Abstract
Functional homomeric and heteromeric ATP-gated P2X receptor channels have been shown to display a characteristic trimeric architecture. Of the seven different isoforms (designated P2X(1)-P2X(7)), P2X(5) occurs in humans primarily as a non-functional variant lacking the C-terminal end of the ectodomain and the outer half of the second transmembrane domain. We show that this truncated variant, which results from the splice-skipping of exon 10, is prone to subunit aggregation because the residual transmembrane domain 2 is too short to insert into the membrane. Alleviation of the negative hydrophobic mismatch by the addition of a stretch of moderately hydrophobic residues enabled formation of a second membrane-spanning domain and strictly parallel homotrimerization. Systematic mutagenesis identified only one transmembrane domain 2 residue, Asp(355), which supported homotrimerization in a side chain-specific manner. Our results indicate that transmembrane domain 2 formation contributes 2-fold to hP2X(5) homotrimerization by tethering the end of the ectodomain to the membrane, thereby topologically restricting conformational mobility, and by intramembrane positioning of Asp(355). While transmembrane domain 2 appears to favor assembly by enabling productive subunit interactions in the ectodomain, Asp(355) seems to assist by simultaneously driving intramembrane helix interactions. Overall, these results indicate a complex interplay between topology, helix-helix interactions, and oligomerization to achieve a correctly folded structure.
Highlights
The question of which molecular determinants lead to the assembly of subunits into P2X receptors has so far been addressed by examining the ability of deletion mutants and chimeric constructs to associate with full-length P2X subunits in a co-immunoprecipitation assay [7]
To further examine the role of both TM2 and the pre-TM2 region in P2X receptor assembly, we utilized the P2X5 isoform, which occurs in humans predominantly as a natural deletion mutant and lacks much of the TM2 and pre-TM2 regions as a result of the splicing-out of exon 10 [8]
Functional Rat P2X5 Receptors Possess a Homotrimeric Architecture—All P2X5 constructs in this study were N-terminal-tagged with a hexahistidyl sequence to allow for protein purification by metal affinity chromatography after expression in X. laevis oocytes
Summary
A deletion mutant terminating 25 amino acids before the start of the TM2 domain was unable to interact with either of the wild type subunits or with itself. This suggested that either TM2 or a region immediately upstream of TM2 carried a critical determinant of specific subunit-subunit interactions, the possibility that the lack of co-assembly was the result of an inadequate secondary or tertiary structure was not ruled out [7]. In this report we sequentially generated a full-length hP2X5 subunit cDNA by progressively and repeatedly inserting codons for 4 – 8 amino acids of the spliced-out exon 10 and determined the assembly state of the resultant constructs by blue native PAGE. We detected a specific role in assembly recognition for only one TM2 residue, Asp355, which is fully conserved among P2X family members and may stabilize helix-helix interactions by satisfying hydrogen bonding groups in the membrane
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