P2X-Receptor Inhibition with Suramin

I demonstrated during this work that FM dyes permeate non-selective through channels located at the apex of cochlear IHCs, most likely through the mechanotransduction channels as proposed by two independent groups (Gale et al., 2001; Meyers et al., 2003). Moreover, Meyers and colleagues found that FM1-43 penetrates cells expressing other non-selective ion channels, like the capsaicin receptor TRPV1 or the ATP-gated P2X receptor (Meyers et al., 2003). Interestingly, the non-selective P2X receptor is specifically expressed at the apex of mammalian cochlear IHCs (Järlebark et al., 2000; Järlebark et al., 2002). As a consequence of the receptor localization FM dye could permeate through these channels in parallel to the mechanotransduction channels. In an attempt to inhibit P2X receptor-mediated styryl dye entry into chick cochlear hair cells, Crumling and colleagues used the pharmacological, non-competitive P2X receptor antagonist suramin (100 M) (Crumling et al., 2009). They observed a block of around 85% of styryl dye signal in hair cells compared to untreated cells, and thus concluded that FM dyes can enter through these channels.

The inhibition of apical located channels would possibly allow the application of FM dyes for vesicle recycling studies in vivo. To test for this I repeated the suramin experiments of Crumling and colleagues on cochlear IHCs of the mouse. After 5 minutes of pre-incubation in 100 M suramin (Sigma-Aldrich) in standard HBSS the organ of Corti was incubated for 1 minute in 5 M FM1-43 dissolved in standard suramin-containing HBSS (100 M). After washing with normal suramin containing HBSS the IHCs were imaged using confocal optics (with the same setup as for normal FM1-43 imaging; see Methods). Interestingly, I got similar results as shown for the chick cochlear hair cells, namely that almost no FM fluorescence appeared inside the IHCs with normal imaging (Figure A 4).

Figure A 4: Effect of the P2X receptor antagonist suramin on FM1-43 entry into cochlear IHCs. FM1-43 fluorescence is almost non-detectable in normal scanning mode (left image;

microscope setup adjusted as for FM1-43 imaging of untreated IHC). Little fluorescence is detectable when imaging with multiple frame-accumulation (4x, right image). Scale bar: 10

m.

However, a careful comparison to control images (Figure 3.17 and Figure 3.18) revealed that also no FM fluorescence signal of neighboring cells or of the stereocilia was observable. A closer view on the 43-suramin dilution revealed that suramin changed the color of FM1-43 from orange in normal HBSS to pink in suramin-containing buffer (Figure A 5A).

Figure A 5: Interaction of FM1-43 with suramin. (A) FM1-43 has an orange-colored appearance in standard HBSS buffer (left). Suramin changed the FM1-43 color to pink (right). (B) The molecular structure of suramin exhibits six sulfonate groups, which make this component a highly negatively charged molecule (the molecular structure of suramin was

downloaded from the homepage of Sigma-Aldrich

http://www.sigmaaldrich.com/structureimages/17/mfcd00210217.gif (downloaded 05.02.2011, 2:07 pm)).

(Figure 2.2).

Spectrophotometer experiments were aimed at analyzing the emission and excitation spectra of FM1-43 in presence or absence of suramin. 1.8 ml of a 2 μm FM1-43 dilution (in H2O) were added to a quartz cuvette (10 mm width) under constant stirring. 200 μl of a 1 mM suramin stock (final concentration 100 μM) or 200 μl H2O (control) were added. A spectrum measurement of dye excitation was performed by measuring dye fluorescence at 570 nm ( 2.5 nm bandwidth; (Henkel et al., 1996a)) over a broad range of excitation wavelength between 300 and 550 nm (2 nm increments; integration time 1 second). Dye emission was measured over a broad range of wavelength between 480 and 700 nm (2 nm increments;

integration time 1 second) at a constant excitation wavelength of 460 nm ( 2.5 nm bandwidth).

Interestingly, suramin addition caused a shift in the peak excitation wavelength from approximately 460 nm to around 530 nm (Figure A 6 A). Despite the shift in the excitation peak, the emission wavelength of FM1-43 surprisingly showed no significant alteration of its spectrum after suramin addition (Figure A 6 B), which demonstrates the direct interaction of the two molecules.

Figure A 6: Normalized excitation and emission spectra of FM1-43 in H2O and suramin.

Spectrum measurement of FM1-43 excitation under control (in ddH₂O) conditions or in 100 M suramin (dissolved in ddH₂O). (A) Emission was measured at 570 nm while the

excitation wavelength was stepwise changed (increments of 2 nm from 300 to 550 nm).

Suramin shifted the peak excitation to a higher wavelength. (B) Dye emission was measured from 470 to 700 nm (increments of 2 nm) at a constant excitation wavelength of 460 nm.

In the next step I used cultured PC12 cells to check whether a direct suramin-dependent change in FM1-43 fluorescence can be observed. To test for this 200 μl FM1-43 (10 μM in PBS) was added to the cells and imaged. Next, to control for a change in fluorescence of

Finally, 200 μl PBS with FM1-43 (10 μM) and suramin (100 μM) was added and several images were captured to document possible changes.

FM1-43 stained the outer leaflet of the plasma membrane of the PC12 cells (Figure A 7). As expected, no change in fluorescence was detected for the control. Interestingly, the addition of FM-suramin resulted in an immediate disappearance of FM fluorescence at the periphery of the cell cluster. Within a few seconds the FM fluorescence faded away inside the cluster (Figure A 7, FM1-43/Suramin #1-#4).

Figure A 7: Suramin effect on FM1-43 membrane staining. FM1-43 alone bound to the plasma membrane of the PC12 cells, clearly visualizing the cell cluster (upper left panel). The application of FM1-43 with suramin (100 μM) resulted in FM fluorescence decrease starting at the periphery of the cell cluster witch reached after a few seconds the center of the cluster (FM1-43/Suramin #1-#4). The application of only FM1-43 revealed no change in membrane bound FM fluorescence (control). Scale bar: 40 µm.

I conclude therefore that FM1-43 interacts on an ionical basis with the sulfonate groups of suramin and consequently FM1-43 is taken of the membrane and becomes almost non-fluorescent (as styryl dyes are only non-fluorescent when inserted into membranes). Suramin-bound FM dye still has a 4-5 fold higher quantum yield compared to the unSuramin-bound aqueous form, as observed from non-normalized spectrophotometer measurements (Figure A 8), indicating that suramin has “membrane simulative” properties for styryl dyes. However, with the quantum yield increasing by 700-fold in membranes, it is clear that sequestration by

Figure A 8: Non-normalized FM1-43 fluorescence spectra. Dye emission was measured from 470 to 700 nm (increments of 2 nm) at a constant excitation wavelength of 460 nm.

Suramin-bound FM1-43 showed higher photon flux than in an aqueous environment.

Taken together, suramin application on hair cells does not inhibit FM dye permeation through the non-selective P2X receptor channels, moreover, suramin interacts directly with the styryl dye, which severely inhibits its native interaction with the plasma membrane and results in the observed block of styryl dye uptake.