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The Journal of Neuroscience, October 1, 2008, 28(40):9939-9952; doi:10.1523/JNEUROSCI.1124-08.2008

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Development/Plasticity/Repair
Aminoglycoside-Induced Phosphatidylserine Externalization in Sensory Hair Cells Is Regionally Restricted, Rapid, and Reversible

Richard J. Goodyear,^1 * Jonathan E. Gale,^2,3 * Kishani M. Ranatunga,¹ Corné J. Kros,¹ and Guy P. Richardson¹

¹School of Life Sciences, University of Sussex, Brighton BN1 9QG, United Kingdom, and ²UCL Ear Institute and ³Department of Physiology, University College London, London WC1X 8EE, United Kingdom

Correspondence should be addressed to Dr. Guy P. Richardson, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK. Email: g.p.richardson{at}sussex.ac.uk

The aminophospholipid phosphatidylserine (PS) is normally restricted to the inner leaflet of the plasma membrane. During certain cellular processes, including apoptosis, PS translocates to the outer leaflet and can be labeled with externally applied annexin V, a calcium-dependent PS-binding protein. In mouse cochlear cultures, annexin V labeling reveals that the aminoglycoside antibiotic neomycin induces rapid PS externalization, specifically on the apical surface of hair cells. PS externalization is observed within 75 s of neomycin perfusion, first on the hair bundle and then on membrane blebs forming around the apical surface. Whole-cell capacitance also increases significantly within minutes of neomycin application, indicating that blebbing is accompanied by membrane addition to the hair cell surface. PS externalization and membrane blebbing can, nonetheless, occur independently. Pretreating hair cells with calcium chelators, a procedure that blocks mechanotransduction, or overexpressing a phosphatidylinositol 4,5-biphosphate (PIP2)-binding pleckstrin homology domain, can reduce neomycin-induced PS externalization, suggesting that neomycin enters hair cells via transduction channels, clusters PIP2, and thereby activates lipid scrambling. The effects of short-term neomycin treatment are reversible. After neomycin washout, PS is no longer detected on the apical surface, apical membrane blebs disappear, and surface-bound annexin V is internalized, distributing throughout the supranuclear cytoplasm of the hair cell. Hair cells can therefore repair, and recover from, neomycin-induced surface damage. Hair cells lacking myosin VI, a minus-end directed actin-based motor implicated in endocytosis, can also recover from brief neomycin treatment. Internalized annexin V, however, remains below the apical surface, thereby pinpointing a critical role for myosin VI in the transport of endocytosed material away from the periphery of the hair cell.

Key words: hair cell; phosphatidylserine; annexin V; aminoglycoside; FM1-43; myosin VI

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Received March 14, 2008; revised July 23, 2008; accepted Aug. 16, 2008.

Correspondence should be addressed to Dr. Guy P. Richardson, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK. Email: g.p.richardson{at}sussex.ac.uk

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