APPS November 2002 Meeting Abstract 2439

Megan Smart¹, Rekha Panchal¹, Ben Gu², James Wiley², Brett Cromer³, David Williams¹, Steven Petrou¹, 1 Department of Physiology, The University of Melbourne, Vic., 2 Department of Medicine, The University of Sydney, NSW, 3 St Vincent's Medical Institute, Carlton, Vic.

The cytolytic P2X7 purinoceptor (595 amino acids in length) is an ATP-gated channel that can undergo conversion to a non-selective pore^1,2. The cytosolic C-terminal tail of the P2X7R is thought to modulate this function. The present study aimed to characterise functional domains within this C-terminal tail. A structure-function analysis of truncated P2X7 receptors and site-directed mutants was performed in HEK-293 cells and Xenopus oocytes. Cells expressing receptors truncated at residue 581 (of 595) have negligible ethidium ion uptake while those expressing the P2X7R truncated at position 582 gave wild type ethidium ion uptake suggesting that pore formation requires over 95% of the C-terminal tail. Channel function was evident even in receptors that were truncated at position 380 indicating that only a small portion of the C-terminal tail is required for channel activity. Surprisingly, a truncation in the region between residues 551-581 resulted in non-functional receptors with no detectable cell surface expression in HEK-293 cells. A more detailed analysis revealed that mutations of single residues could totally abolish both receptor function and cell surface expression of the full length P2X7R suggesting that this region may participate in regulating the membrane trafficking of the P2X7R. While truncation at residues proximal to 550 restored channel function, the pore property was lost indicating that pore formation and membrane trafficking regions are inseparable.