Loss of Slc4a1b Chloride/Bicarbonate Exchanger Function Protects Mechanosensory Hair Cells from Aminoglycoside Damage in the Zebrafish Mutant persephone

Abstract

Mechanosensory hair cell death is a leading cause of hearing and balance disorders in the human population. Hair cells are remarkably sensitive to environmental insults such as excessive noise and exposure to some otherwise therapeutic drugs. However, individual responses to damaging agents can vary, in part due to genetic differences. We previously carried out a forward genetic screen using the zebrafish lateral line system to identify mutations that alter the response of larval hair cells to the antibiotic neomycin, one of a class of aminoglycoside compounds that cause hair cell death in humans. The persephone mutation confers resistance to aminoglycosides. 5 dpf homozygous persephone mutants are indistinguishable from wild-type siblings, but differ in their retention of lateral line hair cells upon exposure to neomycin. The mutation in persephone maps to the chloride/bicarbonate exchanger slc4a1b and introduces a single Ser-to-Phe substitution in zSlc4a1b. This mutation prevents delivery of the exchanger to the cell surface and abolishes the ability of the protein to import chloride across the plasma membrane. Loss of function of zSlc4a1b reduces hair cell death caused by exposure to the aminoglycosides neomycin, kanamycin, and gentamicin, and the chemotherapeutic drug cisplatin. Pharmacological block of anion transport with the disulfonic stilbene derivatives DIDS and SITS, or exposure to exogenous bicarbonate, also protects hair cells against damage. Both persephone mutant and DIDS-treated wild-type larvae show reduced uptake of labeled aminoglycosides. persephone mutants also show reduced FM1-43 uptake, indicating a potential impact on mechanotransduction-coupled activity in the mutant. We propose that tight regulation of the ionic environment of sensory hair cells, mediated by zSlc4a1b activity, is critical for their sensitivity to aminoglcyoside antibiotics.

Author Summary

The majority of hearing loss in humans is caused by death of inner ear mechanosensory hair cells. The aminoglycosides, a family of antibiotics, cause permanent hearing loss and transient vestibular disorders in this fashion. If we can find ways to reduce the hair cell toxicity of these antibiotics, we will be better able to treat tuberculosis, meningitis, and infections arising from chronic conditions like cystic fibrosis. Our lab uses zebrafish as a tool to identify ways to block hair cell death. Zebrafish have hair cells on their surface that are killed by aminoglycosides. We generated a pool of mutants and identified fish that retain hair cells when exposed to aminoglycosides. Here we describe why one of these mutants, persephone, protects hair cells from aminoglycosides. persephone has a mutation in a chloride/bicarbonate exchanger. This mutant protein fails to move chloride across cell membranes. Loss of the protein's activity decreases aminoglycoside entry into hair cells. We speculate that the exchanger mutated in persephone may be a critical regulator of ionic conditions that promote entry of aminoglycosides into hair cells. The persephone mutant furthermore suggests that transiently altering the ionic environment of hair cells may be a useful approach to protect hair cells from aminoglycoside exposure.