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

Published on: May 18, 2009

Point mutations confer loss of ATP-induced human P2X(7) receptor function

R A Worthington1, M L Smart, B J Gu

  • 1Institute for Biomedical Research, The University of Sydney, Australia.

FEBS Letters
|February 20, 2002
PubMed

Insights

Key residues K193 and K311 are essential for ATP binding to the human P2X(7) receptor (hP2X(7)R). Mutations at these sites abolish channel activity, confirming their critical role in receptor function.

Area of Science:

  • Pharmacology
  • Molecular Biology
  • Cell Biology

Background:

  • The human P2X(7) receptor (hP2X(7)R) is an ATP-gated ion channel implicated in various physiological and pathological processes.
  • Understanding the molecular mechanisms of ATP binding to hP2X(7)R is crucial for developing targeted therapeutics.

Purpose of the Study:

  • To identify and characterize the specific amino acid residues essential for ATP binding to the hP2X(7)R.
  • To elucidate the functional consequences of mutations in these key residues on receptor activity.

Main Methods:

  • Site-directed mutagenesis was employed to generate mutant hP2X(7)R constructs (K193A, K311A, P210A).
  • HEK293 cells and Xenopus oocytes were transfected with wild-type and mutant hP2X(7)R.
  • Channel and pore activity were measured using barium uptake, ethidium influx assays, and electrophysiology in response to ATP and BzATP.

Main Results:

  • Mutations K193A and K311A completely abolished barium uptake and ethidium influx in HEK293 cells.
  • The P210A mutation partially reduced these ion flux activities.
  • Electrophysiological recordings in Xenopus oocytes confirmed that K193A and K311A mutations abolished responses to both ATP and BzATP.

Conclusions:

  • Lysine residues at positions 193 (K193) and 311 (K311) are indispensable for ATP binding to the hP2X(7)R.
  • These findings provide critical insights into the ligand-binding domain of the hP2X(7)R and its functional regulation.

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