Journal
JOURNAL OF GENERAL PHYSIOLOGY
Volume 123, Issue 4, Pages 327-340Publisher
ROCKEFELLER UNIV PRESS
DOI: 10.1085/jgp.200409031
Keywords
chloride channels; ion permeation; electrophysiology; mutagenesis; ion channel
Categories
Funding
- NEI NIH HHS [EY014852, R01 EY014852] Funding Source: Medline
- NIGMS NIH HHS [R01 GM060448, GM60448] Funding Source: Medline
Ask authors/readers for more resources
Bestrophins have recently been proposed to comprise anew 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 similar to10% 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. P-SCN/P-CI = 8.2 +/- 1.3 for wild-type and 3.9 +/- 0.4 for S79C. G(SCN)/G(CI), = 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 initiation 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 NITS 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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available