RT Journal Article
SR Electronic
T1 Activation of Trimeric P2X2 Receptors by Fewer than Three ATP Molecules
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 760
OP 766
DO 10.1124/mol.112.080903
VO 82
IS 4
A1 Olga Stelmashenko
A1 Ulyana Lalo
A1 Yue Yang
A1 Laricia Bragg
A1 R. Alan North
A1 Vincent Compan
YR 2012
UL http://molpharm.aspetjournals.org/content/82/4/760.abstract
AB P2X receptors are trimeric membrane proteins. When they bind extracellular ATP, a conformational change occurs that opens a transmembrane ion channel. The ATP-binding pocket is formed in a cleft between two subunits, and a critical amino acid residue for ATP contact is Lys69 (P2X2 numbering). In the present work, we sought to determine whether the binding of fewer than three ATP molecules could open the ion channel. We expressed eight concatenated cDNAs in human embryonic kidney cells, which encoded three serially joined, epitope-tagged, subunits with either Lys or Ala at position 69 (denoted as KKK, KKA, KAK, AKK, KAA, AKA, AAK, and AAA). Western blotting of surface-biotinylated proteins indicated that breakdown of concatemers to individual subunits was minimal. Recording of membrane currents in response to ATP (whole cell and excised outside-out patch) showed that all formed functional channels except AAK, AKA, and AAA. There was no difference in the kinetics of activation and deactivation among KKK, KKA, KAK, and AKK channels, and amplitude of the unitary conductances was in all cases not different from that found after expression of a single wild-type subunit. Currents through KKA and KAK receptors were larger than those observed for AKK receptors. The results indicate that trimeric P2X receptors containing only two intact binding sites can be readily activated by ATP.