d-Tubocurarine and Berbamine: Alkaloids That Are Permeant Blockers of the Hair Cell's Mechano-Electrical Transducer Channel and Protect from Aminoglycoside Toxicity

Abstract

Aminoglycoside antibiotics are widely used for the treatment of life-threatening bacterial infections, but cause permanent hearing loss in a substantial proportion of treated patients. The sensory hair cells of the inner ear are damaged following entry of these antibiotics via the mechano-electrical transducer (MET) channels located at the tips of the hair cell's stereocilia. d-Tubocurarine (dTC) is a MET channel blocker that reduces the loading of gentamicin-Texas Red (GTTR) into rat cochlear hair cells and protects them from gentamicin treatment. Berbamine is a structurally related alkaloid that reduces GTTR labeling of zebrafish lateral-line hair cells and protects them from aminoglycoside-induced cell death. Both compounds are thought to reduce aminoglycoside entry into hair cells through the MET channels. Here we show that dTC (≥6.25 μM) or berbamine (≥1.55 μM) protect zebrafish hair cells in vivo from neomycin (6.25 μM, 1 h). Protection of zebrafish hair cells against gentamicin (10 μM, 6 h) was provided by ≥25 μM dTC or ≥12.5 μM berbamine. Hair cells in mouse cochlear cultures are protected from longer-term exposure to gentamicin (5 μM, 48 h) by 20 μM berbamine or 25 μM dTC. Berbamine is, however, highly toxic to mouse cochlear hair cells at higher concentrations (≥30 μM) whilst dTC is not. The absence of toxicity in the zebrafish assays prompts caution in extrapolating results from zebrafish neuromasts to mammalian cochlear hair cells. MET current recordings from mouse outer hair cells (OHCs) show that both compounds are permeant open-channel blockers, rapidly and reversibly blocking the MET channel with half-blocking concentrations of 2.2 μM (dTC) and 2.8 μM (berbamine) in the presence of 1.3 mM Ca2+ at −104 mV. Berbamine, but not dTC, also blocks the hair cell's basolateral K+ current, IK,neo, and modeling studies indicate that berbamine permeates the MET channel more readily than dTC. These studies reveal key properties of MET-channel blockers required for the future design of successful otoprotectants.

Identification of ion-channel modulators that protect against aminoglycoside-induced hair cell death

“…A compound was designated as a weak blocker if fluorescence was only detected at ×40. The neomycin-protection and gentamicin screen were performed as described previously (26). Briefly, Yo-Pro-1 (Invitrogen, Y3603) (29) prelabeled larvae were incubated in 25 μM test compound with 6.25 μM neomycin sulphate for 1 hour or 50 or 100 μM test compound with 10 μM gentamicin sulphate (MilliporeSigma, G3632) for 5 hours.…”

“…Plates were screened using a ×16 objective. Trunk neuromasts 3-9 were viewed and assessed as described previously (26).…”

“…Cochlear cultures were prepared from pups at P2 as described previously (26,52) and, after 1 day in vitro, were treated with 50 μM test compound and 5 μM gentamicin sulphate (MilliporeSigma, G3632) or augmented levels of extracellular Na + or K + and 5 μM gentamicin sulphate for a further 48 hours before fixation and staining with TRITC-phalloidin (MilliporeSigma, P1951) or double labeling with Texas red-conjugated phalloidin (Invitrogen) and anti-myosin7a (Proteus, 25-6790). Cultures were imaged with a Zeiss Axioplan2 microscope.…”

“…Thus far, however, only 4 compounds discovered using zebrafish larvae have been reported to protect hair cells of the mammalian inner ear from aminoglycoside antibiotics (21,25,26). Two of these, tacrine and PROTO1, provide protection against aminoglycoside-induced hair cell death in cultures of the utricular maculae of 3-to 6-week-old mice (16,21).…”

“…Phenoxybenzamine (25) and berbamine have been shown, respectively, to cause partial and full protection of OHCs in mouse cochlear cultures against aminoglycosides when used at a low concentration. At slightly higher concentrations, however, both can cause OHC death (25,26).…”