Roles for KCC transporters in the maintenance of lens transparency

To determine whether the potassium chloride cotransporter (KCC) family is expressed in the rat lens and to ascertain whether the transporters are involved in the regulation of lens volume and transparency.

RT-PCR was performed on RNA extracted from fiber cells to identify members of the KCC family expressed in the lens. Western blot analysis and immunocytochemistry, using KCC isoform-specific antibodies, were used to verify expression at the protein level and to localize KCC isoform expression. Organ-cultured rat lenses were incubated in isotonic artificial aqueous humor (AAH) that contained either the KCC-specific inhibitor [(dihydronindenyl)oxy] alkanoic acid (DIOA), the KCC activator N-ethylmaleimide (NEM), or the chloride channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) for up to 18 hours. Lens wet weight was monitored, and lens transparency and tissue morphology were recorded with dark-field and confocal microscopy, respectively.

Molecular experiments to characterize KCC isoform expression showed that KCC1, -3, and -4 were all expressed in the lens at both the transcript and protein levels and that KCC2 was not. Immunocytochemistry indicated that the three KCC isoforms exhibited distinct differentiation-dependent expression patterns, with KCC1 and -3 being restricted to the lens cortex, whereas KCC4 was found throughout the entire lens, including the lens core. In the lens cortex, most of the labeling for all KCC isoforms was cytoplasmic, whereas in the lens core, KCC4 labeling was associated with the membrane. Incubation of lenses in 100 microM DIOA for 18 hours caused lenses to increase their wet weight and induced a cortical opacity that was caused by extensive damage to peripheral fiber cells located up to 150 microm in from the lens capsule, whereas deeper fiber cells appeared unaffected by DIOA exposure. Lower concentrations of DIOA (10 microM) revealed that this damage was initiated primarily by the swelling of peripheral fiber cells. In contrast, NPPB-treated lenses exhibited a deeper zone (>100 microm) of cell damage that was initiated by the dilation of the extracellular space between fiber cells. Exposure of lenses to the KCC activator NEM caused cell shrinkage in peripheral fiber cells but extensive cell swelling in deeper fiber cells. Peripheral cell swelling caused a differential recruitment of KCC isoforms from a cytoplasmic pool to the plasma membrane. DIOA-induced cell swelling increased the association of KCC4 with membrane, whereas hypotonic cell swelling dramatically increased the association of KCC1 with the membrane.

The rat lens expresses three KCC transporter isoforms (KCC1, -3, and -4) in a differentiation-dependent manner. Modulation of transporter activity and subcellular localization suggests that multiple KCC transporters mediate KCl efflux in peripheral fiber cells in a dynamic fashion. These results indicate that, in addition to Cl- channels, KCC transporters play a role in mediating a circulating flux of Cl- ions, which contributes to the maintenance of lens transparency through controlling the steady state volume of lens fiber cells.