The P2X4 receptor (P2X4R) is a member of a family of purinergic channels activated by extracellular ATP through three orthosteric binding sites and allosterically regulated by ivermectin (IVM), a broad-spectrum antiparasitic agent. Treatment with IVM increases the efficacy of ATP to activate P2X4R, slows both receptor desensitization during sustained ATP application and receptor deactivation after ATP washout, and makes the receptor pore permeable to NMDG+, a large organic cation. Previously, we developed a Markov model based on the presence of one IVM binding site, which described some effects of IVM on rat P2X4R. Here we present two novel models, both with three IVM binding sites. The simpler one-layer model can reproduce many of the observed time series of evoked currents, but does not capture well the short time scales of activation, desensitization, and deactivation. A more complex two-layer model can reproduce the transient changes in desensitization observed upon IVM application, the significant increase in ATP-induced current amplitudes at low IVM concentrations, and the modest increase in the unitary conductance. In addition, the two-layer model suggests that this receptor can exist in a deeply inactivated state, not responsive to ATP, and that its desensitization rate can be altered by each of the three IVM binding sites. In summary, this study provides a detailed analysis of P2X4R kinetics and elucidates the orthosteric and allosteric mechanisms regulating its channel gating.

• MeSH
• Adenosine Triphosphate metabolism   MeSH
• Algorithms MeSH
• Ion Channel Gating drug effects   physiology   MeSH
• HEK293 Cells MeSH
• Ivermectin metabolism   MeSH
• Humans MeSH
• Markov Chains MeSH
• Patch-Clamp Techniques MeSH
• Receptors, Purinergic P2X4 drug effects   metabolism   physiology   MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• Ivermectin MeSH
• Receptors, Purinergic P2X4 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
• Names of Substances
• Adenosine Triphosphate MeSH
• Purinergic P2X Receptor Agonists MeSH
• Alanine MeSH
• Ivermectin MeSH
• Receptors, Purinergic P2X4 MeSH