TY - JOUR
T1 - TRPA1-mediated accumulation of aminoglycosides in mouse cochlear outer hair cells
AU - Stepanyan, Ruben S.
AU - Indzhykulian, Artur A.
AU - Catalinavélez-Ortega, A.
AU - Boger, Erich T.
AU - Steyger, Peter S.
AU - Friedman, Thomas B.
AU - Frolenkov, Gregory I.
N1 - Funding Information:
We thank Drs. Kelvin Y. Kwan and David P. Corey for providing us with Trpa1 knockout mice and Mrs. Stephanie Edelmann for maintaining mouse colony and genotyping the mice. We also thank Drs. David P. Corey, Bradley K. Taylor, Nuria Gavara, and Lisa L. Cunningham for critical reading of our manuscript. This work was supported by National Organization for Hearing Research Foundation (to RS) and NIDCD/NIH (DC009434 to GIF), as well as NIDCD Intramural funds (DC00048 to TBF) and NIDCD/NIH (DC004555 to PSS).
PY - 2011/12
Y1 - 2011/12
N2 - 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 asmechanotransduction 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 withmouse 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, inOHCs 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.
AB - 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 asmechanotransduction 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 withmouse 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, inOHCs 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.
KW - 4-hydroxynonenal
KW - Gentamicin
KW - Organ of Corti
KW - Ototoxicity
KW - Reactive oxygen species
KW - Transient receptor potential A1 channel
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UR - http://www.scopus.com/inward/citedby.url?scp=84855678573&partnerID=8YFLogxK
U2 - 10.1007/s10162-011-0288-x
DO - 10.1007/s10162-011-0288-x
M3 - Article
C2 - 21879401
AN - SCOPUS:84855678573
SN - 1525-3961
VL - 12
SP - 729
EP - 740
JO - JARO - Journal of the Association for Research in Otolaryngology
JF - JARO - Journal of the Association for Research in Otolaryngology
IS - 6
ER -