Purinergic Signalling

, Volume 8, Issue 2, pp 317–325

Conserved ectodomain cysteines are essential for rat P2X7 receptor trafficking

  • Marie Jindrichova
  • Pavlo Kuzyk
  • Shuo Li
  • Stanko S. Stojilkovic
  • Hana Zemkova
Original Article

Abstract

The P2X7 receptor (P2X7R) is a member of the ATP-gated ion channel family that exhibits distinct electrophysiological and pharmacological properties. This includes low sensitivity to ATP, lack of desensitization, a sustained current growth during prolonged receptor stimulation accompanied with development of permeability to large organic cations, and the coupling of receptor activation to cell blebbing and death. The uniquely long C-terminus of P2X7R accounts for many of these receptor-specific functions. The aim of this study was to understand the role of conserved ectodomain cysteine residues in P2X7R function. Single- and double-point threonine mutants of C119–C168, C129–C152, C135–C162, C216–C226, and C260–C269 cysteine pairs were expressed in HEK293 cells and studied using whole-cell current recording. All mutants other than C119T-P2X7R responded to initial and subsequent application of 300-μM BzATP and ATP with small amplitude monophasic currents or were practically nonfunctional. The mutagenesis-induced loss of function was due to decreased cell-surface receptor expression, as revealed by assessing levels of biotinylated mutants. Coexpression of all double mutants with the wild-type receptor had a transient or, in the case of C119T/C168T double mutant, sustained inhibitory effect on receptor trafficking. The C119T-P2X7R mutant was expressed on the plasma membrane and was fully functional with a slight decrease in the sensitivity for BzATP, indicating that interaction of liberated Cys168 with another residue rescues the trafficking of receptor. Thus, in contrast to other P2XRs, all disulfide bonds of P2X7R are individually essential for the proper receptor trafficking.

Keywords

Purinergic P2X receptors ATP-gated channels Disulfide bonds Trafficking BzATP 

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Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Marie Jindrichova
    • 1
  • Pavlo Kuzyk
    • 1
  • Shuo Li
    • 2
  • Stanko S. Stojilkovic
    • 2
  • Hana Zemkova
    • 1
  1. 1.Department of Cellular and Molecular Neuroendocrinology, Institute of PhysiologyAcademy of Sciences of the Czech RepublicPragueCzech Republic
  2. 2.Section on Cellular Signaling, Program in Developmental Neuroscience, NICHDNIHBethesdaUSA

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