The role of ClC-3 in volume-activated chloride currents and volume regulation in bovine epithelial cells demonstrated by antisense inhibition.
1. A chloride current with mild outward rectification was induced in the native bovine non-pigmented ciliary epithelial (NPCE) cells by a 23 % hypotonic solution. The current showed no or little inactivation at depolarized steps. 2. ATP blocked 88 and 61 % of the outward and inward components of the volume-activated chloride current (ICl,vol) with an IC50 of 5.3 and 9.6 mM, respectively. 3. The volume-activated chloride current was decreased and the activation of the current was delayed by inhibiting endogenous ClC-3 expression using a ClC-3 antisense oligonucleotide. The inhibition of the current as a function of antisense concentration was asymptotic with a maximum about 60 %. The remaining current was probably not derived from ClC-3 and was inhibited by ATP. 4. ClC-3 expression in the bovine NPCE cells was verified by immunofluorescence studies. ClC-3 immunofluorescence was distributed throughout the cells but with the predominant location within the nucleus. The expression of ClC-3 protein was diminished by the ClC-3 antisense oligonucleotide with the greatest diminution occurring in the nuclear region. 5. The size of the volume-activated chloride current was positively correlated with the ClC-3 immunofluorescence level. 6. Regulatory volume decrease of the NPCE cells was reduced by ClC-3 antisense oligonucleotide. 7. We conclude that endogenous ClC-3 is associated with the volume-activated chloride current and is involved in cell volume regulation, but that it can only contribute towards a proportion of the current in NPCE cells. 8. The nuclear predominance of ClC-3 immunofluorescence in NPCE cells, the absence of basal activity of chloride current and the marked pharmacological differences between IClC-3 and ICl,vol argue against ClC-3 being the only, or even the main, volume-activated chloride channel in NPCE cells.
Wang
L
L
School of Biosciences, Cardiff University, Cardiff CF1 3US, UK.
Chen
L
L
Jacob
T J
TJ
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D018118:Chloride Channels / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D002712:Chlorides / Q000378:metabolism*; D002924:Ciliary Body / Q000166:cytology* / Q000502:physiology; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D007038:Hypotonic Solutions; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology*
D018118:Chloride Channels; D002712:Chlorides; C115376:ClC-3 channel; D007038:Hypotonic Solutions; D020319:Oligodeoxyribonucleotides, Antisense; D000255:Adenosine Triphosphate