Inward-rectifier chloride currents in Reissner's membrane epithelial cells

Abstract

Sensory transduction in the cochlea depends on regulated ion secretion and absorption. Results of whole-organ experiments suggested that Reissner's membrane may play a role in the control of luminal Cl(-). We tested for the presence of Cl(-) transport pathways in isolated mouse Reissner's membrane using whole-cell patch clamp recording and gene transcript analyses using RT-PCR. The current-voltage (I-V) relationship in the presence of symmetrical NMDG-Cl was strongly inward-rectifying at negative voltages, with a small outward current at positive voltages. The inward-rectifying component of the I-V curve had several properties similar to those of the ClC-2 Cl(-) channel. It was stimulated by extracellular acidity and inhibited by extracellular Cd2+, Zn2+ and intracellular ClC-2 antibody. Channel transcripts expressed include ClC-2, Slc26a7 and ClC-Ka, but not Cftr, ClC-1, ClCa1, ClCa2, ClCa3, ClCa4, Slc26a9, ClC-Kb, Best1, Best2, Best3 or the beta-subunit of ClC-K, barttin. ClC-2 is the only molecularly-identified channel present that is a strong inward rectifier. This study is the first report of conductive Cl(-) transport in epithelial cells of Reissner's membrane and is consistent with an important role in endolymph anion homeostasis.

Figure 1. Strong inward-rectifier and smaller outward Cl⁻ currents

A, Step pulses were applied from −140 mV to +40 mV, returning to the holding voltage −100 mV and repeated every 10 s. B, The mean current voltage relationship was obtained from 24 cells.

Figure 2. Dependence of inward-rectifier Cl⁻ currents on pH

The voltage protocol consisted of holding at −100 mV for 13 s with a 2 s pulse at +40 mV. All effects of pH were reversible. A, The activation of the current at −100 mV by external acidification from pH 7.3 to pH 6.8. B, The inhibition of the current at −100 mV by external alkalinization from pH 7.3 to pH 7.8.

Figure 3. Dependence of inward-rectifier Cl⁻ currents on inhibition by Zn²⁺ and Cd²⁺

Representative recordings; voltage protocol as in Figure 2. All effects of Zn²⁺ and Cd²⁺ were reversible. A, The inhibition of the current at −100 mV by 50 µM Zn²⁺. B, The inhibition of the current at −100 mV by 500 µM Cd²⁺.

Figure 4. Inhibition of inward-rectifier Cl⁻ currents by ClC-2 antibody

Summary I-V relationships; voltage protocol as in Figure 1. Currents recorded with antibody (3 µg/ml) raised against an intracellular epitope of ClC-2 added to the pipette solution (Anti-ClC-2 Ab; up triangles) were significantly reduced at negative membrane voltages compared to those serving as “Control” with heat-inactivated antibody (down triangles).