MAQ PI3K inhibitor was previously employed to photocontrol the voltage-gated Shaker potassium channel ( Banghart et al., 2004). We introduced single cysteine mutations as attachment sites at a series of different positions in portions of the first and second pore loops (P1 and P2) of TREK1 (Figure 1A) and expressed the channel
in HEK293 cells. MAQ was applied in the external solution to each of these cysteine-substituted mutants and photoswitching was tested by measuring the modulation of the current when illumination was switched back and forth from 500 nm to 380 nm. We first examined cysteine substitutions at residue N122 in P1 and K231 in P2 of TREK1, since these are homologous to the optimal site for photoblock by MAQ in the Shaker channel (Shaker 422) (Banghart et al., 2004). While both sites showed photomodulation, they had a different dependence on light, i.e., on the configuration of the MAQ photoswitch. TREK1(K231C-MAQ) produced photoblock in the trans state (500 nm illumination), as found in Shaker ( Banghart et al., 2004), but TREK1(N122C-MAQ) produced photoblock in the cis state (380 nm illumination) ( Table 1). The opposite photoswitching at the two attachment positions indicates P1 and P2 differ structurally and that P2 more closely resembles the P loop of Shaker. This is interesting in view of the levels of homology of the conserved C-terminal portion
of the P regions, where TREK1′s P1 and P2 have 17% and 23% identity Metformin mw (55% and
57% similarity), respectively, to the P of Shaker, and a unique long loop precedes TREK’s P1 ( Figure S1 available online). Photomodulation was also seen at two other MAQ attachment sites in P1 (Table 1). The strongest photomodulation was at S121C (Table 1), which displayed 64% ± 3% (n = 14) block under 380 nm light and was unblocked by isomerization to trans under illumination at 500 nm ( Figure 1C). Since MAQ thermally relaxes into the trans state, TREK1(S121C-MAQ) has the advantage that the channel is unblocked and can function normally in the dark. Cysteine-substituted versions of TREK1 that can be photoblocked by MAQ could be introduced into neurons by transfection, but this would add the heterologous protein to the native either protein and result in overexpression. One way around this would be to generate a genetic knockin that replaces the native TREK1 with a version of TREK1 that is identical except for the single cysteine substitution. However, knockin production is lengthy and costly. We therefore sought an alternative easier strategy for introducing the MAQ photoswitch into native channels. We developed a subunit replacement strategy to obtain optical control over a neuron’s native TREK1 channels (Figure 2A). As shown earlier, deletion of the TREK1 carboxy-terminal tail (TREK1ΔC) results in retention of the channel in the endoplasmic reticulum (Chemin et al., 2005).