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Received for publication May 17, 2006.
Revised July 13, 2006.
Accepted for publication July 13, 2006.
Lysine residues near each end of the receptor ectodomain (in rat P2X2 Lys69 and Lys308) have been implicated in ATP binding to P2X receptors. We recorded membrane currents from human embryonic kidney cells expressing P2X subunits, and found that lysine to alanine substitutions at equivalent positions in the P2X3 receptor (Lys63 and Lys299) also prevented channel function. Heteromeric P2X2/3 receptors are formed when P2X2 and P2X3 subunits are expressed together; they can be distinguished by their relatively sustained response to 
-methylene-ATP. By co-expression of wild type P2X3 and mutated P2X2 subunit, we found that the heteromeric P2X2/3 channel functioned normally when either lysine in the P2X2 subunit was mutated to alanine (i.e. [K69A] or [K308A]), but not when both lysines were mutated to alanine (i.e. [K69A, K308A]). However, co-expression of wild type P2X2 with a mutated P2X3 subunit ([K68A] or [K299A]) produced no functional heteromers. The rescue of the single lysine mutant P2X2 subunit by wild type P2X3 (but not the converse) suggests that the heteromeric channel contains one P2X2 and two P2X3 subunits, and that the receptor functions essentially normally so long as two subunits are not mutated. The failure to rescue function in the P2X2 subunit with both lysines mutated by wild type P2X3 suggests that these residues from two different subunits interact in agonist binding or channel opening.
Key words:
Purinergic, Purinergic, Func. analysis receptor/ion channel mutants
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