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Whole-Cell Patch Clamping of Isolated Fiber Cells Confirms that Spatially Distinct Cl^– Influx and Efflux Pathways Exist in the Cortex of the Rat Lens

¹From the Departments of Physiology and ²Optometry and Vision Science, School of Medical Sciences, University of Auckland, Auckland, New Zealand.

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 Gd³⁺, 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.