable tissues and are involved in many physiological and pathophysiological processes such as platelet aggregation, contraction of smooth muscle, immu- ne responses, cell proliferation and apoptosis or neurotransmission. In mammals, seven P2X subunits (P2X1-P2X7) have been identified. They differ mainly in distribution, pharmacological profile and kinetics of ATP-induced responses. The sub- type P2X7 is the most specific in the P2X family and widely differs from other P2X subtypes.

• Keywords
• extracelulární ATP, purinergní P2X rodina, buněčná proliferace a apoptóza,
• MeSH
• Adenosine Triphosphate biosynthesis   MeSH
• Purinergic P2X Receptor Agonists pharmacokinetics   pharmacology   MeSH
• Apoptosis physiology   MeSH
• Extracellular Space MeSH
• Cell Physiological Phenomena MeSH
• Humans MeSH
• Intercellular Signaling Peptides and Proteins MeSH
• Cell Proliferation MeSH
• Receptors, Purinergic P2X * analysis   biosynthesis   physiology   isolation & purification   classification   metabolism   drug effects   MeSH
• Receptors, Purinergic P2X1 blood   MeSH
• Receptors, Purinergic P2X3 physiology   MeSH
• Receptors, Purinergic P2X4 MeSH
• Receptors, Purinergic P2X7 immunology   MeSH
• Signal Transduction MeSH
• Check Tag
• Humans MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH