Experimental Characterization and Mathematical Modeling of P2X7 Receptor Channel Gating

Abstract

The P2X7 receptor is a trimeric channel with three binding sites for ATP, but how the occupancy of these sites affects gating is still not understood. Here we show that naïve receptors activated and deactivated monophasically at low and biphasically at higher agonist concentrations. Both phases of response were abolished by application of Az10606120, a P2X7R-specific antagonist. The slow secondary growth of current in the biphasic response coincided temporally with pore dilation. Repetitive stimulation with the same agonist concentration caused sensitization of receptors, which manifested as a progressive increase in the current amplitude, accompanied by a slower deactivation rate. Once a steady level of the secondary current was reached, responses at high agonist concentrations were no longer biphasic but monophasic. Sensitization of receptors was independent of Na+ and Ca2+ influx and about 30-min washout was needed to reestablish the initial gating properties. T15E- and T15K-P2X7 mutants showed increased sensitivity for agonists, responded with monophasic currents at all agonist concentrations, activated immediately with dilated pores, and deactivated slowly. The complex pattern of gating exhibited by wild-type channels can be accounted for by a Markov state model that includes negative cooperativity of agonist binding to unsensitized receptors caused by the occupancy of one or two binding sites, opening of the channel pore to a low conductance state when two sites are bound, and sensitization with pore dilation to a high conductance state when three sites are occupied.

Conserved ectodomain cysteines are essential for rat P2X7 receptor trafficking

“…The lack of I 2 response also indicates that these mutants do not dilate. In further contrast to WT receptor [38,50], SS1-SS3 double mutants did not show facilitation/sensitization of receptors during repetitive agonist application (data not shown). The SS4 and SS5 double mutants were practically nonfunctional in response to both agonists in 300-μM concentration (Fig.…”

“…Dose-response data points were fitted by a three-parameter logistic equation using a nonlinear curve-fitting program that derives the EC 50 and Hill coefficient values of the produced curves (SigmaPlot 2000 v9.01; SPSS Inc., Chicago, IL). The current amplitude was measured using the program CLAMP-FIT 9 (Axon Instruments).…”

“…If not otherwise stated, brief (2-10 s) applications of different concentrations of agonists were used to evoke inward currents, and one or two responses were recorded from one cell to prevent the sensitization of the receptor after repeated agonist applications [51]. To estimate the concentration producing 50% of the maximal response (EC 50 ) values, the responses from different cells were pooled, and dose response curves were constructed with mean values.…”

“…The maximum amplitude of current (I max ) was assessed following the application of 1-mM BzATP, and the EC 50 …”

“…Functional P2X7Rs are homotrimers [28] abundantly expressed in cells of immunological origin or function, in particular macrophages [43], mast cells [7], and microglia [49], as well as in some types of cancer [31,42]. The receptor exhibits low sensitivity to ATP, does not desensitize, and gradually develops permeability to organic cations, causing a sustained current growth accompanied by cell blebbing and death [1,12,32,45,50]. The C-terminus of P2X7R is longest among P2XRs and was suggested to account for many of these receptor-specific characteristics [2,41,45].…”