Optical Control of Endogenous Proteins with a Photoswitchable Conditional Subunit Reveals a Role for TREK1 in GABAB Signaling

Department of Molecular and Cell Biology and Helen Wills Neuroscience Institute, 271 Life Sciences Addition, University of California, Berkeley, Berkeley, CA 94720, USA.
Neuron (Impact Factor: 15.05). 06/2012; 74(6):1005-14. DOI: 10.1016/j.neuron.2012.04.026
Source: PubMed


Selective ligands are lacking for many neuronal signaling proteins. Photoswitched tethered ligands (PTLs) have enabled fast and reversible control of specific proteins containing a PTL anchoring site and have been used to remote control overexpressed proteins. We report here a scheme for optical remote control of native proteins using a "photoswitchable conditional subunit" (PCS), which contains the PTL anchoring site as well as a mutation that prevents it from reaching the plasma membrane. In cells lacking native subunits for the protein, the PCS remains nonfunctional internally. However, in cells expressing native subunits, the native subunit and PCS coassemble, traffic to the plasma membrane, and place the native protein under optical control provided by the coassembled PCS. We apply this approach to the TREK1 potassium channel, which lacks selective, reversible blockers. We find that TREK1, typically considered to be a leak channel, contributes to the hippocampal GABA(B) response.

41 Reads
  • Source
    • "Activation of Gaqand Gao-coupled receptors and protein kinases, such as protein kinase C and protein kinase A, inhibit the activity of TREK1 channels (Enyedi and Czirjak, 2010). However, there is an enhancement of TREK1 activity in response to the activation of Gai-coupled receptors including GABA B receptors (Cain et al., 2008; Sandoz et al., 2012). A number of clinically important drugs also affect the activity of TREK1, including the neuroprotective agent riluzole (Duprat et al., 2000), the antipsychotic agent chlorpromazine (Patel et al., 1998), and the antidepressant agent fluoxetine (Kennard et al., 2005). "
    [Show abstract] [Hide abstract]
    ABSTRACT: TREK1 potassium channels are members of the two pore domain (K2P) potassium channel family and contribute to background potassium conductances in many cell types where their activity can be regulated by a variety of physiological and pharmacological mediators. Fenamates such as FFA, MFA, NFA and diclofenac and the related experimental drug, BL-1249 enhance the activity of TREK1 currents and we show that BL-1249 is the most potent of these compounds. Alternative translation initiation produces a shorter, N-terminus truncated form of TREK1 with a much reduced open probability and a proposed increased permeability to sodium compared to the longer form. We show that both forms of TREK1 can be activated by fenamates and that a number of mutations which affect TREK1 channel gating occlude the action of fenamates but only in the longer form of TREK1. Furthermore, fenamates produce a marked enhancement of current through the shorter, truncated form of TREK1 and reveal a K(+)-selective channel, like the long form. These results provide insight into the mechanism of TREK1 channel activation by fenamates and, given the role of TREK1 channels in pain, they suggest a novel analgesic mechanism for these compounds.
    Molecular pharmacology 02/2014; 85(5). DOI:10.1124/mol.113.091199 · 4.13 Impact Factor
  • Source
    • "The Girkindependent component of the GABA B R-induced current was suppressed by 8Br-cAMP, arguing that this conductance is modulated via the Gi/o-dependent inhibition of adenylyl cyclase activity and subsequent decline in intracellular cAMP. In light of the 8Br-cAMP sensitivity and recent studies (Deng et al., 2009; Sandoz et al., 2012 "
    [Show abstract] [Hide abstract]
    ABSTRACT: Repeated cocaine exposure triggers adaptations in layer 5/6 glutamatergic neurons in the medial prefrontal cortex (mPFC) that promote behavioral sensitization and drug-seeking behavior. While suppression of metabotropic inhibitory signaling has been implicated in these behaviors, underlying mechanisms are unknown. Here, we show that Girk/KIR3 channels mediate most of the GABAB receptor (GABABR)-dependent inhibition of layer 5/6 pyramidal neurons in the mPFC and that repeated cocaine suppresses this pathway. This adaptation was selective for GABABR-dependent Girk signaling in layer 5/6 pyramidal neurons of the prelimbic cortex (PrLC) and involved a D1/5 dopamine receptor- and phosphorylation-dependent internalization of GABABR and Girk channels. Persistent suppression of Girk signaling in layer 5/6 of the dorsal mPFC enhanced cocaine-induced locomotor activity and occluded behavioral sensitization. Thus, the cocaine-induced suppression of GABABR-Girk signaling in layer 5/6 pyramidal neurons of the prelimbic cortex appears to represent an early adaptation critical for promoting addiction-related behavior.
    Neuron 10/2013; 80(1):159-170. DOI:10.1016/j.neuron.2013.07.019 · 15.05 Impact Factor
  • Source
    • "(C,D) Whole-cell recording from HEK293T cell expressing either TREK1-PCS alone (C) or co-expressed with WT (D) and labeled with MAQ. Alternating illumination at 500 nm (green) and 380 nm (magenta) reversibly blocks and unblocks constant outward current, as seen at different holding potentials (Sandoz et al., 2012). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Optogenetic tools were originally designed to target specific neurons for remote control of their activity by light and have largely been built around opsin-based channels and pumps. These naturally photosensitive opsins are microbial in origin and are unable to mimic the properties of native neuronal receptors and channels. Over the last 8 years, photoswitchable tethered ligands (PTLs) have enabled fast and reversible control of mammalian ion channels, allowing optical control of neuronal activity. One such PTL, maleimide-azobenzene-quaternary ammonium (MAQ), contains a maleimide (M) to tether the molecule to a genetically engineered cysteine, a photoisomerizable azobenzene (A) linker and a pore-blocking quaternary ammonium group (Q). MAQ was originally used to photocontrol SPARK, an engineered light-gated potassium channel derived from Shaker. Potassium channel photoblock by MAQ has recently been extended to a diverse set of mammalian potassium channels including channels in the voltage-gated and K2P families. Photoswitchable potassium channels, which maintain native properties, pave the way for the optical control of specific aspects of neuronal function and for high precision probing of a specific channel's physiological functions. To extend optical control to natively expressed channels, without overexpression, one possibility is to develop a knock-in mouse in which the wild-type channel gene is replaced by its light-gated version. Alternatively, the recently developed photoswitchable conditional subunit technique provides photocontrol of the channel of interest by molecular replacement of wild-type complexes. Finally, photochromic ligands also allow photocontrol of potassium channels without genetic manipulation using soluble compounds. In this review we discuss different techniques for optical control of native potassium channels and their associated advantages and disadvantages.
    Frontiers in Molecular Neuroscience 04/2013; 6:6. DOI:10.3389/fnmol.2013.00006 · 4.08 Impact Factor
Show more


41 Reads
Available from