Deduction of the PAC channel states upon proton and/or C77304 binding.

√: site occupied; ×: site unoccupied; C: pore closed; O: pore opening; C0, C1, C2, C3, C4, and C5 represent different closed states of the PAC channel, and O1 and O2 represent 2 open states. The proton binding site on the PAC channel was defined as site 0, and the 2 C77304 binding sites on it were denoted as site 1 and site 2, which mediate the activation and inhibition effect of the compound, respectively. There are 8 possible combinations of the occupancy situations of site 0, site 1, and site 2 by proton and/or the compound (ranging from no site being occupied to all 3 sites being occupied). Taking into consideration the 2 pore states (open or closed), there exist theoretically 16 different channel states. Several experimentally determined constraints reduced the channel states: (i) C77304 binding to site 1 alone cannot drive the channel opening; (ii) C77304 binding to site 2 requires the channel in its open state, which means site 0 must be bound with protons if site 2 was to be occupied; (iii) the channel would be trapped in the closed state as long as site 2 is bound with C77304; and (iv) the pore of the PAC channel is highly unlikely to be opened without protons binding with site 0. These constraints excluded 8 possible channel states in this table (highlighted with shadows) and only 8 channel states are therefore practically achievable.
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