Abstract

The wild-type P2X₂ purinergic receptor (P2X_2aR) and its splice form lacking the intracellular Val³⁷⁰-Gln⁴³⁸ C-terminal sequence (P2X_2bR) respond to ATP stimulation with comparable EC₅₀ values and peak current/calcium responses but desensitize in a receptor-specific manner. P2X_2aR desensitizes slowly and P2X_2bR desensitizes rapidly. We studied the effects of different agonists, and of substituting the ectodomain, on the pattern of calcium signaling by P2X_2aR and P2X_2bR. Both receptors showed similar EC₅₀values (estimated from the peak calcium response) and IC₅₀values (estimated from the rate of calcium signal desensitization) for agonists, in the order 2-MeS-ATP ≤ ATP ≤ ATPγS < BzATP ≪ αβ-meATP, and the IC₅₀ values for agonists were shifted to the right compared with their EC₅₀ values. Furthermore, the ATP-induced receptor-subtype specific pattern of desensitization was mimicked by high- but not by low-efficacy agonists, suggesting a ligand-specific desensitization pattern. To test this hypothesis, we generated chimeric P2X_2aR and P2X_2bR containing the Val⁶⁰-Phe³⁰¹ectodomain sequence of P2X₃R and Val⁶¹-Phe³¹³ ectodomain sequence of P2X₇R instead the native Ile⁶⁶-Tyr³¹⁰ sequence. The mutated P2X_2a+X₃R and P2X_2b+X₃R exhibited comparable EC₅₀ values for ATP, BzATP, and αβ-meATP in the submicromolar concentration range and desensitized in a receptor-specific and ligand-nonspecific manner. On the other hand, the chimeric P2X₂+X₇R exhibited decreased sensitivity for ATP and desensitized in a receptor-nonspecific manner. These results suggest that efficacy of agonists for the ligand-binding domain of P2X₂Rs reflects the strength of desensitization controlled by their C-terminal structures.