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Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels
16:36

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Published on: May 18, 2009

Inter-subunit disulfide cross-linking in homomeric and heteromeric P2X receptors

Benjamin Marquez-Klaka1, Jürgen Rettinger, Annette Nicke

  • 1Department of Neurochemistry, Max-Planck-Institute for Brain Research, Deutschordenstr. 46, 60528 Frankfurt, Germany.

European Biophysics Journal : EBJ
|April 23, 2008
PubMed

Insights

Mutations in P2X receptors reveal conserved residue proximity in P2X2 and P2X1/2 subtypes, impacting ATP binding site and channel function. This finding is less evident in P2X3 and P2X4 receptors.

Area of Science:

  • Molecular biology
  • Neuroscience
  • Biochemistry

Background:

  • P2X receptors are ATP-gated ion channels crucial for cellular signaling.
  • Seven subunits (P2X1-7) assemble into functional homotrimeric or heterotrimeric channels.
  • Previous studies identified specific residues (K68, F291) in P2X1 involved in disulfide cross-linking and ATP binding.

Purpose of the Study:

  • To investigate the conservation of residue proximity, analogous to P2X1's K68 and F291, in other P2X receptor subtypes.
  • To explore the functional consequences of cross-linking in P2X1/2, P2X3, and P2X4 receptors.

Main Methods:

  • Site-directed mutagenesis to introduce cysteine residues in P2X1, P2X2, P2X3, and P2X4 subunits.
  • Pairwise expression of mutant subunits in Xenopus laevis oocytes.
  • Non-reducing SDS-PAGE analysis to detect disulfide-linked dimers.
  • Functional assays of cross-linked heteromers and recovery with DTT treatment.

Main Results:

  • Specific dimer formation observed between P2X2(K69C) and P2X2(F289C) mutants.
  • Heteromeric P2X1(K68C)/P2X2(F289C) dimers formed, but not P2X1(F291C)/P2X2(K69C).
  • Cross-linked P2X1/2 heteromers exhibited reduced function, restored by DTT; cross-linking was less efficient in P2X3 and P2X4 mutants.

Conclusions:

  • The spatial proximity of residues analogous to P2X1's K68 and F291 is conserved in P2X2 receptors.
  • This proximity is present at one interface in heteromeric P2X1/2 receptors, suggesting a conserved structural feature for ATP binding.
  • The analogous residue positions appear redundant for P2X3 and P2X4 receptor function.

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