Direct observation of ATP-induced conformational changes in single P2X ₄ receptors

The ATP-gated P2X₄ receptor is a cation channel, which is important in various pathophysiological events. The architecture of the P2X ₄ 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.