Heteromultimeric P2X_1/2 Receptors Show a Novel Sensitivity to Extracellular pH

Abstract

Rat P2X 1 and P2X 2 subunits were coexpressed in defolliculated Xenopus oocytes and the resultant P2X receptors studied under voltage-clamp conditions. Extracellular ATP elicited biphasic inward currents, involving an initial rapidly inactivating (P2X 1 -like) component and a later slowly inactivating (P2X 2 -like) component. The maximum amplitude of P2X 1 -like ATP responses was increased in some cells by lowering extracellular pH (from 7.5 to 6.5), whereas P2X 2 -like responses and those of homomeric rP2X 1 and rP2X 2 receptors were not changed by this treatment. Concentration-response (C/R) curves for ATP for pH-enhanced P2X 1 -like responses were biphasic, and clearly distinct from monophasic ATP C/R curves for homomeric rP2X 1 and rP2X 2 receptors. Under acidic (pH 5.5 and 6.5) and alkaline (pH 8.5) conditions, ATP C/R curves for P2X 1 -like responses showed increases in agonist potency and efficacy, compared with data at pH 7.5, but the same was not true of homomeric rP2X 1 and rP2X 2 receptors. ATP C/R

Mouse Leydig cells express multiple P2X receptor subunits

“…The expression of purinergic receptors P2X [1][2][3][4][5][6][7] in Leydig cells was detected by Western blot analysis. Since a large number of high purity Leydig cells are needed for these experiments, we used 20 testes in each preparation.…”

“…Proteins were then transferred to nitrocellulose membranes (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA) by Bio-Rad Trans-Blot SD semidry transfer cell (Bio-Rad Laboratories Inc., Richmond, CA, USA). Membranes were blocked with 5% BSA in TBST (100 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% Tween 20) for 1 h, and then incubated for 1 h with anti-P2X [1][2][3][4][5][6][7] (1:400) polyclonal antibodies (Alomone Labs Ltd., Jerusalem, Israel) diluted in 5% BSA in TBST. After washing, membranes were incubated for 1 h with goat anti-rabbit IGg conjugated to horseradish peroxidase, diluted in TBST.…”

“…The coverslips were incubated 120 min with the same primary antibodies anti-P2X [1][2][3][4][5][6][7] (1:200; rabbit) and anti-calreticulin (Chemicon International, Temecula, California, USA; 1:200; chicken) diluted with 0.01 M PBS, 0.5% Triton X-100, 1% BSA. Then, the cells were incubated with secondary antibodies Alexa Fluor 488 (goat, anti-rabbit) and 594 (goat, anti-chicken; Molecular Probes Inc., Eugene, USA) for 60 min, both in a dilution of 1:800.…”

“…Seven P2X receptor subunits (P2X 1-7 ) were cloned and characterized in vertebrates, and each of them can form two type of assemblies, homomeric, P2X 1 [50], P2X 2 [3], P2X 3 [8], P2X 4 [2], P2X 5 [18], P2X 7 [46], and P2X 6 [23], or heterotrimeric P2X 2/3 [31], P2X 1/5 [48], P2X 4/6 [30], P2X 2/6 [27], P2X 1/2 [5], P2X 1/4 [38], and P2X 4/7 [19]. Each subunit is composed by two transmem-brane regions (TM1 and TM2) forming the channel pore, a large extracellular domain, which contains the ATP binding site and both N and C termini located intracellularly [14,48,22].…”

Northern ring conformation of methanocarba‐adenosine 5′‐triphosphate required for activation of P2X receptors

“…These subunits can combine to form either homo-or hetero-multimeric receptors. So far, eleven functional P2X receptor multimers have been identified [North, 2002;Brown et al, 2002], but with each functional ion channel consisting of at least 3 subunits, there could be more.…”