Bestrophin-1 Enables Ca2+-activated Cl− Conductance in Epithelia

Abstract

Epithelial cells express calcium-activated Cl؊ channels of unknown molecular identity. These Cl ؊ channels play a central role in diseases such as secretory diarrhea, polycystic kidney disease, and cystic fibrosis. -activated Cl Ϫ channels (CaCC) 2 are present in almost every cell type examined. Although this type of channel has been studied extensively, the molecular identity of CaCC in epithelial tissues remains a mystery (1, 2). The channels are of small conductance, show outward rectification upon moderate increases in intracellular Ca 2ϩ , and have an anion selectivity of I Ϫ Ͼ Cl Ϫ (3, 4). Both direct activation of the channel by increases in intracellular Ca 2ϩ and indirect activation through Ca 2ϩ -dependent phosphorylation by calmodulin kinase II have been described (2, 5). Small conductance CaCC in excised membrane patches of human pancreatic CFPAC cells have been shown to be regulated by Ca 2ϩ , calmodulin kinase II, and inositol 3,4,5,6-tetrakisphosphate (6). CaCC are inhibited by compounds like DIDS and niflumic acid, although these pharmacological tools lack specificity (7).Molecular candidates for CaCC have been proposed in the past. A family of Ca 2ϩ -activated Cl Ϫ channels (CLCA) has been identified (8, 9). These proteins are involved not only in chloride conductance and epithelial secretion but also in cell-cell adhesion, apoptosis, and cell cycle control. However, detailed structure-function analysis is still missing, and these proteins have a surprisingly low Ca 2ϩ sensitivity (1). Thus, CLCA could be part of a higher Cl Ϫ channel complex (10). The ClC-3 channel has been shown to be regulated by calmodulin kinase II (11). However, ClC-3 is a cytosolic Cl Ϫ channel, located in endosomes, and ClC-3 knock-out animals apparently do not lack CaCC (1).Previous studies demonstrated that bestrophin-1, the product of the vitelliform macular dystrophy (VMD2) gene, is able to form CaCC (12). Mutations in the VMD2 gene cause earlyonset autosomal dominant macular dystrophy of the retina, the so-called Best disease (13). Previous studies detected this channel in the basolateral membrane of the retinal pigment epithelium (14), where it controls the light-peak amplitude in the electrooculogram. Expression of BEST1 was reported to be limited to the retinal pigment epithelium, although it has also been detected in cultured airway epithelial cells (15,16). For the first time, we will provide evidence that endogenously expressed BEST1 causes Ca 2ϩ -activated Cl Ϫ conductance in epithelial tissues. EXPERIMENTAL PROCEDURESUssing Chamber Recordings-Mice (C57BL/6, Charles Rivers Laboratories) were killed under CO 2 narcosis by cervical dislocation. Tissues were put immediately into an ice-cold buffer solution containing 145 mmol/liter NaCl, 3.8 mmol/liter KCl, 5 mmol/liter D-glucose, 1 mmol/liter MgCl 2 , 5 mmol/liter HEPES, and 1.3 mmol/liter calcium gluconate (pH 7.4). After mounting into a perfused micro-Ussing chamber, apical and basolateral surfaces of the epithelium were perfused continuously with buffer solu...

Transport and Function of Chloride in Vascular Smooth Muscles

“…The putative role of bestrophin as the CaCC is questioned though by the observation that a large fraction of heterologous expressed bestrophin appears intracellularly [175,177,179]. This contradicts its function as a membrane channel.…”

“…Several other groups have recently used siRNA technique in cell culture and found an association between the bestrophin proteins and I Cl(Ca) [179,181,185,195,196,197]. We have found that downregulation of bestrophin-3 in mesenteric small artery VSMCs with siRNA suppressed the cGMP-dependent I Cl(Ca) without affecting the co-expressed classical I Cl(Ca) [112].…”

“…Based on the protein homology in the ion selectivity filters to ligand-gated anion channels such as the GABA and glycine receptors, bestrophin was cloned as a putative Cl – channel [177]. Subsequent studies by several independent laboratories identified bestrophins as candidates for CaCCs [177,178,179]. Heterologous expression of one bestrophin isoform in different cell types produces similar I Cl(Ca) [67,175], but different bestrophin isoforms produce Cl – currents with different characteristics [177].…”

“…Heterologous expression of one bestrophin isoform in different cell types produces similar I Cl(Ca) [67,175], but different bestrophin isoforms produce Cl – currents with different characteristics [177]. Bestrophins have been shown to respond with I Cl(Ca) after G-protein-coupled receptor stimulation [175,179,180,181]. Bestrophins are relatively sensitive to [Ca 2+ ] i with a half-maximal activation in the physiological range of about 200 n m [177,182,183].…”

Loss of TMEM16A Causes a Defect in Epithelial Ca2+-dependent Chloride Transport

“…While cAMP-dependent Cl Ϫ secretion by CFTR is well examined, detailed analysis of epithelial Ca 2ϩ -dependent Cl Ϫ secretion is hampered by the lack of a molecular counterpart. Although bestrophins may form Ca 2ϩ -dependent Cl Ϫ channels and facilitate Ca 2ϩ -dependent Cl Ϫ secretion in epithelial tissues (10,11), they are unlikely to form secretory Cl Ϫ channels in the apical cell membrane, because Ca 2ϩ -dependent Cl Ϫ secretion is still present in epithelia of mice lacking expression of bestrophin (12). Bestrophins may rather have an intracellular function by facilitating receptor mediated Ca 2ϩ signaling and activation of membrane localized channels (13).…”