Kinetics of P2X7 Receptor-Operated Single Channels Currents

Abstract

Human P2X7 receptors were expressed in Xenopus laevis oocytes and single channels were recorded using the patch-clamp technique in the outside-out configuration. ATP4- evoked two types of P2X7 receptor-mediated single channel currents characterized by short-lived and long-lived openings. The short- and long-lasting open states had mean open times of approximately 5 and approximately 20 ms and slope conductances near -60 mV of 9 and 13 pS, respectively. The open probabilities of the short and long openings were strongly [ATP4-]-dependent with EC50 values of approximately 0.3 mM and approximately 0.1 mM ATP4-, respectively. The channel kinetics did not change significantly during sustained P2X7 receptor activation for several minutes, as was also observed in recordings in the cell-attached patch-clamp configuration. Activation and deactivation of the short openings followed exponential time courses with time constants in the range of 20 ms, and displayed a shallow [ATP4-] dependence of the activation process. The kinetics of the short channel openings at negative membrane potentials fitted well to a linear C-C-C-O model with two ATP4- binding steps at equal binding sites with a dissociation constant Kd of 139 microM.

Localization of the gate and selectivity filter of the full-length P2X7 receptor

“…In contrast, in extended single-channel recordings, we were unable to detect a single-channel correlate of pore dilation (17,18). An unexpected explanation for the discrepancy between macroscopic and microscopic currents came from the seminal demonstration that V rev shifts may reflect not pore dilatation but rather profound changes in the concentration of intracellular ions associated with prolonged P2XR activation (19).…”

“…We combined membrane current recordings in the whole-cell mode and in various excised patch clamp configurations by probing with various cysteine-reactive methanethiosulfonate (MTS) reagents to assess the accessibility of cysteine residues that were substituted in the TM2-lined channel from both sides of the membrane. We determined the single-channel properties of the MTS-responsive mutants to clarify the molecular mechanisms underlying the macroscopic channel modifications (17). In addition, we biochemically assessed the accessibility of substituted cysteine residues to a bulky cysteinereactive fluorescent maleimide dye to identify cysteine residues that are accessible but not functionally responsive to MTS reagents.…”

Interaction of Purinergic P2X4 and P2X7 Receptor Subunits

“…It was calculated that non-specific bystander effects occur at a fluorescence molecule density of ≥2000 molecules/µm 2 (Clayton and Chattopadhyay, 2014). Given a surface area A of Xenopus oocytes of about 2 × 10 7 µm 2 (Lin-Moshier and Marchant, 2013), a single channel current amplitude i at −40 mV of ∼0.4 pA, an open probability P o (1 mM ATP) of about 0.2 (Riedel et al, 2007), and a maximal P2X7-mediated wholecell current I of 2000 nA (Figure 4), the molecule density D = I/(iPoA) = 1.25 molecules/µm 2 for fluorescence-labeled P2X7 receptors, or 3.75 P2X7 subunits/µm 2 , far higher than the critical expression density for bystander FRET. Furthermore, the lack of FRET signals for our negative controls argue against non-specific FRET.…”

The P2X7 Receptor Channel: Recent Developments and the Use of P2X7 Antagonists in Models of Disease

“…Single channel recordings of P2X7 channels have also been used to investigate how this ion channel operates. Markwardt and colleagues measured a single channel conductance of 9-13 pS for P2X7 in Xenopus laevis oocytes (Riedel et al, 2007a) and have developed extensive kinetic models for the transitions between open and closed states. These kinetic models were recently extended to a 16-state Markov model, taking into account both low-high affinity agonist binding and sensitization/desensitization states (Khadra et al, 2013).…”