Unravelling the intricate cooperativity of subunit gating in P2X2 ion channels

Christian Sattler,Klaus Benndorf,Christopher Unzeitig,Thomas Eick,Andrea Schweinitz,Sabine Hummert,Eckhard Schulz,Ralf Schmauder,Frank Schwede

doi:10.1038/s41598-020-78672-w

Abstract

Ionotropic purinergic (P2X) receptors are trimeric channels that are activated by the binding of ATP. They are involved in multiple physiological functions, including synaptic transmission, pain and inflammation. The mechanism of activation is still elusive. Here we kinetically unraveled and quantified subunit activation in P2X2 receptors by an extensive global fit approach with four complex and intimately coupled kinetic schemes to currents obtained from wild type and mutated receptors using ATP and its fluorescent derivative 2-[DY-547P1]-AET-ATP (fATP). We show that the steep concentration-activation relationship in wild type channels is caused by a subunit flip reaction with strong positive cooperativity, overbalancing a pronounced negative cooperativity for the three ATP binding steps, that the net probability fluxes in the model generate a marked hysteresis in the activation-deactivation cycle, and that the predicted fATP binding matches the binding measured by fluorescence. Our results shed light into the intricate activation process of P2X channels.

Highlights

Ionotropic purinergic (P2X) receptors are trimeric channels that are activated by the binding of ATP
The crystal structure of the zebrafish P2X4 channel has proven a trimeric architecture and revealed that the shape of a single subunit resembles that of a dolphin[6,7], which was later confirmed for P2X3 receptors[8]
All recordings subjected to the global fit analysis were performed in whole HEK293 cells at − 50 mV to stay in the physiological range of voltages and to obtain currents of reasonable amplitude

Summary

Introduction

Ionotropic purinergic (P2X) receptors are trimeric channels that are activated by the binding of ATP. ATP is bound to binding sites between two neighbored subunits, distant by as much as 40 Å from the extracellular boundary of the transmembrane domain, and cradled by the dolphin head, upper body, lower body, left flipper and dorsal fin. It is presently not fully clear how the signal of ligand binding propagates and opens the channel pore. We extended these analyses by combining electrophysiological time-dependent data with the corresponding orthogonal data of ligand binding which enabled us to specify kinetic schemes in considerable detail and to identify complex types of cooperativity among the subunits in CNGA224,25 and HCN2 channels[26]

Methods

Results

Conclusion