Abstract
P2X(7) receptors are ATP-gated cation channels composed of three identical subunits, each having intracellular amino and carboxyl termini and two transmembrane segments connected by a large ectodomain. Within the P2X family, P2X(7) subunits are unique in possessing an extended carboxyl tail. We expressed the human P2X(7) subunit as two complementary fragments, a carboxyl tail-truncated receptor channel core (residues 1-436 or 1-505) and a tail extension (residues 434-595) in Xenopus laevis oocytes. P2X(7) channel core subunits efficiently assembled as homotrimers that appeared abundantly at the oocyte surface, yet produced only approximately 5% of the full-length P2X(7) receptor current. Co-assembly of channel core subunits with full-length P2X(7) subunits inhibited channel current, indicating that the lack of a single carboxyl tail domain is dominant-negative for P2X(7) receptor activity. Co-expression of the tail extension as a discrete protein increased ATP-gated current amplitudes of P2X(7) channel cores 10-20-fold, fully reconstituting the wild type electrophysiological phenotype of the P2X(7) receptor. Chemical cross-linking revealed that the discrete tail extension bound with unity stoichiometry to the carboxyl tail of the P2X(7) channel core. We conclude that a non-covalent association of crucial functional importance exists between the carboxyl tail of the channel core and the tail extension. Using a slightly shorter P2X(7) subunit core and subfragments of the tail extension, this association could be narrowed down to include residues 409-436 and 434-494 of the split receptor. Together, these results identify the tail extension as a regulatory gating module, potentially making P2X(7) channel gating sensitive to intracellular regulation.
Highlights
The P2X7 receptor, an ATP-gated cation channel, is expressed predominantly in immune cells, glial cells, and epithelial cells
Macroscopic human P2X7 (hP2X7) Receptor Current Kinetics Are Strongly Affected by Truncation, but Are Restored by Co-expression of the Truncated Carboxyl Tail—Fig. 1, B–F, shows typical traces of currents induced by 1 mM free ATP (ATP4Ϫ) and recorded by the two-electrode voltage-clamp method from intact X. laevis oocytes expressing the indicated hP2X7 constructs
Two observations indicate that this stimulating effect involves the carboxyl tail-truncated hP2X7 receptor cores: (i) expression of the hP2X7434–595 tail alone did not lead to ATP-activated currents, and (ii) currents mediated by the wild type hP2X7 receptors were not significantly affected by co-expression of the carboxyl tail
Summary
The P2X7 receptor, an ATP-gated cation channel, is expressed predominantly in immune cells (such as macrophages and lymphocytes), glial cells, and epithelial cells (for recent reviews, see Refs. 1 and 2). Co-expression of the hP2X71– 436 receptor core with the hP2X7434 –595 carboxyl tail as discrete polypeptides increased the ATP-induced current amplitude ϳ15-fold, reaching a value near that of the non-split full-length hP2X7 (Fig. 1, D, G, and H).
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