In-silico insights into aminoglycoside intake by TMC1

Aminoglycosides are small-molecule antibiotics that are widely used as treatment against a variety of bacterial infections. A side-effect of aminoglycosides use is dose-dependent hearing loss, often referred to as cochleotoxicity. This is hypothesized to be due to the inability of the inner-ear sensory hair cells to clear these molecules out leading to permanent hair-cell death. Multiple experiments involving manipulations of components of the hair-cell mechanotransduction machinery, including tip links and transmembrane channel-like 1 (TMC1) and TMC2 proteins, have shown that an important pathway for aminoglycoside intake by hair-cells is their transduction channel. Recent studies have also shown that TMC1 and TMC2 proteins are the pore forming components of the hair-cell mechanotransduction machinery. Although experiments suggest that these positively charged aminoglycoside molecules can enter hair cells through TMCs, the mechanism and the energetics of this process have not been studied. Here we use AlphaFold2 models of the human TMC1 protein with and without calcium- and integrin-binding protein 2 (CIB2) in the presence of Kanamycin A (KanA) along with microsecond-long voltage simulations to study the pore opening mechanism and the interactions between KanA and pore residues. We also present the free-energy surface of the process obtained using metadynamics simulations (a biased sampling method) which quantifies the energetics of this pore-crossing event. The open-pore conformations that were generated because of the KanA crossing event were further simulated with voltage to calculate the conductance of the channel in its putative open state and to assess the role of CIB2 in its conduction. These results give qualitative and a quantitative insights into the mechanism of pore opening and KanA crossing through the inner-ear transduction channel.