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Purpose: To test the hypothesis that lens fiber cells use different combinations of transport proteins to mediate Cl influx and efflux in order to regulate their steady state volume., Methods: Cells were isolated from rat lenses by enzymatic dissociation in the presence of Gd(3+), and short and long fiber cells were assigned to peripheral efflux and deeper influx zones, respectively. Electrical properties were of isolated cells, and whole lenses were analyzed by using whole-cell patch clamping and intracellular microelectrodes, respectively, before and after exposure to hyposmotic challenge and/or the addition of [(dihydronindenyl)oxy] alkanoic acid (DIOA)., Results: Cells from the influx zone were dominated by an outwardly rectifying Cl(-) conductance, and exposure to hyposmotic challenge increased this conductance. Cells isolated from the efflux zone were dominated by K(+) conductance(s) with only a minimal contribution from the Cl(-) conductance. Exposure of cells that exhibited a minimal baseline Cl(-) conductance to hyposmotic challenge caused swelling and a transient increase in Cl(-) current. In other cells that initially lacked a Cl(-) conductance, hyposmotic challenge caused swelling, but no increase in outward current. However, the subsequent addition of DIOA exacerbated swelling and activated a Cl(-) current. Under isosmotic conditions, addition of DIOA also induced cell swelling and the transient activation of a Cl(-) current. In whole lenses, exposure to hyposmotic challenge increased the contribution of an anion conductance to the membrane potential., Conclusions: In peripheral cells, Cl(-) efflux is primarily mediated by potassium chloride cotransporters (KCCs) and its activity can be upregulated by hyposmotic challenge. In addition, these cells also contain a Cl(-) channel that exhibits a variable baseline activity level and that can be recruited to effect regulatory volume decrease if the KCC transporters are inhibited.

Purpose: 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.