EXPRESSION OF VOLTAGE-ACTIVATED POTASSIUM KCNQ CHANNELS IN MOUSE RETINA.

SPITZMAUL, G.; GERMAN, O. L.; CARIGNANO, C.; BARILÁ, E.; JENTSCH, T.

Rio de Janeiro

Congreso; 9th World Congress International Brain Research Organization; 2015

KCNQ channels (Kv7) form a subfamily of voltage-gated K+ channel with 5 members in mammals (KCNQ1-KCNQ5). These channels, in particular KCNQ2/3, are molecular correlates of the M-current, a current that is already active at resting potentials and which can be modulated by muscarinic receptors and which has a profound influence on neuronal excitability. Moreover, these channels provide K+ current in epithelia (sensory and nonsensory)such as cochlea, intestine, lung cell lines and post-synapses like vestibular organ. Several human genetic diseases are associated with mutations in KCNQ channels: cardiac arrhythmia, in part with deafness (KCNQ1); neonatal epilepsy (KCNQ2 and KCNQ3); and progressive hearing loss (KCNQ4). Over the past years several mouse models were generated to investigate localization, function and mutation-associated pathologies of KCNQ channels in detail. We used in present work, transgenic mice for KCNQ4 and -5 channels. Recent publications showed the expression of KCNQ4 and -5 in primate eye and also a possible link between Kcnq5 gen and refractive errors. For these reasons, we investigate the expression and function of these channels in WT, Kcnq4-/- and Kcnq5-/- mice. Using a KO-controlled immunohistochemistry, we found a weak labeling of KCNQ4 in retinal pigmented epithelium (RPE) cells, which is enhanced in albino mice. However, fluorescence was detected after an amplification of the primary signal by a biotin deposition-based method. Opposite to what was reported in other species, KCNQ5 channel subunit was not found in mouse retina or RPE cells. A non-specific signal for KCNQ5 was present in both genotypes (WT and Kcnq5-/-) when we used a normal staining protocol that was lost in retina after antigen retrieval. However,the signal was still present in vestibular organ of wild-type mice (control tissue). Aditionally, we performed KCNQ3 staining in WT and albino mice (without KO control). KCNQ3 was found in fibers of the outer plexiform layer and the inner segment of the retina. We also performed patch-clamp analysis of WT mouse retinal neurons. Experiments were done with retinal slices of adult mice and with cultured retinal neurons obtained from P2 mice. Neurons were cultured for several days to allow in vitro maturation and were then subjected to patch-clamp analysis. In retinal slices, we detected potassium currents in bipolar neurons. However, this current could not be inhibited by XE-991, a KCNQ channel blocker, agreeing with our inability to detect KCNQ channels in these cells by immunocytochemistry. On the other hand, with isolated immature neurons in culture, we observed only very small voltage-activated currents. Thus, at this stage of maturation of cultured neurons, the expression of voltage-gated potassium currents is very low. Our results suggest that KCNQ4 channel may participate in the homeostasis of K+ in the subretinal space while KCNQ3 may regulate the excitability of retinal neurons in mature retinas.