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

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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 (IV) 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 IV 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.

Introduction

The transduction of sound into neural activity depends on the creation and maintenance of a luminal fluid, endolymph, in the inner ear that is high in K+ concentration ([K+]) and low in both [Na+] and [Ca2+] [21]. However, there is little difference in [Cl] (∼120 to 130 mM) between endolymph and the basolateral fluid, perilymph, in spite of the large transepithelial endocochlear potential (EP) of +80 to +100 mV [21]. The EP and perilymphatic [Cl] predict (via the Nernst equation) an extremely high endolymphatic [Cl] of ∼2600 mM based on simple passive electrochemical diffusion. Dysfunction of Cl regulation would be expected to lead to large osmotic disturbances that would result in luminal volume changes and the consequent disruption of normal hearing. Gross volume changes have been associated with pathological states such as Meniere’s syndrome (swelling) and Schiebe’s deformity (shrinking).

On that basis, it has long been thought that some epithelial cells lining the cochlear duct may actively absorb Cl from endolymph to maintain its [Cl] near that of perilymph, and radiotracer experiments in the intact cochlea point to Reissner’s membrane as a mediator of Cl transport [19]. Reissner’s membrane is an epithelial monolayer (with a discontinuous mesothelial layer on the basolateral side) that forms much of the boundary of the cochlear lumen. The present study was undertaken to resolve at the single cell level whether there are significant Cl conductive pathways in Reissner’s membrane epithelial cells that could support its putative role in endolymph Cl homeostasis.

Section snippets

Methods

Tissues were obtained for RNA isolation and for electrophysiology following protocols approved by the Institutional Animal Care and Use Committee of Kansas State University, as described earlier [17]. The compositions of the solutions for electrophysiological recordings were (in mM) pipette 150 NMDG-Cl, 1 MgCl2, 0.273 CaCl2, 1 EGTA, 10 Hepes, pH 7.3, ∼300 mOsm, 100 nM free Ca2+[29] and bath 150 NMDG-Cl, 1 MgCl2, 0.7 CaCl2, 10 Hepes, 5 glucose, pH 7.3, ∼300 mOsm. All solutions for patch clamp were

Results

Whole-cell patch clamp recordings from Reissner’s membrane epithelial cells were made under conditions where Cl was the only major permeable ion. The Cl currents were characterized by (a) strongly inward-rectifying currents with slow activation at negative voltages and (b) weakly outward-rectifying currents (Fig. 1). The prominent inward-rectifying currents were similar to those described for ClC-2 and were investigated further in more detail.

We tested the effects of agents (external pH, Cd2+

Discussion

The contribution of Cl transporters to the support of auditory and vestibular neural processes has recently been reviewed [21]. However, the present paper is the first report of a significant involvement of conductive Cl pathways in Reissner’s membrane epithelium. We identified by means of gene array, RT-PCR and electrophysiology several channels that carry Cl. The molecular identities of the channels that carry the observed currents were not unambiguously determined, but candidate genes

Conclusion

In summary, we have identified a complex Cl current in Reissner’s membrane epithelial cells that may be carried by multiple transport proteins. Cl is known to play a critical role in sensory outer hair cell tuning and amplification through its involvement with the motor protein, prestin [22], [23], [28], although the influence of luminal (endolymphatic) [Cl] is not known. Our findings support a possible role of Reissner’s membrane in Cl homeostasis of endolymph in the support of hearing.

Acknowledgments

This work was supported by National Institutes of Health Grants R01-DC-00212 and P20-RR-017686. We thank Dr. Philine Wangemann for advice and helpful discussions. The advice and assistance of Donald Harbidge, Joel Sanneman, Dr. Hiromitsu Miyazaki, Dr. Takayuki Kudo, Dr. Hyoung-mi Kim and Dr. Kalidou Ndiaye are greatly appreciated.

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