Direct Observation of ATP-Induced Conformational Changes in Single P2X4 Receptors

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Abstract
The ATP-gated P2X4 receptor is a cation channel, which is important in various pathophysiological events. The architecture of the P2X4 receptor in the activated state and how to change its structure in response to ATP binding are not fully understood. Here, we analyze the architecture and ATP-induced structural changes in P2X4 receptors using fast-scanning atomic force microscopy (AFM). AFM images of the membrane-dissociated and membrane-inserted forms of P2X4 receptors and a functional analysis revealed that P2X4 receptors have an upward orientation on mica but lean to one side. Time-lapse imaging of the ATP-induced structural changes in P2X4 receptors revealed two different forms of activated structures under 0 Ca2+ conditions, namely a trimer structure and a pore dilation-like tripartite structure. A dye uptake measurement demonstrated that ATP-activated P2X4 receptors display pore dilation in the absence of Ca2+. With Ca2+, the P2X4 receptors exhibited only a disengaged trimer and no dye uptake was observed. Thus our data provide a new insight into ATP-induced structural changes in P2X4 receptors that correlate with pore dynamics. ATP is not only a source of intracellular energy but can act as an intercellular signal by binding membrane receptors. Purinergic receptors, which bind with nucleotides including ATP are known as P2 receptors and are divided into two types: ion channel-type P2X receptors and metabotropic-type P2Y receptors. P2X receptors are thought to undergo conformational changes in response to ATP binding, leading to the opening of transmembrane channels, through which cations enter the cells. A growing body of evidence shows that P2X receptors control various physiological and pathophysiological cellular responses. However, the receptor structure and the conformational changes it experiences upon stimulation remained to be clarified. Here, we employed an atomic force microscope (AFM) to observe P2X receptor behavior at the single channel level. We chose to analyze the P2X4 receptor, because it is known to increase the transmembrane pore size (i.e., pore dilation) in the absence of extracellular calcium. Activated P2X4 receptor exhibited a trimeric topology with a pore-like structure in the center. When calcium was present the receptor exhibited a trimer without a pore structure at its center. These structural changes corresponded well with the changes of ion permeability of P2X4 receptor.