Cl- influx into rat cortical lens fiber cells is mediated by a Cl- conductance that is not ClC-2 or -3.
OBJECTIVE
Exposure of organ-cultured lenses to Cl(-) channel blockers under isotonic conditions induces a localized cortical zone of extracellular space dilations. The purpose of this study was to investigate whether elongated lens fiber cells from this zone contain an anion conductance that mediates Cl(-) influx and whether two chloride channel isoforms known to be expressed in the lens (ClC-2 and -3) are responsible.


METHODS
Fiber cells were isolated by enzymatic dissociation in the presence of Gd(3+) and Co(2+) and their electrical properties analyzed by whole-cell patch clamping. Cells from the zone of extracellular space dilations were selected for analysis on the basis of cell length. RT-PCR and immunocytochemistry were used to determine whether ClC-2 or -3 channel isoforms are expressed in fiber cells located in the zone of extracellular space dilations.


RESULTS
Cells from the zone of extracellular space dilations were typically >120 microm in length and exhibited an outwardly rectifying Cl(-) conductance that was blocked by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) and displayed an anion selectivity sequence of I(-) > Cl(-) >> gluconate. ClC-2 and -3 were found to be expressed at the transcript and protein level in lens fiber cells, but subsequent immunocytochemical studies indicated that expressed proteins did not colocalize with cell membranes in the zone of extracellular space dilations, being predominately cytoplasmic in nature.


CONCLUSIONS
Taken together, the data indicate that extracellular space dilations are due to the inhibition of a Cl(-) channel(s) that normally mediates Cl(-) influx into cortical lens fiber cells under isotonic conditions. The molecular identity of this channel remains to be determined.
Webb
Kevin F
KF
Department of Physiology, School of Medical Sciences, University of Auckland, Auckland, New Zealand.
Merriman-Smith
B Rachelle
BR
Stobie
Jonelle K
JK
Kistler
Joerg
J
Donaldson
Paul J
PJ
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D000090007:CLC-2 Chloride Channels; D002470:Cell Survival; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology; D002712:Chlorides / Q000378:metabolism*; D005110:Extracellular Space; D007150:Immunohistochemistry; D007904:Lens Cortex, Crystalline / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D008564:Membrane Potentials; D018408:Patch-Clamp Techniques; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000090007:CLC-2 Chloride Channels; D018118:Chloride Channels; D002712:Chlorides; C115376:ClC-3 channel; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid