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