Abstract
P2X receptors for ATP are a family of ligand-gated cation channels. There are 11 conserved positive charges in the extracellular loop of P2X receptors. We have generated point mutants of these conserved residues (either Lys --> Arg, Lys --> Ala, Arg --> Lys, or Arg --> Ala) in the human P2X(1) receptor to determine their contribution to the binding of negatively charged ATP. ATP evoked concentration-dependent (EC(50) approximately 0.8 microm) desensitizing responses at wild-type (WT) P2X(1) receptors expressed in Xenopus oocytes. Suramin produced a parallel rightward shift in the concentration response curve with an estimated pK(B) of 6.7. Substitution of amino acids at positions Lys-53, Lys-190, Lys-215, Lys-325, Arg-202, Arg-305, and Arg-314 either had no effect or only a small change in ATP potency, time course, and/or suramin sensitivity. Modest changes in ATP potency were observed for mutants at K70R and R292K/A (20- and 100-fold decrease, respectively). Mutations at residues K68A and K309A reduced the potency of ATP by >1400-fold and prolonged the time course of the P2X(1) receptor current but had no effect on suramin antagonism. Lys-68, Lys-70, Arg-292, and Lys-309 are close to the predicted transmembrane domains of the receptor and suggest that the ATP binding pocket may form close to the channel vestibule.
Highlights
Summary of data for wild-type and mutant P2X1 receptors pEC50 is the Ϫlog10 of the ATP EC50 value. 50% time corresponds to the time taken from peak current to 50% decay of the current after an application of ATP at EC90
Point mutations of conserved positively charged amino acids in the extracellular loop of the P2X1 receptor have indicated that residues Lys-68, Lys-70, Lys-309, and Arg-292 contribute to the binding of ATP
Mutation of these residues prolonged the time course of the P2X1 receptor-mediated current, indicating that they may have some effect on the kinetics of channel opening
Summary
Cloning and Mutagenesis of the Human P2X1 Receptor—Oligonucleotide primers designed to amplify positions 43–1631 of the published human P2X1 cDNA sequence [23] (EMBL A47363) were utilized for reverse transcription-PCR1 on a surgical sample of human bladder. After three 5-min washes in TBST, visualization of the protein bands was achieved with an ECL (Plus) kit (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. In order to confirm that synthesized mutant receptors had been processed correctly and transported to the cell membrane of the oocyte, biotinylation of all surface proteins with Sulfo-NHS-LC-Biotin (Pierce) was used. This compound is impermeable to the cell membrane and, in intact cells, will only biotinylate proteins present on the cell surface. Oocytes were washed to remove contaminating proteins and incubated in 0.5 mg/ml Sulfo-NHS-LC-Biotin in ND96 buffer for 30 min. After washing extensively in TBST, biotinylated protein bands on the membrane were visualized with ECL (Plus) (Amersham Pharmacia Biotech)
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