Charge and lipophilicity are required for effective block of the hair-cell mechano-electrical transducer channel by FM1-43 and its derivatives

The styryl dye FM1-43 is widely used to study endocytosis but behaves as a permeant blocker of the mechano-electrical transducer (MET) channel in sensory hair cells, loading rapidly and specifically into the cytoplasm of hair cells in a MET channel-dependent manner. Patch clamp recordings of mouse outer hair cells (OHCs) were used to determine how a series of structural modifications of FM1-43 affect MET channel block. Fluorescence microscopy was used to assess how the modifications influence hair-cell loading in mouse cochlear cultures and zebrafish neuromasts. Cochlear cultures were also used to evaluate otoprotective potential of the modified FM1-43 derivatives. Structure-activity relationships reveal that the lipophilic tail and the cationic head group of FM1-43 are both required for MET channel block in mouse cochlear OHCs; neither moiety alone is sufficient. The extent of MET channel block is augmented by increasing the lipophilicity/bulkiness of the tail, by reducing the number of positive charges in the head group from two to one, or by increasing the distance between the two charged head groups. Loading assays with zebrafish neuromasts and mouse cochlear cultures are broadly in accordance with these observations but reveal a loss of hair-cell specific labelling with increasing lipophilicity. Although FM1-43 and many of its derivatives are generally cytotoxic when tested on cochlear cultures in the presence of an equimolar concentration of the ototoxic antibiotic gentamicin (5 µM), at a 10-fold lower concentration (0.5 µM), two of the derivatives protect OHCs from cell death caused by 48 h-exposure to 5 µM gentamicin.


Data analysis for the mouse dye loading assay
The same mixed-effects model was fitted to fluorescence intensity mouse data, based on the identity of the compound and natural logarithm-transformed time as interacting fixed effects, since only one concentration was assessed.Unlike the fluorescence intensity analysis in zebrafish, since individual cochlear cultures were followed through time, longitudinal data were accounted for by inclusion of first order autoregressive correlation between time points grouped by replicate.

Analysis of loading properties of FM1-43 and its derivatives in zebrafish lateral line hair cells as a function of time and concentration and mouse cochlear cultures as a continuous time series
The ability of 13550 and its derivatives to load into the hair cells was assessed as a function of both time and concentration for zebrafish lateral line hair cells, and as a function of time for mouse cochlear culture hair cells at the single concentration that was used in this study (i.e., 0.3 µM) (Supplementary Figure S1, supported by Supplementary Table S1, zebrafish, and Supplementary Table S2, mouse).The fluorescence of compound 13550, like commercially available FM1-43, shows a strong dependence on both time and concentration in zebrafish, and on time in mouse hair cells.By comparison, compounds 14885, 13670, and 13698, that either failed to load or showed a significant reduction in fluorescence compared to 13550, displayed similar loading behaviour in both mouse OHCs and zebrafish neuromasts.Compounds 13957 and 13668, both of which showed a significant increase in fluorescence compared to 13550, also load in a similar manner in mouse and zebrafish.Compounds 16327 and 13667 behaved in a similar manner to 13550 in mouse OHCs, but they showed a reduction in fluorescence in zebrafish.Compounds 13551, 13669, and 28552 showed an increase in mouse, compared to 13550, but a decrease in zebrafish.The remaining compounds are in broad alignment regarding loading compared to the parent compounds, although this is time and concentration dependent.None of the compounds showed a significantly faster rate of uptake than 13550, though 14957 did show a stronger response to increasing concentration.Intercept is the comparison of 3 μM of each derivative with 3 μM of 13550 for 5 minutes (as main text Figure 7A).Derivatives that are significantly different to 13550 are highlighted in green.Concentration is the gradient of the fitted line (linear on a log-scale) through all concentrations tested for each derivative compared to the gradient for 13550.Derivatives that are significantly different to 13550 are highlighted in yellow.Time is the gradient of the line (linear on a log-scale) through all time points tested for each derivative compared to the 13550 gradient (GLMM).
Derivatives that are significantly different to the 13550 are highlighted in purple.Red boxes surround those derivatives that were significantly brighter than 13550.
Statistical analysis comparing fluorescence intensity (arbitrary units) in mouse cochlear culture of derivatives of FM1-43 (groups I-VI) to the parent compound (13550).Est(imates) are the natural logarithm of the proportional change compared to 13550, with their Standard Errors (SE), z-values, and p-values are shown for: Intercept which is the comparison of 0.3 μM of each derivative with 0.3 μM of 13550 for 5 minutes (as main text Figure 7B).Derivatives that are significantly different to the 13550 are highlighted in green.

Table S3 :
Time which is the gradient of the line through all time points tested for each derivative compared to the 13550 gradient (GLMM).Derivatives that are significantly different to the 13550 highlighted in purple.Red boxes surround those derivatives that were significantly brighter than 13550.Statistical analysis of Outer Hair Cell counts following treatment with derivatives of FM1-43.Estimated means (Est.) are the natural logarithm of the proportional difference compared to Controls (panels A and C) and Gentamicin alone (panels B and D) at treatment concentrations of 5 μM (panels A and B) and 0.5 μM (panels C and D), with their Standard Errors (SE).Z-values, and p-values are also shown from a Poisson GLMM.