Identification of ectodomain regions contributing to gating, deactivation, and resensitization of purinergic P2X receptors.
The P2X receptors (P2XRs) are a family of ligand-gated channels activated by extracellular ATP through a sequence of conformational transitions between closed, open, and desensitized states. In this study, we examined the dependence of the activity of P2XRs on ectodomain structure and agonist potency. Experiments were done in human embryonic kidney 293 cells expressing rat P2X2aR, P2X2bR, and P2X3R, and chimeras having the V60-R180 or V60-F301 ectodomain sequences of P2X3R instead of the I66-H192 or I66-Y310 sequences of P2X2aR and P2X2bR. Chimeric P2X2a/V60-F301X3R and P2X2b/V60-F301X3R inherited the P2X3R ligand-selective profile, whereas the potency of agonists for P2X2a/V60-R180X3R was in between those observed at parental receptors. Furthermore, P2X2a/V60-F301X3R and P2X2a/V60-R180X3R desensitized in a P2X2aR-specific manner, and P2X2b/V60-F301X3R desensitized with rates comparable with those of P2X2bR. In striking contrast to parental receptors, the rates of decay in P2X2a/V60-F301X3R and P2X2b/V60-F301X3R currents after agonist withdrawal were 15- to 200-fold slower. For these chimeras, the decays in currents were not dependent on duration of stimuli and reflected both continuous desensitization and deactivation of receptors. Also, participation of deactivation in closure of channels inversely correlated with potency of agonists to activate receptors. The delay in deactivation was practically abolished in P2X2a/V60-R180X3R-expressing cells. However, the recovery from desensitization of P2X2a/V60-F301X3R and P2X2a/V60-R180X3R was similar and substantially delayed compared with that of parental receptors. These results indicate that both ectodomain halves participate in gating, but that the C and N halves influence the stability of open and desensitized conformation states, respectively, which in turn reflects on rates of receptor deactivation and resensitization.
Section on Cellular Signaling, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-4510, USA.
Hana
H
Zemkova
Mu-Lan
ML
He
Taka-aki
TA
Koshimizu
Stanko S
SS
Stojilkovic
D000255:Adenosine Triphosphate / Q000502:physiology; D002460:Cell Line; D002678:Chimera; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D007668:Kidney; D008024:Ligands; D011487:Protein Conformation; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000502:physiology*; D058476:Receptors, Purinergic P2X2; D058477:Receptors, Purinergic P2X3; D013329:Structure-Activity Relationship; D014162:Transfection
D008024:Ligands; C546401:P2RX2 protein, human; C546407:P2RX3 protein, human; C546403:P2rx2 protein, rat; C546410:P2rx3 protein, rat; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2; D058476:Receptors, Purinergic P2X2; D058477:Receptors, Purinergic P2X3; D000255:Adenosine Triphosphate