Journal
JOURNAL OF GENERAL PHYSIOLOGY
Volume 128, Issue 3, Pages 247-259Publisher
ROCKEFELLER UNIV PRESS
DOI: 10.1085/jgp.200609581
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Funding
- NEI NIH HHS [R01 EY014852, EY014852] Funding Source: Medline
- NIGMS NIH HHS [GM60448, R01 GM060448] Funding Source: Medline
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Mutations in human bestrophin-1 (VMD2) are genetically linked to several forms of retinal degeneration but the underlying mechanisms are unknown. Bestrophin-1 (hBest1) has been proposed to be a Cl-channel involved in ion and fluid transport by the retinal pigment epithelium (RPE). To date, however, bestrophin currents have only been described in overexpression systems and not in any native cells. To test whether bestrophins function as Ca2+-activated Cl- (CaC) channels physiologically, we used interfering RNA (RNAi) in the Drosophila S2 cell line. S2 cells express four bestrophins (dbest1-4) and have an endogenous CaC current. The CaC current is abolished by several RNAi constructs to dbest1 and dbest2, but not dbest3 or dbest4. The endogenous CaC current was mimicked by expression of dbest1 in HEK cells, and the rectification and relative permeability of the current were altered by replacing F81 with cysteine. Single channel analysis of the S2 bestrophin currents revealed an similar to 2-pS single channel with fast gating kinetics and linear current -voltage relationship. A similar channel was observed in CHO cells transfected with dbest1, but no such channel was seen in S2 cells treated with RNAi to dbest1. This provides definitive evidence that bestrophins are components of native CaC channels at the plasma membrane.
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