Anion Permeation in Ca 2 1 - activated Cl 2 Channels

Ca 2 1 -activated Cl channels (Cl Ca Cs) are an important class of anion channels that are opened by increases in cytosolic [Ca 2 1 ]. Here, we examine the mechanisms of anion permeation through Cl Ca Cs from Xenopus oocytes in excised inside-out and outside-out patches. Cl Ca Cs exhibited moderate selectivity for Cl over Na: P Na / P Cl 5 0.1. The apparent affinity of Cl Ca Cs for Cl was low: K d 5 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 . ClO 4 . I . N 3 . Br . Cl . formate . HCO 3 . acetate 5 F . gluconate. The conductance sequence was as follows: N 3 . Br . Cl . N(CN) 2 . I . SCN . COOH . ClO 4 . acetate . HCO 3 5 C(CN) 3 . gluconate. Permeant anions block in a voltage-dependent manner with the following affinities: C(CN) 3 . SCN 5 ClO 4 . N(CN) 2 . I . N 3 . Br . HCO 3 . 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 Ca Cs 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 Ca 2 1 depended on the permeant anion at low [Ca 2 1 ] (100–500 nM). Apparently, occupancy of the pore by a permeant anion increased the affinity of the channel for Ca 2 1 . 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 5 z 9.2. The channel may be blocked by OH 2 ions, or protons may titrate a site in the pore necessary for ion permeation. These data demonstrate that the permeation properties of Cl Ca Cs are different from those of CFTR or ClC-1, and provide insights into the nature of the Cl Ca C pore. key words: voltage clamp • chloride current • selectivity • pH • anomalous mole fraction I N T R O D U C T I O N Ca 2 1 -activated Cl channels (Cl Ca Cs) 1 serve a number of important well defined physiological functions in a variety of cell types. These functions include epithelial secretion (Begenisich and Melvin, 1998; Morris, 1999), olfactory transduction (Kurahashi and Yau, 1994), regulation of vascular tone (Hirakawa et al., 1999), neuronal excitability (Frings et al., 1999), and fast block to polyspermy in certain eggs (Jaffe and Cross, 1986). Despite the physiological importance of Ca 2 1 -activated Cl current (I Cl.Ca ), the channels underlying this current remain incompletely understood at both the molecular and biophysical levels. Although a family of putative Cl Ca Cs has been cloned (Cunningham et al., 1995; Gruber et al., 1998, 1999; Gandhi et al., 1998; Ji et al., 1998), the behavior of this cloned “CLCA” channel does not resemble Cl Ca Cs from many native cells including those we have studied in Xenopus oocytes (Hartzell et al., 1997; Machaca and Hartzell, 1998, 1999a,b; Kuruma and Hartzell, 1999, 2000). However, there may be several subtypes of Ca 2 1 -activated chloride channels, and the CLCA family may comprise one subtype. The observations that Cl Ca C single-channel conductances range from 1 to 50 pS and that I Cl.Ca in different cell types can be regulated differently suggests that there is more than one type of channel (see Kuruma and Hartzell, 2000). We have been interested in I Cl.Ca in Xenopus oocytes because this cell type has long served as a model system for studying Cl Ca Cs and because the channels in Xenopus oocytes in many ways resemble those in cardiac muscle (Collier et al., 1996), smooth muscle (Hirakawa et al., 1999), secretory epithelial cells (Begenisich and Melvin, 1998), and neurons (Frings et al., 1999). As one of the prime examples of Cl Ca Cs, it is important to understand thoroughly the gating, permeation, and regulation of the Xenopus oocyte channel. We and others have previously characterized its gating properties and regulation by Ca 2 1 and voltage (Kuruma and Hartzell, 1999, 2000; Machaca and Hartzell, 1999a; Callamaras and Parker, 2000), but the mechanisms of anion permeation through this channel have not yet been thoroughly investigated. There appear to be several common features of anion-selective channels that have been studied. First, anion channels are relatively nonselective among anions. Address correspondence to H. Criss Hartzell, Department of Cell Biology, Emory University School of Medicine, 1648 Pierce Drive, Atlanta, GA 30322-3030. Fax: (404) 727-6256; E-mail: [email protected] 1 Abbreviations used in this paper: CFTR, cystic fibrosis transmembrane regulator; Cl Ca C, Ca 2 1 -activated Cl 2 channel; ClC, voltagegated Cl 2 channel family; I Cl.Ca , Ca 2 1 -activated Cl 2 current; GHK, Goldman-Hodgkin-Katz.