Unravelling the intricate cooperativity of subunit gating in P2X2 ion channels.

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.

Relating ligand binding to activation gating in P2X2 receptors using a novel fluorescent ATP derivative.

Ionotropic purinergic receptors (P2X receptors) are non-specific cation channels that are activated by the binding of ATP at their extracellular side. P2X receptors contribute to multiple functions, including the generation of pain, inflammation, or synaptic transmission. The channels are trimers and structural information on several of their isoforms is available. In contrast, the cooperation of the subunits in the activation process is poorly understood. We synthesized a novel fluorescent ATP derivative, 2-[DY-547P1]-AET-ATP (fATP) to unravel the complex activation process in P2X2 and mutated P2X2 H319K channels with enhanced apparent affinity by characterizing the relation between ligand binding and activation gating. fATP is a full agonist with respect to ATP that reports the degree of binding by bright fluorescence. For quantifying the binding, a fast automated algorithm was employed on human embryonic kidney cell culture images. The concentrations of half maximum occupancy and activation as well as the respective Hill coefficients were determined. All Hill coefficients exceeded unity, even at an occupancy <10%, suggesting cooperativity of the binding even for the first and second binding step. fATP shows promise for continuative functional studies on other purinergic receptors and, beyond, any other ATP-binding proteins.