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.

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    • "To photo stimulate action potentials, a Shaker pore mutation was used to convert the K + channel into a non-selective cation channel (Chambers et al. 2006). Later, MAL-AZO-QA was used to control the two-pore TREK1 channel in transfected hippocampal neurons (Sandoz et al. 2012). Cysteines were engineered into homologous sites on each pore loop. "
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    • "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). "
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    • "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 "
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