Anion Permeation in Ca²⁺-Activated Cl⁻ Channels

Ca²⁺-activated Cl channels (Cl_CaCs) are an important class of anion channels that are opened by increases in cytosolic [Ca²⁺]. Here, we examine the mechanisms of anion permeation through Cl_CaCs from Xenopus oocytes in excised inside-out and outside-out patches. Cl_CaCs exhibited moderate selectivity for Cl over Na: P_Na/P_Cl = 0.1. The apparent affinity of Cl_CaCs for Cl was low: K_d = 73 mM. The channel had an estimated pore diameter >0.6 nm. The relative permeabilities measured under bi-ionic conditions by changes in E_rev were as follows: C(CN)₃ > SCN > N(CN)₂ > ClO₄ > I > N₃ > Br > Cl > formate > HCO₃ > acetate = F > gluconate. The conductance sequence was as follows: N₃ > Br > Cl > N(CN)₂ > I > SCN > COOH > ClO₄ > acetate > HCO₃ = C(CN)₃ > gluconate. Permeant anions block in a voltage-dependent manner with the following affinities: C(CN)₃ > SCN = ClO₄ > N(CN)₂ > I > N₃ > Br > HCO₃ > Cl > gluconate > formate > acetate. Although these data suggest that anionic selectivity is determined by ionic hydration energy, other factors contribute, because the energy barrier for permeation is exponentially related to anion hydration energy. Cl_CaCs exhibit weak anomalous mole fraction behavior, implying that the channel may be a multi-ion pore, but that ions interact weakly in the pore. The affinity of the channel for Ca²⁺ depended on the permeant anion at low [Ca²⁺] (100–500 nM). Apparently, occupancy of the pore by a permeant anion increased the affinity of the channel for Ca²⁺. The current was strongly dependent on pH. Increasing pH on the cytoplasmic side decreased the inward current, whereas increasing pH on the external side decreased the outward current. In both cases, the apparent pKa was voltage-dependent with apparent pKa at 0 mV = ∼9.2. The channel may be blocked by OH⁻ ions, or protons may titrate a site in the pore necessary for ion permeation. These data demonstrate that the permeation properties of Cl_CaCs are different from those of CFTR or ClC-1, and provide insights into the nature of the Cl_CaC pore.