Discussion aminoglycoside project Discussion on MET 2 Discussion on TRP channels 7 Discussion on ROS 9 Discussion on Mitochondria 13 Discussion on Apoptosis 20Discussion on METIt has been shown by Marcotti et al. that aminoglycosides enter inner ear haircells through mechanoelectrical transduction (MET) channels  and a theoretical model with which the rate of aminoglycoside uptake rate can be calculated has been developed . Apart from the electrophysiology data by Marcotti et al. there are many studies that corroborate the finding that aminoglycosides enter inner ear hair cells via the MET channel.
A small proportion of the MET channels in normal hair cells is open in the absence of any stimuli . This resting current is absent in hair cells from mice suffering from various forms of hereditary deafness, e.g. mice expressing a mutated form of myosin VIIA show both vestibular and cochlear dysfunction and lack resting transduction currents ADDIN EN.CITE . It has been demonstrated by Kros et al. that myosin VIIA is required for the normal gating of the MET channel ADDIN EN.CITE . The myosin VIIA mutation impairs the uptake of gentamicin by hair cells . In other words uptake of aminoglycosides in hair cells which lack resting transducer currents (all MET channels being closed) is limited.
FM1-43 is a styryl pyridinium dye that enters inner ear hair cells through open MET channels  and behaves like a permeant blocker ADDIN EN.CITE .
Hair cells take up FM1-43 but surrounding (non-transducing) cells show little of nor labelling . Analogously, within the cochlea haircells are damaged by aminoglycosides whereas surrounding (non-transducing) cells remain unharmed . It was found that FM1-43 competes with aminoglycosides for entry into the haircells and the ototoxicity of neomycin is reduced in the presence of FM1-43 ADDIN EN.CITE . This means that aminoglycosides and FM1-43 both target the MET channel. When haircells are treated with EGTA the tiplinks are broken and the MET channels close. In the presence of EGTA the loading with FM1-43 by haircells is inhibited ADDIN EN.CITE . Haircells from mice which are homozygous for the myosin VIIA mutation Myo7a6J do not load with FM1-43 ADDIN EN.CITE . Initially during a dye pulse FM1-43 strongly labels the hairbundles ADDIN EN.CITE . It is clear from these observations that results obtained from studies with FM1-43 can give insights into aminoglycoside uptake by haircells.
It has been reported that aminoglycosides enter haircells via receptor mediated endocytosis . Endocytosis cannot explain why within the cochlea only the haircells degenerate in the presence of aminoglycosides  whilst surrounding cells remain unharmed. Also, hair cells which express mutated myosin (important for normal MET gating) do not take up aminoglycosides . Undoubtedly aminoglycosides will enter the haircells via endocytosis to a small extent, but these studies show that aminoglycosides predominantly enter the haircells via the MET channel.What about hair cells that do not take up AGs?
There are some studies where no aminoglycoside uptake into haircells was observed. Dulon et al.  showed that triturated haircells are not affected by the presence of 5 mM gentamicin for 6 hours. Trituration is a very harsh isolation method for hair cells and in all likelihood a large proportion of the hairbundles were destroyed during this procedure.The hairbundle is the location of the MET channels, triturating the haircells probably destroyed the stereocilia and these haircells were not transducing.
These observations are in stark contrast with the study done by Kotecha and Richardson  where an organotypic culture of the cochlea was used. The haircells in this preparation are transducing and exposure with 1 mM of gentamicin for 1 hour completely destroyed the haircells.
In another study by Williams et al. it can be clearly seen that isolating OHCs can lead to the loss of stereocilia. See figure 1 in . These haircells will not transduce.
Adapted from Figure 1 in The study by Dulon et al.  employs essentially the same method of isolating hair cells, which probably leads to hairbundle damage.
In another study by Zajic and Schacht  isolated outer hair cells appear to have compromised stereocilia (see figures 1-5 in ).The recent observation that aminoglycosides enter hair cells through the MET channel can explain the apparent discrepancies between the Kotecha and Richardson study and the results obtained in the Schacht laboratory.AG uptake via MET channels can explain a lot of observations
Exposure to aminoglycosides leads to a phased degeneration of haircells, first the OHCs then the IHCs [13, 14]. This can be explained by the fact that an outer hair cells has more stereocilia (~81) than an inner hair cell (~48) .
This means that an OHC has more MET channels than an IHC and therefore more pathways for AG uptake. If OHCs load up quicker with aminoglycosides than IHCs it can be expected that OHCs degenerate quicker.
Are there any studies which show that aminoglycoside uptake in OHCs is faster than IHCs? FM1-43 uptake in OHCs is quicker than IHCs ADDIN EN.CITE .Hair cell loss due to AGs starts in the basal coil and progresses apically .
This is not due to a concentration gradient of AGs within the cochlea because this same pattern is observed in organotypic cultures that are incubated with a uniform concentration of AGs . This means that this pattern of sensitivity is based on inherent properties of the hair cells. Basal OHCs have larger transducer currents than apical OHCs  and OHCs have larger transducer currents than IHCs . The uptake rate of AGs will be higher in the basal coil than in the apical coil due to the differences in conductance.
Clinically the effects of aminoglycosides are characterized by hearing loss initially at high frequencies which corresponds to hair cell damage in the basal coil .
In vivo studies have shown that immature hair cells take up less gentamicin than mature hair cells. Also, mature cells die faster than immature cells upon exposure to AGs . One possible explanation could be a difference in MET current amplitude between immature and mature hair cells. However, transduction current amplitude stays constant during maturation .
The endocochlear potential in mice is only fully developed the 12th day after birth ( REF. In immature animals the driving force for positively charged molecules like AGs is lower than in mature animals, this could explain the differential sensitivity between immature and mature hair cells in vivo.
FM1-43 labeling is much stronger in the basal coil than in the apical coil ADDIN EN.CITE .
As described above, FM1-43 enters the hair cells via the MET channel. The differences in labeling can be explained by the fact that basal coil hair cells have larger transducer currents than apical coil hair cells.
The uptake of FM1-43 is temperature dependent, i.e. at 4 (C the uptake rate is reduced ADDIN EN.CITE . This could indicate a temperature dependence of the opening of MET channels possibly due to a dependence on myosin ATPase.
Gentamicin gets taken up quicker by OHCs in the presence of background
noise as opposed to in animals maintained under noise-attenuated
conditions . This suggests that the MET channels were open during the background noise allowing more AGs in than under noise-attenuated conditions.The rate of FM1-43 loading into basal coil HCs is much faster than in apical coil HCs ADDIN EN.CITE . This again can be explained by the differences in MET current amplitudes with basal coil HCs displaying larger MET conductances than apical coil conductances.When incubated with gentamicin it first labels the stereocilia of hair cells before diffusing through the cell . This suggests that the location where the AGs get taken up by the haircells is situated within the stereocilia. This corroborates the hypothesis that AGs enter via the MET channel as demonstrated by Marcotti et al. .
Spermine is a polyamine which is naturally present in cells  and displays effects on hair cells very similar to those of AGs and FM1-43.
Spermine causes haircell damage without affecting any of the surrounding cells . This indicates that hair cells have a very specific uptake mechanisms for polyamines which suggests involvement of the MET channel.
Spermine causes damage to OHCs comparable to DHS and amikacin .
It is important to take spermine into account because it directly targets mitochondria. I will discuss in the discussion section on Mitochondria.Aminoglycosides preferentially target inner ear hair neurons and proximal tubule kidney cells. Although undoubtedly some slow uptake of aminoglycosides through endocytosis will take place, it is striking that other cell types in the direct surroundings of inner ear hair cells are not affected by the presence of aminoglycosides. (What about non-kidney cell types in the direct surroundings?)
This suggests that inner ear hair cells and kidney proximal tubule cells share some common uptake system for aminoglycosides. As shown above, there is convincing evidence that AGs enter the hair cells via the MET channel. What evidence is there that AGs enter the kidney cells via MET channels as well?
In order to address this question we will first investigate whether or not kidney proximal tubule cells display mechano-electrical transduction.
Do kidney cells display mechanoelectrical transduction?Proximal tubule k