Mouse Bestrophin-2 Is a Bona fide Cl− Channel

Abstract

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.

Bestrophin-2 is a candidate calcium-activated chloride channel involved in olfactory transduction

“…This permeability sequence corresponds to the Eisenman ''weak field strength'' lyotropic series (42), indicating that in both channels, the interaction between the permeant ion and the channel is weak, and permeability is related to the ease with which the permeant ion leaves the bulk solution and enters the channel. Our data are in general agreement with previous measurements (21,22,39).…”

“…In fact, currents were half-maximal at a Ca concentration of 0.4 M for mBest2, similar to previous results (19,21), whereas native currents required a higher Ca concentration, 4.7 M, in general agreement with micromolar values measured in rat (39) and mouse (17). Doseresponse relations obtained from isolated frog olfactory cilia also showed a similar K 1/2 of 4.8 M (4).…”

“…4 and 39, and a slight outward rectification in mBest2 currents. Previous reports found a more linear currentvoltage relation for mBest2 currents (21,22).…”

“…We have found that mBest2 is the only bestrophin expressed in the OE. Site-directed mutagenesis experiments have demonstrated (21,22,26) that the putative second transmembrane domain of mBest2 participates in forming the channel pore, and therefore mBest2 appears to be at least part of the pore. However, it might also be possible that mBest2 forms a heteromultimer with other channel subunits, or that it has associated proteins that may, for example, modify Ca sensitivity and inactivation properties.…”

“…The biophysical properties of mBest2 have been recently investigated with the patch-clamp technique, showing that mBest2 by itself forms Ca-activated Cl channels in heterologous systems and is a structural component of the ion-conducting pore. Bestrophin-2 can thus be considered a bona fide Cl channel (21,22,26,32).…”