The potential energy calculations on resting and open states of zfP2X4 receptors revealed that AR1induced allosteric changes led to energy releases during conformational transition between the resting and open states. In contrast, MD simulations together with PCA analysis showed that AR2 and 3 can only induce outward and upward movements of head domain, respectively. Therefore, AR2 and AR3 would not trigger downstream allosteric changes. These observations provided a conceivable explanation for why only AR1 rather than AR2 and 3 is able to trigger channel activation of P2X4 receptors. The DGbind together with the downstream allosteric changes-induced energy releases may enable AR1 to efficiently overcome the energy barrier for the channel gating. Meanwhile, based on the observation that conformational fluctuations of head domain, especially the downward motion, greatly enhance the preference of ABP for AR1, we proposed that the downward motion of head domain facilitated by AR1 may further increase the preference of ABP for AR1. It looks like an induced-fit/positive feedback mechanism when AR1 occurs. Taken together, the contact between ATP and site S1 promoted downward motion of head domain correlated well with conformational changes of ABP and subsequently increased preference of ABP for AR1. Once ATP comes in contact with site S1, it will initiate an ‘induced-fit’/positive-feedback mechanism, with the increased free energy release and the downstream allosteric changes-induced energy releases acting as the driving force. In contrast, AR2 and 3 are not capable of facilitating the downward motion of head domain and therefore, would not evoke such positive feedback. The comparisons between closed and open structures, and the equilibrated averaged structure after MD simulations would not only facilitate studying ligand-recognition of P2X4 receptors, but also will provide some clues to understanding of channel gating mechanism. The movements observed in MD simulations exhibited high a similarity to bound-ATP induced conformational changes of ABP in open crystal structure. For instance, the structural rearrangements of K70, K68, R289 and R143, and closing movements of loop139-146 and loop169-183. However, the rightward movement of loop169-183 and the down movement of head domain during MD simulations were more evident than those of ATP-bound open structure. On the contrary, the upward movement of head domain in MD data was not as obvious as that of open structure. These observations confirmed the crucial role of inherent dynamics of head domain in both AR and channel gating of P2X4 receptors. However, the complex dynamics in the process of channel gating well beyond the inherent dynamics of head domain, during which bound-ATP evoked coordinated movements of multiple domains, especially for the head and dorsal fin domains, are crucial for this process.