Molecular and electrophysiological characterization of P2X receptors in human monocytes

Abstract

"In order to characterize the general properties and the presence of P2X receptors in single human monocytes we used RT-PCR, flow cytometry, patch-clamp and the two-electrode voltage-clamp techniques. A high percentage of monocytes expressed the canonical P2X1, its splicing variant P2X1del and P2X4 mRNAs. P2X7 transcript was found in a minimal fraction of cells. P2X1 receptor immunoreactivity was observed in most monocytes but lower immunoreactivity was found for P2X4 and P2X7. Our data suggest heterogeneous expression of P2X receptors in monocytes and is supported by the electrophysiological data obtained. Currents mediated by P2X1 (EC50=1.9±0.8 μM) and P2X1del (EC50 >1000 μM) channels, expressed in Xenopus leavis oocytes, have different ATP sensitivity and kinetics. Both currents mediated by P2X1 and P2X1del channels kept increasing during the continuous presence of high ATP concentrations. Currents mediated by the native P2X1 receptors in human monocytes showed an EC50=6.3±0.2 μM. Currents have kinetics that resemble those observed for P2X1 and P2X1del receptors in oocytes. Co-expression of both receptors showed an EC50= 4.65±1.34 μM and different properties to the observed for each receptor expressed alone. This study is the first to demonstrate heterogeneous expression of P2X receptors in human monocytes and their distribution among monocyte subpopulations classified by their expression of CD14 and CD16. Our study is the first in demonstrate the expression of P2X1 receptor and its splicing variant P2X1del in most human monocytes. We also, for the first time, described functional homomeric P2X1del channels and showed that currents mediated by P2X1 or P2X1del increased in amplitude when activated with high ATP concentrations in a similar fashion to those channels that increase their conductance under similar conditions, such as P2X7, P2X2, and P2X4 channels."

"Usando técnicas de electrofisiología y biología molecular se analizó la expresión de los receptores P2X en monocitos humanos. Se encontró el mRNA de la subunidad P2X1 y su variante de splicing llamada P2X1del, así como del receptor P2X4 en un alto porcentaje de monocitos, en cambio el receptor P2X7 se encontró en un porcentaje mínimo. El análisis en células únicas y electrofisiológico sugiere la expresión heterogénea de subunidades P2X en los monocitos. Las corrientes mediadas por los canales P2X1 (EC50=1.9±0.8 µM) Y P2X1del (EC50 >1000 µM) expresados en ovocitos de Xenopus laevis presentaron diferente cinética y sensibilidad al ATP. Ambas corrientes mediadas por estos receptores mostraron incremento en la amplitud de la corriente bajo un estimulo prolongado con altas concentraciones de ATP. La activación de receptores P2X1 nativos en monocitos humanos mostró una EC50=6.3±0.2 µM. Estas corrientes presentaron características similares a las observadas cuando se expreso a los receptores P2X1 y P2X1del en ovocitos. La co-expresión del P2X1 y P2X1del mostro una EC50=4.5±1.34 µM y propiedades diferentes a las observadas en ovocitos que expresaron solo a uno de los receptores. Nuestro estudio muestra por primera vez la expresión heterogénea de los receptores P2X y su distribución entre las sub- poblaciones de monocitos humanos, la formación de canales funcionales al expresar al receptor P2X1del, y el incremento en la amplitud de corriente en los receptores P2X1y P2X1del homoméricos cuando se estimulan con concentraciones altas de ATP por tiempos prolongados, de manera similar a los canales P2X7, P2X2 y P2X4."