Aminoglycoside uptake and clearance in mammalian cells

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DESCRIPTION (provided by applicant): Aminoglycosides like gentamicin are antibiotics used for treating serious infections. However, these drugs also kill inner ear sensory hair cells, causing deafness and vestibular deficits. Endocytosis of aminoglycosides into cells is insufficiently rapid to account for their swift appearance in the cytoplasm and observed cytotoxicity. Recently, this laboratory described a rapid, non-endocytotic uptake mechanism for aminoglycoside entry, modulated by TRPV channel regulators. Aminoglycosides can also rapidly enter hair cells through transduction channels and apical endocytosis. The hair cell apex is bathed in endolymph. If the endolymph pathway is critical for aminoglycosides to enter hair cells, how do aminoglycosides enter the endolymph from the vasculature? A TRPV channel, TRPV4, is expressed at the luminal membrane of marginal and endothelial cells in the stria vascularis. TRPV4 could allow aminoglycosides to enter endothelial cells from the blood, and exit marginal cells into endolymph. We will use aortal endothelial cells and kidney tubule cells as models of endothelial uptake and marginal cell clearance of aminoglycosides in the inner ear, and verify that TRPV4 channel is bi-directionally aminoglycoside-permissive. We will determine if marginal cells in strial explants clear aminoglycosides and if this mechanism is TRPV4-dependent. Finally, we will conduct in vivo gentamicin injection experiments and determine if strial endothelial cells uptake gentamicin, allowing the drug to pass through stria vascularis, and if this process is also dependent on TRPV4 channel.