(A) Sub-scheme describing calcium and voltage allosteric interaction for closed channels. The VSD transit between two resting (R) and active (A) configuration governed by the equilibrium constant , where each Ca2+ site undergoes unbound (U) - Ca2+ bound (B) transitions governed by the equilibrium constant . The allosteric factor accounts for the coupling between the calcium and voltage sensors (CTD-VSD). (B–C) VSD kinetic models in the presence of Ca2+ according to CTD-VSD interaction schemes I and II (Figure 3A–B), respectively, where the vertical transitions (R-A) represent the VSD movement and the horizontal transitions (R-R and A-A) are Ca2+-binding reactions when the VSD is in the resting or active conformation. For Scheme I (B), each VSD can undergo R0-A0 or R1-A1 transitions depending on the unbound or bound state of the Ca2+ site in the α-subunit, respectively. Thus, the R1-A1 equilibrium is defined by increased -fold (). For Scheme II (C), the R0-A0 to R4-A4 transitions represent the VSD equilibrium with 0, 1, 2, 3, and four occupied Ca2+ sites in the channel. Thus, for each Ca2+ bound the equilibrium constant increase -fold reaching to when the four Ca2+ sites are occupied. The thickness of the arrows indicates the probability of transitions. (D) General sub-scheme of the CTD-VSD interaction including two Ca2+ sites for each CTD (RCK1 and RCK2 sites). For each RCK1 and RCK2 site the unbound-Ca2+ bound transitions are governed by the equilibrium constants and . The factor describes the cooperativity between the sites within the same α-subunit; and the and factors define the allosteric coupling between the RCK1 and RCK2 sites and the VSD, respectively. (E) Schematic representation of VSD kinetic model according to the extended version of the Scheme I (B) and Scheme II (C) accounting for both RCK1 and RCK2 Ca2+-sites on each α-subunit. The VSD transitions in Scheme I only depend on the unbound or bound state of the RCK sites within the same α-subunit: unliganded (R0,0-A0,0), RCK1 site occupied (R1,0-A1,0), RCK2 site occupied (R0,1-A0,1) and both sites occupied (R1,1-A1,1). For sake simplicity, Scheme II, only depicts VSD transitions described for the Scheme I. In this case, x and y represent the number of RCK1 sites occupied and number of RCK2 sites occupied, respectively, in the other three α-subunits (x = from 0 to 3 and y = from 0 to 3).