We took interest in the regulation of TREK1 by Gi-coupled GPCRs, since several transmitter-gated versions of these are found in the hippocampus (Padgett and Slesinger, 2010). Postsynaptically,
hippocampal GABAB receptors can inhibit calcium channels (Mintz and Bean, 1993), but they are primarily known to enhance the potassium channels that underlie the slow inhibitory postsynaptic potential (IPSP). The slow IPSP is known to involve G protein-coupled inwardly rectifying potassium (Kir3) channels (Lüscher et al., 1997). Baclofen is generally used to study the GABAB response (Dutar and Nicoll, 1988). Using baclofen, Koyrakh and colleagues showed evidences for an additional unidentified GABAB channel target (Koyrakh et al., 2005). Our PCS approach enables us to identify this channel as TREK1. As selleck kinase inhibitor with Kir3 channels, TREK1 is also postsynaptic (Sandoz et al., 2008), where it is complexed with the postsynaptic machinery via interaction with AKAP150 (Sandoz et al., 2006). This is the second case
where a 2P potassium channel has been implicated in GABAergic signaling, since TREK2 appears to mediate a different and much slower IPSP in entorhinal cortex (Deng et al., 2009). These findings suggest that 2P potassium channels may have a broad role in synaptic signaling in the brain. It breaks with the traditional Selleckchem Small molecule library notions that Kir3 channels are the sole targets of postsynaptic GABAB receptors and that 2P-potassium channels serve simply as leak channels in the hippocampus. Our PCS approach offers an affordable and powerful strategy for identifying the molecular basis of unknown ionic currents and for obtaining a pharmacological foothold in multisubunit signaling proteins. Cysteine mutations were introduced into mTREK1 cDNA in the pIRES2EGFP
expression vector using the QuickChange mutagenesis kit (Agilent). The PCR protocol used was 1 cycle (95°, 30 s), 16 cycles (95°, 30 s; 55°, 1 min; 68°, 12 min). TREK1-PCS has been made by PCR and introduced in pIRES2EGFP expression vector. HEK293 Cells were transiently cotransfected using Lipofectamine 2000 (Invitrogen) with TREK1 mutants or TREK1-PCS. For Levetiracetam coexpression, TREK1 or TREK1-PCS are cotransfected with a ratio of 1:3 to 1:5 with 1.6 μg of DNA total per 18-mm-diameter coverslip. Hippocampal neurons were transfected using the calcium phosphate method. Each 12 mm coverslip received 1.1 μg of TREK1-PCS DNA and 0.2 μg of Tomato DNA. HEK293 cells were maintained in DMEM with 5% FBS on poly-L-lysine-coated glass coverslips. Dissociated hippocampal neurons were obtained from postnatal rats (P0-1) and plated at 75,000 cells/coverslip on poly-L-lysine-coated glass coverslips (12 mM). Neurons were maintained in media containing MEM supplemented with 5% fetal bovine serum, B27 (Invitrogen), and GlutaMAX (Invitrogen). HEK293 cell electrophysiology was performed 24–72 hr after transfection solution containing (in mM): 145 mM NaCl, 4 mM KCl, 1 mM MgCl2, 2 mM CaCl2, and 10 mM HEPES.