This value is in agreement with the amount of residual baclofen-induced current in the Kir3.1 knockout mouse and in Kir3.2 knockout mouse (26%), as well as in the Kir3.2/Kir3.3 double knockout mice (15%) (Koyrakh et al., 2005). The relative contribution Dabrafenib mw of Kir3 and TREK1 channels depends, in part, on membrane

voltage. Since Kir3 is an inward rectifier and TREK1 is an outward rectifier, a holding potential of −55mV favors the TREK1 channel component, while at more negative potentials, such as −70mV, the Kir3 component will be favored. Photoswitched tethered ligands have opened the door to the selective, rapid and reversible optical control of membrane signaling through proteins that are normally insensitive to light (Szobota and Isacoff, 2010 and Fehrentz et al., 2011). We have developed a scheme for targeting optical control via a PTL to native proteins without the requirement for genetic knockin. The approach is to express a PCS that contains an anchoring site for the photoswitch as well as a mutation that retains the subunit inside the cell. The engineered subunit does not traffic and so has no function unless native subunits SCH 900776 cell line are present,

coassemble with it, and carry it to the cell surface. The PCS/WT complex is rendered light-sensitive once an externally applied membrane-impermeant photoswitch is attached to the PCS, something possible only at the plasma membrane. To generate Cytidine deaminase a PCS requires the fulfillment of three conditions. First, it is necessary to identify an appropriate site for cysteine modification on the extracellular face of the protein complex where the PTL will be covalently anchored so that its photoisomerization between trans and

cis states results in liganding, and hence modulation of signaling, in one isomer state but not the other. Second, this cysteine-substituted subunit must be mutated in a manner that eliminates its cell-surface trafficking as a homomultimer, but which allows for its surface targeting when it is coassembled with the wild-type subunit. Finally, the function of the heteromeric complex between the cysteine-substituted, trafficking-deficient (PCS) subunit and the wild-type subunit must be efficiently gated by PTL(s) on the PCS subunit(s). We describe here how these conditions can be met. The first step in developing a PCS—that of anchoring a PTL for photocontrol—has been successfully accomplished in a variety of proteins with a variety of ligands. The two main approaches have been steric block of an active site (either a pore of an ion channel or a catalytic site of an enzyme) or allosteric regulation by the PTL attached to a receptor’s ligand binding domain (Gorostiza and Isacoff, 2008, Szobota and Isacoff, 2010 and Fehrentz et al., 2011).