Na⁺ influx triggers bleb formation on inner hair cells

Large blebs form rapidly on apical membranes of sensory inner hair cells (IHCs), when the organ of Corti is freshly isolated from adult guinea pigs. Bleb formation had two distinguishable phases. Initially, we identified small particles labeled with fluorescent annexin V; these rapidly coalesced into larger aggregates. After particle aggregation, a single membrane bleb emerged from cuticular plate at the vestigial kinocilium location, eventually reaching ∼10 μm maximum spherical diameter; blebs this size often detached from IHCs. Development of blebs was associated with elevated concentration of intracellular Na⁺; blocking Na⁺ influx through mechanotransduction and ATP channels in the apical pole of IHCs or by replacement of Na⁺ with N-methyl-D-glucamine prevented Na⁺ loading and bleb formation. Depletion of intracellular ATP, blocking cAMP synthesis, inhibition of vesicular transport with brefeldin A, or inhibition of phosphatidylinositol 3-kinase with 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran- 4-one (LY-294002) significantly reduced bleb formation in the presence of a Na⁺ load. Neither the mechanism of blebbing nor the size growth of the IHC blebs was associated with cellular apoptosis or necrosis. Bleb formation was not significantly reduced by disassembling microtubules or decreasing intracellular hydrostatic pressure. Moreover, no polymerized actin was observed in the lumen of blebs. We conclude that IHC bleb formation differs from classic blebbing mechanisms and that IHC blebs arise from imbalance of endocytosis and exocytosis in the apical plasma membrane, linked to Na⁺ loading that occurs in vitro.