Characterization of Ca²⁺ influx through recombinant P2X receptor in C6BU‐1 cells

Abstract

1 The e ects of exogenous adenosine 5'-triphosphate (ATP) and a,b-methylene ATP (a,bmeATP) on C6BU-1 cells transfected with P2X 2 and P2X 3 subtypes, separately or together (P2X 2+3 ), were investigated using fura-2¯uorescence recording and whole-cell patch clamp recording methods. 2 Untransfected C6BU-1 cells showed no intracellular Ca 2+ ([Ca 2+ ] i ) increase in response to depolarizing stimulation with high K + or stimulation with ATP. There was no current induced by ATP under voltage clamp conditions in untransfected C6BU-1 cells. ATP caused Ca 2+ in¯ux only from extracellular sources in C6BU-1 cells transfected with the P2X subtypes, suggesting that the C6BU-1 cell line is suitable for the characterization of Ca 2+ in¯ux through the P2X subtypes. 3 In C6BU-1 cells transfected with the P2X 2 subtype, ATP (more than 10 mM) but not a,bmeATP (up to 100 mM) evoked a rise in [Ca 2+ ] i . 4 In the cells transfected with the P2X 3 subtype, current responses under voltage clamp conditions were observed at ATP concentrations higher than 0.1 mM of a,bmeATP were required. This discrepancy in the concentration dependence of the agonist responses with respect to the [Ca 2+ ] i rise and the current response was seen only with the P2X 3 subtype. In addition, the agonist-induced rise in [Ca 2+ ] i was observed only after the ®rst application because of desensitization of this subtype. 5 In C6BU-1 cells co-transfected with P2X 2 and P2X 3 , ATP at 1 mM evoked a [Ca 2+ ] i rise. This responsiveness was higher than that of the other subtype combinations tested. The e ciency of expression was improved by co-transfection with P2X 2 and P2X 3 , when compared to transfection with the P2X 3 subtype alone. The desensitization of the P2X 2+3 was apparently slower than that of the P2X 3 subtype alone. Therefore, this combination could respond to the repeated application of agonists each time with a [Ca 2+ ] i rise. 6 These results suggest that the P2X 2 and P2X 3 subtypes assemble a heteromultimer and that this heterogeneous expression acquires more e ective Ca 2+ dynamics than that by homogenously expressed P2X 2 or P2X 3

P2X and NMDA receptor involvement in temporomandibular joint-evoked reflex activity in rat jaw muscles

“…However, the inability of TNP-ATP to antagonize the effects of the higher dose of α,β-me-ATP raises the possibility that the higher dose of α,β-me-ATP could have evoked non-specific pharmacological effects. 4) two consecutive TMJ injections of α,β-me-ATP produced a rapid desensitizing effect, which is a well-documented feature of ATP and its agonists in the case of P2X 1 and P2X 3 receptors but not heteromeric P2X 2/3 receptor subtypes (Bland-Ward and Humphrey, 1997; Lewis et al 1995; Ueno et al 1998, 1999; see Burnstock, 2006; North and Surprenant, 2000). .…”

Characterization of Multiple P2X Receptors in Cultured Normal Human Epidermal Keratinocytes

“…P2X 2 and P2X 4-7 receptors are characterized by insensitivity to ab-meATP and a slowly desensitizing current. Although homomeric P2X 2 receptors are insensitive to ab-meATP, heteromeric P2X 2/3 receptors are characterized by their sensitivity to ab-meATP and a slowly desensitizing current (Lewis et al, 1995;Ueno et al, 1998). Additionally, the responses of the currents are categorized by their sensitivity to the P2X antagonist PPADS.…”

A Protein Kinase C Site Highly Conserved in P2X Subunits Controls the Desensitization Kinetics of P2X2 ATP-gated Channels

“…Modulation of the desensitization rate of neurotransmitter-gated channels is recognized as a potentially important mechanism for modulation of neuronal excitability (14). P2X receptors are nonselective cation channels with high permeability to calcium ions (15,16), so the subtypespecific desensitization phenotype of ATP-mediated currents has a significant impact on the levels of intracellular calcium and subsequent activation of calcium-dependent effectors. Studies on the relationship between the slowly desensitization kinetics of P2X 2 receptor and its primary sequence have emphasized the requirement for several structural features including the transmembrane domains and their intracellular flanking regions (17), the negatively charged residue Asp 349 located in the second transmembrane domain (18), and the unique C-terminal tail of the P2X 2A splicing variant (19 -21).…”