Impulse activity of corneal cold nerve terminals modulation by ATP-gated ion channel permeant photoswitches

[Purpose]: In retinas lacking photoreceptors, photoisomerizable small molecules (photoswitches) enter retinal ganglion cells, thus modulating their electrical activity by acting on native Kv and HCN ion channels and conferring them sensitivity to a particular light wavelength. The aim of the present study was to explore the effect of the synthetic photoswitch DENAQ on the electrical activity of peripheral nerve terminals of trigeminal sensory neurons innervating the cornea of the guinea pig, and to define the mechanisms used by DENAQ to enter into the nerve endings.
[Methods]: Nerve terminal impulse activity of cold terminals was recorded in excised guinea-pig corneas pre-incubated with DENAQ (2µM-2mM); a set of corneas were incubated with either a P2X or TRPV1 channel antagonist before incubation with DENAQ. Non-incubated corneas served as control. Spontaneous activity (SA) of cold thermoreceptors at 34oC was recorded under dim light. Then a light stimulating protocol consisting of 5 light pulses (460 nm, 125 mW/cm2, 15s-duration) applied at 15s intervals was applied to define the photoswitching effect of DENAQ on SA. Presence of P2X and TRPV1 channels in corneal cold nerves was ascertained by immunohistochemistry.
[Results]: SA of corneal cold nerve endings was reduced after pre-incubation with DENAQ compared with control (3.1±0.5 imps/s vs 7.6±0.8 imp/s, 200µM DENAQ vs control respectively, p<0.001). In addition, SA was further reduced by 50% during the 460nm light-on periods (p<0.001). This photoswitching effect of DENAQ was dose-dependent. Illumination with 460 nm light did not affect SA in control corneas. Photoswitching effect of DENAQ was abolished by pre-treatment with non-selective P2X antagonists (suramine, TNT-ATP) and selective P2X3 antagonist purotoxin 1 but not by selective P2X7 antagonist A740003 nor by TRPV1 blocker capsazepine. A fraction of TRPM8+ cold nerves was positive for P2X3 receptor. TRPV1 was also functional in part of corneal cold terminals.
[Conclusions]: The non-membrane permeable photoswitch DENAQ enters to corneal cold sensory nerve endings preferentially through ATP-gated P2X cation channels, mainly P2X3. Once inside, DENAQ photoisomerization to cis configuration when exposed to 460 nm blue light hyperpolarizes the nerve terminal and decreases significantly the spontaneous activity of corneal cold thermoreceptors, opening a new approach to treat ocular pain and dry eye.