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Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time
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P2X receptor intermediate activation states have altered nucleotide selectivity.

Liam E Browne1, R Alan North

  • 1Faculties of Medical and Human Sciences, and Life Sciences, University of Manchester, Manchester, M13 9PT, United Kingdom.

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
|September 13, 2013
PubMed
Summary

ATP binding to purinergic P2X receptors induces conformational changes. This binding makes the receptors sensitive to other nucleotides, revealing new activation mechanisms for these crucial nervous system proteins.

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Area of Science:

  • Neuroscience
  • Molecular Biology
  • Biochemistry

Background:

  • Purinergic P2X receptors are vital membrane proteins in the nervous system.
  • They regulate primary afferent transmission and central respiratory control.
  • These receptors possess three extracellular ATP binding sites.

Purpose of the Study:

  • To investigate the conformational changes in P2X receptors upon ATP binding.
  • To explore how ATP binding affects the receptor's sensitivity to other nucleotides.
  • To understand the functional implications of these conformational shifts.

Main Methods:

  • Expression of rat P2X7 receptor in human embryonic kidney cells.
  • Measurement of membrane currents before and after photo-affinity labeling with BzATP.
  • Photo-affinity labeling using ultraviolet light to tether the agonist.

Main Results:

  • Tethered BzATP resulted in a persistent current, even after washout.
  • The receptor became responsive to CTP and ADP, which are typically ineffective agonists.
  • Similar nucleotide sensitivity changes were observed with P2X2 receptors upon ATP exposure.

Conclusions:

  • ATP binding to P2X receptors induces a conformational change to an intermediate closed state.
  • This conformational change enhances the receptor's sensitivity to pyrimidine and diphosphate nucleotide analogs.
  • The findings elucidate novel mechanisms of P2X receptor activation and regulation.