Moreover, following exposure to MAQ, alternating illumination between 380 nm and 500 nm produced no change in the basal current in TREK1ΔC-S121C-transfected cells (Figure 2B). Because the cytoplasmic N-terminal domain and the first transmembrane segment (M1) of TREK1 are sufficient to dimerize with the full-length check details TREK1 channel (Veale et al., 2010), we hypothesized that TREK1ΔC would dimerize with the wild-type TREK1 channel (WT) and produce a functional channel (Figure 2A). In contrast with the lack of photomodulation of current

in MAQ-labeled cells expressing TREK1ΔC(S121C) alone (Figure 2B), coexpression of TREK1ΔC(S121C) with WT in HEK293 cells yielded a TREK1 current that was strongly photomodulated (Figure 2C). This indicates that TREK1ΔC(S121C) assembles with the WT subunits and that the heteromeric channel goes to the cell surface, where the TREK1ΔC(S121C) subunit is labeled by

the charged, membrane-impermeant MAQ endowing the channel with regulation by light via photoisomerization of MAQ. From here on, we refer to the TREK1ΔC(S121C) subunit that contains the cysteine photoswitch attachment site and that is retained internally unless coassembled with a WT native subunit as the TREK1 photoswitchable conditional subunit (TREK1-PCS). For the approach to work as intended, the heteromeric TREK1-PCS/WT BKM120 channel would need to retain normal functions of the TREK1 channel. We tested the TREK1-PCS/WT heteromeric channel to determine whether it was regulated by external and L-NAME HCl internal stimuli in the same way as WT. To do this, we examined the sensitivity to stimuli of total WT current in cells expressing WT alone and compared this to the photoblocked current component from cells coexpressing the TREK1-PCS along with WT, where the light-sensitive current is attributed solely to the heteromeric TREK1-PCS/WT

channel labeled with MAQ on the TREK1-PCS. TREK1 channels are inhibited by external acidification, due, it has been proposed, to titration of a histidine residue in P1 (Cohen et al., 2008 and Sandoz et al., 2009), an effect that has been attributed to C-type inactivation (Bagriantsev et al., 2011, Cohen et al., 2008 and Sandoz et al., 2009). We found that the light-gated current obtained from MAQ-labeled HEK293T cells coexpressing the TREK1-PCS and WT subunit is also inhibited by external acidification (Figure 3A). This inhibition of the photogated current in the TREK1-PCS/WT heterodimer was 53.6% ± 8% (n = 8), similar to the 60.6% ± 5% (n = inhibition of total current in WT alone (p > 0.7, t test). We next investigated the regulation of the TREK1-PCS/WT heterodimer channel by internal modification induced by GPCR activation. Gi-coupled receptors have been shown to enhance TREK1 current (Cain et al., 2008).