Received for publication October 13, 2005.
Revised February 7, 2006.
Accepted for publication February 8, 2006.

Carboxy-terminal Splicing Enhances Physical Interactions between the Cytoplasmic Tails of Purinergic P2X Receptors

Abstract

Purinergic P2X receptors are ion-conducting channels composed of three subunits, each having two transmembrane domains and intracellular amino (N) and carboxyl (C) termini. Although alternative splicing extensively modifies the C-terminal sequences of P2X subunits, the direct influence of such post- transcriptional modifications on receptor architecture and function remains poorly understood. Here we focused on mouse pituitary P2X₂ receptors. In this tissue, progressive splicing of the P2X_2a C- terminus generated two functional subunit variants, P2X_2b and P2X_2e, which exhibited accelerated desensitization rates and attenuated calcium signals when the receptors were in homomeric states. To measure the inter-subunit interaction in living cells, the efficient transfer of bioluminescent resonance energy between luciferase and fluorescent proteins attached to the N- or C-subunit termini of these subunits was used. The constitutive interactions between the full-length C-termini of P2X_2a receptor were detected by a significant increase in fluorescence/luminescence intensity ratio compared with negative controls. Moreover, interactions between C- termini and between C- and N-termini of adjacent subunits were significantly enhanced in homomeric and heteromeric receptors containing P2X_2b or P2X_2e subunits. Finally, deletion of two amino acids at the splicing junction, but not at the C- terminal end of the P2X_2b receptor, resulted in the enhancement of channel desensitization and luminescence resonance energy transfer. These results indicate that C-terminal structure plays a critical role in the cytoplasmic inter-subunit interactions and suggest that the extent of subunit interactions prior to ATP application could contribute to the subsequent channel activity and conformation changes associated with agonist-dependent desensitization.

Key words: Purinergic, Ion channel regulation, Ca imaging, Fluorescence techniques

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