A Protein Kinase C Site Highly Conserved in P2X Subunits Controls the Desensitization Kinetics of P2X2 ATP-gated Channels

Éric Boué-Grabot,Vincent Archambault,Philippe Séguéla

doi:10.1074/jbc.275.14.10190

Éric Boué-Grabot, Vincent Archambault + Show 1 more

Open Access

https://doi.org/10.1074/jbc.275.14.10190

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Abstract

P2X receptors are nonselective cation channels gated by extracellular ATP. Recombinant mammalian P2X subunits assemble in homomeric ionotropic ATP receptors that differ by their agonist sensitivity and desensitization rate in heterologous expression systems. Using site-directed mutagenesis and voltage clamp recording in Xenopus oocytes, we identified the highly conserved protein kinase C site TX(K/R) located in the intracellular N terminus of P2X subunits as a critical determinant of kinetics in slowly desensitizing (time constant, >1 min) rat P2X(2) receptors. Mutant receptors P2X(2)T18A, T18N, and K20T devoid of this consensus site exhibited quickly desensitizing properties (time constant, <1 s). In contrast with wild-type receptors, mutant P2X(2) receptors with truncated C terminus exhibited variable cell-specific kinetics with quickly desensitizing currents converted to slowly desensitizing currents by phorbol ester-mediated stimulation of protein kinase C. Phosphorylation of Thr(18) was demonstrated directly by immunodetection using specific monoclonal antibodies directed against the phosphothreonine-proline motif. Our data indicate that both phosphorylation of the conserved threonine residue in the N-terminal domain by protein kinase C and interaction between the two cytoplasmic domains of P2X(2) subunits are necessary for the full expression of slowly desensitizing ATP-gated channels.

Highlights

Fast ionotropic responses to extracellular ATP are mediated by the activation of ATP-gated channels or P2X receptors present on the surface of various cell types
The electrophysiological characterization of recombinant homomeric and heteromeric P2X receptors expressed in heterologous systems led to their grouping in three functional categories based on their sensitivity to ATP and on their desensitization properties: 1) P2X1 [3] and P2X3 [4, 5] assemble in quickly desensitizing homomeric receptors highly sensitive to ATP and ␣␤-methylene ATP (EC50 around 1 ␮M); 2) P2X2 [6], P2X4 [7, 8], P2X5 [9], P2X2ϩ3 [5], P2X1ϩ5 [10, 11], and P2X4ϩ6 [12] receptors are less sensitive to ATP (EC50 around 10 ␮M) and desensitize at slow to moderate rate; 3) P2X7 re
Studies on the relationship between the slowly desensitization kinetics of P2X2 receptor and its primary sequence have emphasized the requirement for several structural features including the transmembrane domains and their intracellular flanking regions [17], the negatively charged residue Asp349 located in the second transmembrane domain [18], and the unique C-terminal tail of the P2X2A splicing variant (19 –21)

Summary

Introduction

Fast ionotropic responses to extracellular ATP are mediated by the activation of ATP-gated channels or P2X receptors present on the surface of various cell types. Using site-directed mutagenesis and electrophysiological characterization of recombinant P2X receptors in Xenopus oocytes, we report here the identification of a phosphothreonine part of the highly conserved protein kinase C site located intracellularly in the N terminus of P2X subunits as a critical determinant for the expression of slowly desensitizing P2X2 ATP-gated channels.

Results

Conclusion