BzATP triethylammonium salt

Amino acid residues in the P2X7 receptor that mediate differential sensitivity to ATP and BzATP.[Pubmed:17032903]

Mol Pharmacol. 2007 Jan;71(1):92-100.

Agonist properties of the P2X7 receptor (P2X7R) differ strikingly from other P2X receptors in two main ways: high concentrations of ATP (> 100 microM) are required to activate the receptor, and the ATP analog 2',3'-O-(4-benzoyl-benzoyl)ATP (BzATP) is both more potent than ATP and evokes a higher maximum current. However, there are striking species differences in these properties. We sought to exploit the large differences in ATP and BzATP responses between rat and mouse P2X7R to delineate regions or specific residues that may be responsible for the unique actions of these agonists at the P2X7R. We measured membrane currents in response to ATP and BzATP at wild-type rat and mouse P2X7R, at chimeric P2X7Rs, and at mouse P2X7Rs bearing point mutations. Wild-type rat P2X7R was 10 times more sensitive to ATP and 100 times more sensitive to BzATP than wild-type mouse P2X7R. We found that agonist EC50 values were determined solely by the ectodomain of the P2X7R. Two segments (residues 115-136 and 282-288), when transposed together, converted mouse sensitivities to those of rat. Point mutations through these regions revealed a single residue, asparagine284, in the rat P2X7R that fully accounted for the 10-fold difference in ATP sensitivity, whereas the 100-fold difference in BzATP sensitivity required the transfer of both Lys127 and Asn284 from rat to mouse. Thus, single amino acid differences between species can account for large changes in agonist effectiveness and differentiate between the two widely used agonists at P2X7 receptors.

Serum constituents can affect 2'-& 3'-O-(4-benzoylbenzoyl)-ATP potency at P2X(7) receptors.[Pubmed:11264244]

Br J Pharmacol. 2001 Apr;132(7):1501-8.

1. 2'-& 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) is the prototypic agonist for P2X(7) receptors. In this study we demonstrate that bovine serum albumin (BSA) can affect the potency of BzATP at P2X receptors. 2. BzATP potency (pEC(50)) to stimulate ethidium accumulation in cells expressing recombinant P2X7 receptors varied between 6.5 and 4, depending upon the species orthologue studied and ionic conditions employed. BSA (0.1 - 1 mg ml(-1)) and foetal bovine serum (FBS, 1 - 10% v v(-1)) inhibited responses to BzATP but only when the BzATP pEC(50) exceeded 5. 3. BSA did not block ATP-stimulated ethidium accumulation, suggesting its effects were independent of P2X(7) receptor blockade. 4. BSA did not cause breakdown of nucleotides, although FBS (10% v v(-1)) exhibited appreciable nucleotidase activity and caused significant breakdown of ATP. 5. In the presence of BSA, lipids such as 11-((5-dimethylaminonaphthalene-1-sulphonyl)amino)undecanoic acid (DAUDA) and arachidonic acid (AA) markedly increased BzATP potency. Lipids had no affect on ATP potency in the presence of BSA and had little effect on responses to BzATP in the absence of BSA. 6. These results suggested that the reduction in BzATP potency by BSA was due to BzATP binding to BSA and that lipids prevented this binding. Consistent with this hypothesis, BzATP inhibited binding of the fluorescent lipid, DAUDA, to BSA. 7. In conclusion, BSA and lipids can markedly affect BzATP potency at P2X(7) receptors but this is probably a consequence of BzATP binding to BSA. This finding has important implications when using BzATP in vivo or in the presence of albumin.

Pharmacological and molecular characterization of P2X receptors in rat pelvic ganglion neurons.[Pubmed:9831914]

Br J Pharmacol. 1998 Oct;125(4):771-81.

1. The presence and characteristics of P2X receptors on neurons of the rat major pelvic ganglia (MPG) have been studied using whole cell voltage-clamp, in situ hybridization and immunohistochemistry. 2. Rapid application of ATP (100 microM) to isolated rat MPG neurons induced moderately large inward currents (0.33-5.3 nA) in 39% of cells (108/277). The response to ATP occurred very rapidly, with an increase in membrane conductance, and desensitized slowly. 3. The concentration-response curve for ATP yielded an EC50 of 58.9 microM. The agonist profile was ATP> or =2MeSATP=ATPgammaS>BzATP, while alpha,beta-MeATP, beta,gamma-MeATP, UTP and ADP were all inactive at concentrations up to 100 microM. 4. The response to ATP was antagonized by suramin (pA2=5.6), reactive blue-2 (IC50=0.7 microM) and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). 5. Lowering the pH from 7.4 to 6.8 produced a marked potentiation (to 339% of control) of the responses to ATP (30 microM), while raising the pH to 8.0 attenuated the responses (to 20% of control). The EC50s for ATP were 28.8, 58.9 and 264 microM at pH 6.8, 7.4 and 8.0, respectively. 6. Co-application of ATP with Zn2+ produced a marked enhancement of the responses to ATP, with an EC50 of 9.55 microM. In the presence of Zn2+ (30 microM), the EC50 for ATP was decreased to 4.57 microM. 7. In situ hybridization revealed that the P2X receptor transcripts levels in rat MPG neurons are P2X2>P2X4>P2X1, P2X3, P2X5 and P2X6. The immunohistochemical staining revealed a small number of neurons with strong P2X2 immunoreactivity. 8. In conclusion, our results indicate that there are P2X receptors present on MPG neurons. The pharmacological characteristics of these receptors, the in situ hybridization and immunohistochemical evidence are consistent with them being of the P2X2 subtype, or heteromultimers. with P2X2 being the dominant component.