Modelling the permeation of mechanically sensitive ion channels in auditory hair cells and its implications for preventing drug-induced ototoxicity

The experiments presented in this thesis are aimed at better understanding the ion and drug permeation properties of the mechano-electrical transduction (MET) channels of the hair cells responsible for hearing. Aminoglycosides (AGs) are potent antibiotics prescribed worldwide in the treatment of gram-negative infections such as sepsis, neonatal infections, and those associated with cystic fibrosis. AGs permeate into hair cells of the inner ear via the MET channels, which are large, non-selective mechanosensitive cation channels at the tips of the stereocilia. Once inside, AGs cause death of hair cells with varying degrees of severity depending on the drug family member and location of the cell along the length of the cochlea, with basal outer hair cells (OHCs) dying more readily than apical OHCs and inner hair cells (IHCs). By experimentally modelling the interaction between the MET channel and drugs that block it, we can determine electrical properties of the channel and calculate entry rates of the drugs into the cells. I present evidence for a correlation between the toxicity of three AGs (gentamicin, kanamycin and amikacin) and their entry rate through the MET channel in OHCs, with gentamicin being the most readily permeable and amikacin being the least. Furthermore, I have found that all four drugs (including the fluorescently conjugated gentamicin-Texas Red) that I have modelled permeate at a higher rate through individual channels in basal OHCs than those in apical OHCs. I have also probed the roles of calcium, maturation, and driving force in drug permeation, and addressed aspects of the genetics of the channel and how these may relate to our model. Lastly, I present evidence for the existence of volume-regulated anion channels (VRACs) in the membranes of OHCs, which could potentially be an alternative route of entry for ototoxic compounds.