Intracellular calcium measurements as a method in studies on activity of purinergic P2X receptor channels

Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-4510 Submitted 29 January 2003 ; accepted in final form 22 April 2003 Extracellular nucleotide-activated purinergic receptors (P2XRs) are a family of cation-permeable channels that conduct small cations, including Ca 2+ , leading to the depolarization of cells and subsequent stimulation of voltage-gated Ca 2+ influx in excitable cells. Here, we studied the spatiotemporal characteristics of intracellular Ca 2+ signaling and its dependence on current signaling in excitable mouse immortalized gonadotropin-releasing hormone-secreting cells (GT1) and nonexcitable human embryonic kidney cells (HEK-293) cells expressing wild-type and chimeric P2XRs. In both cell types, P2XR generated depolarizing currents during the sustained ATP stimulation, which desensitized in order (from rapidly desensitizing to nondesensitizing): P2X 3 R > P2X 2b + X 4 R > P2X 2b R > P2X 2a + X 4 R > P2X 4 R > P2X 2a R > P2X 7 R. HEK-293 cells were not suitable for studies on P2XR-mediated Ca 2+ influx because of the coactivation of endogenously expressed Ca 2+ -mobilizing purinergic P2Y receptors. However, when expressed in GT1 cells, all wild-type and chimeric P2XRs responded to agonist binding with global Ca 2+ signals, which desensitized in the same order as current signals but in a significantly slower manner. The global distribution of Ca 2+ signals was present independently of the rate of current desensitization. The temporal characteristics of Ca 2+ signals were not affected by voltage-gated Ca 2+ influx and removal of extracellular sodium. Ca 2+ signals reflected well the receptor-specific EC 50 values for ATP and the extracellular Zn 2+ and pH sensitivities of P2XRs. These results indicate that intracellular Ca 2+ measurements are useful for characterizing the pharmacological properties and messenger functions of P2XRs, as well as the kinetics of channel activity, when the host cells do not express other members of purinergic receptors. ATP-gated receptor channels; inward currents; intracellular calcium signals; desensitization-inactivation; voltage-gated calcium influx; localized and global calcium signals Address for reprint requests and other correspondence: S. Stojilkovic, Section on Cellular Signaling, ERRB/NICHD/NIH, Bldg. 49, Rm. 6A-36, 49 Convent Drive, Bethesda, MD 20892-4510 (E-mail: stankos{at}helix.nih.gov ).