Reflections on the Applications of Immunohistochemistry to Understanding the Maintenance of Electrochemical Gradients in the Inner Ear

Abstract

The author of the accompanying classic paper from the Journal of Histochemistry and Cytochemistry (Crouch JJ, Sakaguchi N, Lytle C, Schulte BA. Immunohistochemical localization of the Na-K-Cl Co-transporter (NKCC1) in the Gerbil Inner Ear. Journal of Histochemistry & Cytochemistry 1997;45(6):773-778) comments on how the immunohistochemical techniques used in the study provided critical new information that helped define the cellular and molecular mechanisms involved in the generation and maintenance of electrochemical gradients in the ear, particularly the presence of the Na,K,2Cl symporter (NKCC) in the inner ear. (J Histochem Cytochem 71: 509–510, 2023)

Maintaining an appropriate ionic equilibrium between cells and extracellular fluids is essential to cellular and organ function. Ion homeostasis is regulated mainly at the cell membrane by a wide variety of transporters and channels specialized to selectively move different species of ions across the plasmalemma. Different cell types have adapted the type, quantity, and membrane distribution of these transport proteins to perform specialized functions such as nerve conduction, muscle contraction, and exocrine secretion.

The mammalian cochlea and vestibular system are complex structures that regulate hearing and balance. Both depend on highly organized and interdependent networks of specialized epithelial and connective tissue cells for normal physiological activity. In the cochlea, a high positive direct current resting potential or endocochlear potential (EP) and large K⁺ gradients between endolymph and perilymph serve to drive K⁺ through apical transduction channels in sensory hair cells and initiate auditory nerve activity. At the time this article was published, our laboratory had mapped the precise cellular distribution of both the alpha and beta subunit isoforms of the Na,K-ATPase pump in the gerbil and mouse inner ear. Although it was firmly established that this transporter was essential to the production of these electrochemical gradients, it was also clear that Na,K-ATPase alone could not explain the establishment of such large gradients. Fortunately, available pharmacological evidence strongly suggested the presence of another transport protein, the Na,K,2Cl symporter (NKCC), in the inner ear. Clinical treatment of edema caused by organ failure with loop diuretics such as furosemide, which are highly selective inhibitors of NKCC, was known to cause hearing loss or deafness. Experimental studies on animals linked these hearing losses to temporary or permanent ablation of the EP. However, despite the obvious importance of NKCC to auditory function, nothing was known about its tissue and cellular distribution in the inner ear.

This reprinted article provided important new information essential to defining the specific cellular and molecular mechanisms involved in the generation and maintenance of the large electrochemical gradients in the ear. First using reverse transcription polymerase chain reaction (RT-PCR), we established that the secretory isoform (NKCC1) was the only isoform present in the inner ear. We then mapped its distribution to marginal cells in the stria vascularis as well as to three subpopulations of fibrocytes in the cochlear lateral wall. An essential element of this study was the use of ultrastructural immunohistochemical labeling to establish the precise location of NKCC1 in the basolateral plasmalemma of strial marginal cells and its lack of expression in the cell membrane of the adjacent intermediate cells, which would have led to a very different conclusion. These findings paved the way for subsequent physiological studies showing that the low [K⁺] in the intrastrial space where the EP is generated, is regulated by the K⁺ uptake activity of both Na,K-ATPase and NKCC1 in the marginal cell basolateral membrane. A second major finding was the co-localization of NKCC1 and Na,K-ATPase in the cell membrane of subpopulations of lateral wall fibrocytes which form a syncytial network with strial basal and intermediate cells. This further solidified the prevailing theory pioneered by our laboratory that K⁺ effluxed from sensory hair cells is actively recycled back to the scala media via this syncytium and provided another piece to the puzzle into the precise molecular mechanisms involved in this process.

Since the publication of this article, ¹ many studies have validated the essential role of NKCC1 to inner ear function. Deafness and balance disorders have been reported in mouse strains with mutations in Slc12a2, the gene that encodes NKCC1, and similar deficits occur in mice with genetic modifications that inactivate this cotransporter. More recently in 2020, mutations in SLC12A2 have been associated with autosomal dominant nonsyndromic hearing loss and cochlear-vestibular defects in humans. Fortunately, these human gene variants appear to be very rare. However, a few studies have reported diminished immunostaining for NKCC1 in strial marginal cells and lateral wall fibrocytes in aged animals with hearing losses. Thus, the possibility that more common variants in SLC12A2 with milder but compounding effects with age, should not be overlooked.