TRPA1-Mediated Accumulation of Aminoglycosides in Mouse Cochlear Outer Hair Cells

Abstract

Aminoglycoside ototoxicity involves the accumulation of antibiotic molecules in the inner ear hair cells and the subsequent degeneration of these cells. The exact route of entry of aminoglycosides into the hair cells in vivo is still unknown. Similar to other small organic cations, aminoglycosides could be brought into the cell by endocytosis or permeate through large non-selective cation channels, such as mechanotransduction channels or ATP-gated P2X channels. Here, we show that the aminoglycoside antibiotic gentamicin can enter mouse outer hair cells (OHCs) via TRPA1, non-selective cation channels activated by certain pungent compounds and by endogenous products of lipid peroxidation. Using conventional and perforated whole-cell patch clamp recordings, we found that application of TRPA1 agonists initiates inward current responses in wild-type OHCs, but not in OHCs of homozygous Trpa1 knockout mice. Similar responses consistent with the activation of nonselective cation channels were observed in heterologous cells transfected with mouse Trpa1. Upon brief activation with TRPA1 agonists, Trpa1-transfected cells become loaded with fluorescent gentamicin-Texas Red conjugate (GTTR). This uptake was not observed in mocktransfected or non-transfected cells. In mouse organ of Corti explants, TRPA1 activation resulted in the rapid entry of GTTR and another small cationic dye, FM1-43, in OHCs and some supporting cells, even when hair cell mechanotransduction was disrupted by pre-incubation in calcium-free solution. This TRPA1-mediated entry of GTTR and FM1-43 into OHCs was observed in wild-type but not in Trpa1 knockout mice and was not blocked by PPADS, a non-selective blocker of P2X channels. Notably, TRPA1 channels in mouse OHCs were activated by 4-hydroxynonenal, an endogenous molecule that is known to be generated during episodes of oxidative stress and accumulate in the cochlea after noise exposure. We concluded that TRPA1 channels may provide a novel pathway for the entry of aminoglycosides into OHCs.

Fluorescent aminoglycosides reveal intracellular trafficking routes in mechanosensory hair cells

“…A number of alternative entry mechanisms have been proposed in addition to the MET channel or through endocytosis. It has been suggested that AGs enter through other channels including transient receptor potential channels (75)(76)(77). The sodium glucose transporter SLC5A2 may also play a role in AG transport in the kidney (78).…”

Permeation of Fluorophore-Conjugated Phalloidin into Live Hair Cells of the Inner Ear Is Modulated by P2Y Receptors

“…Styryl dyes enter hair cells via the mechanotransduction channel, endocytotic vesicles, and P2X channels (Balak et al 1990;Nishikawa and Sasaki 1996;Griesinger et al 2002;Meyers et al 2003;Griesinger et al 2004;Marcotti et al 2005;Kaneko et al 2006;Crumling et al 2009). Ototoxic aminoglycosides also enter hair cells through multiple routes including the mechanotransduction channel, endocytosis, chloride/bicarbonate exchangers, and TRPA1 channels (Alharazneh et al 2011;Stepanyan et al 2011;Hailey et al 2012). This is the first reported evidence of living hair cells taking up phalloidin.…”

Aminoglycoside-Induced Oxidative Stress: Pathways and Protection

“…Interestingly, the pattern of a differential gradient for basal to apical uptake did not persist beyond 3 h. More recently, attention has focused on ion channels in hair cells (Marcotti et al 2005 ). Transient Receptor Potential channels (trpv1, trpv4, trpa1, and trpp1) are nonselective cation channels that appear to permit aminoglycoside entry (Stepanyan et al 2011 ). Mechanoelectrical transduction (MET) channels are necessary for aminoglycoside ototoxicity (Alharazneh et al 2011 ).…”