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Identification of a Residue Important in Anion Binding and Conduction
Address correspondence to Criss Hartzell, Department of Cell Biology and The Center for Neurodegenerative Diseases, 615 Michael St., 535 Whitehead Biomedical Research Building, Emory University School of Medicine, Atlanta, GA 30322-3030. Fax: (404) 727-6256; email: criss{at}cellbio.emory.edu
Bestrophins have recently been proposed to comprise a new family of Cl- channels. Our goal was to test whether mouse bestrophin-2 (mBest2) is a bona fide Cl- channel. We expressed mBest2 in three different mammalian cell lines. mBest2 was trafficked to the plasma membrane as shown by biotinylation and immunoprecipitation, and induced a Ca2+-activated Cl- current in all three cell lines (EC50 for Ca2+ = 230 nM). The permeability sequence was SCN-: I-: Br-: Cl-: F- (8.2: 1.9: 1.4: 1: 0.5). Although SCN- was highly permeant, its conductance was 
10% that of Cl- and SCN- blocked Cl- conductance (IC50 = 12 mM). Therefore, SCN- entered the pore more easily than Cl-, but bound more tightly than Cl-. Mutations in S79 altered the relative permeability and conductance for SCN- as expected if S79 contributed to an anion binding site in the channel. PSCN/PCl = 8.2 ± 1.3 for wild-type and 3.9 ± 0.4 for S79C. GSCN/GCl = 0.14 ± 0.03 for wild-type and 0.94 ± 0.04 for S79C. In the S79 mutants, SCN- did not block Cl- conductance. This suggested that the S79C mutation altered the affinity of an anion binding site for SCN-. Additional evidence that S79 was located in the conduction pathway was provided by the finding that modification of the sulfhydryl group in S79C with MTSET+ or MTSES- increased conductance significantly. Because the effect of positively and negatively charged MTS reagents was similar, electrostatic interactions between the permeant anion and the channel at this residue were probably not critical in anion selectivity. These data provide strong evidence that mBest2 forms part of the novel Cl- conduction pathway in mBest2-transfected cells and that S79 plays an important role in anion binding in the pore of the channel.
Key Words: chloride channels • ion permeation • electrophysiology • mutagenesis • ion channel
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