To confirm that these were bona

To confirm that these were bona fide interactions, we investigated whether the potassium channel subunits or Tmem263 formed complexes with GLT-1 or DAT after immunoprecipitation. Lysates of HEK-293 cells previously cotransfected with HA-GLT-1 or HA-DAT, and either YFP-Kv7.3, YFP-Kv7.2 or YFP-Kv7.5 were immunoprecipitated with anti-HA MPI-0479605 (Fig. 1A). Only YFP-Kv7.3 formed stable complexes with HA-GLT-1, although a weak band was visible for YFP-Kv7.2 in lysates obtained with solubilization in standard RIPA buffer (Fig. 1A). Similar results were obtained for HA-DAT (Fig. 1B). In these conditions, Kv7.5 did not co-immunoprecipitate with either transporter, and no bands were detected in the controls where HA-GLT-1 or HA-DAT were omitted (Fig. 1A/1B, lanes 6–8). However, we were unable to co-immunoprecipitate Tmem263 either with HA-GLT-1 or HA-DAT under different solubilization conditions (not shown), indicating that it is a weak or transient interaction, if real. To investigate the possible existence of a spatial association of the transporters with the potassium channels in neurons, we co-transfected primary cortical cultures with either HA-GLT-1 or mCherry-DAT and a mixture of YFP-Kv7.2 plus YFP-Kv7.3. Both potassium channel subunits were included since co-expression of Kv7.2 and Kv7.3 leads to greater surface expression (Schwake et al., 2000) and generation of much larger currents (Wang et al., 1998) than when either subunit is expressed alone. As expected, potassium channels were enriched in the axons, especially in the axonal initial segment (AIS), while the transporters were more evenly distributed in the axonal and somatodendritic compartments (supplementary Fig. 1). In the somatodendritic compartment, GLT-1 was enriched is dendritic spines (supplementary Fig. 1A), while DAT was uniformly distributed (supplementary Fig. 1D). Along the axonal shaft, GLT-1 and Kv7.2/7.3 tended to localize in clusters (Fig. 2A and B), and a similar pattern was observed for DAT and Kv7.2/7.3 (Fig. 3A and B). To quantitatively analyze whether transporter clusters co-localized with those formed by the potassium channel, we analyzed at least 30 fluorescence images that were enhanced by deconvolution prior to determining the respective Pearson and Manders' coefficients using the ImageJ JACoP plugin software (Bolte and Cordelieres, 2006). The Pearson coefficients were 0.61 ± 0.03 and 0.69 ± 0.01 for GLT-1/Kv7.2/7.3 and DAT/Kv7.2/7.3, respectively, both values clearly above the cutoff of 0.5 that is generally accepted as valid co-localization (Zinchuk and Zinchuk, 2008). Similarly, Manders’ coefficients (M1 and M2), which represent the fraction of overlap between two molecule types with values ranging from to 1, were obtained after thresholding the images. For GLT-1/Kv7.2/7.3, M1 and M2 values were 0.60 ± 0.04 and 0.51 ± 0.04, respectively, whereas for DAT/Kv7.2/7.3 the values were 0.68 ± 0.4 and 0.59 ± 0.3, respectively. Indeed, more visual impression of co-localization could be obtained by representing pixel fluorescence intensity profiles along the axonal shafts of each individual color staining, showing substantial overlap among the plots of each color channel (Figs. 2C and 3C, images presented in gray tones). To investigate if these proteins also interacted functionally, first we assayed the effect of coexpression of GLT-1 or DAT with Tmem263 or Kv7.2/7.3 on the uptake of [3H]glutamate or [3H]dopamine. While uptake of both glutamate and dopamine were significantly stimulated by the presence of the potassium channel, Tmem263 had no detectable effect (Fig. 4). To analyze the bioenergetic causes of the stimulatory effect of Kv7.2/7.3 on GLT-1 and DAT, we tested the hypothesis that the depolarizing effect of sodium ions, either entering the cells with glutamate or dopamine, might be counteracted by the activity of M-currents. To that end, we first repeated these uptake experiments in the presence of XE-991, a selective inhibitor of Kv7 potassium channels. As the stimulatory effect was prevented in the presence of the channel blocker (Fig. 4), next we performed electrophysiological measurements to detect the existence of a rapid coupling mechanism between the transporters and potassium channel. The neuronal cell line HT22 was co-transfected with both YFP-Kv7.2/YFP-Kv7.3 and HA-GLT-1 or mCherry-DAT, and co-transfected cells were electrophysiologically registered in whole cell patch-clamp configuration.