Molecular Biology and Gene Expression
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 co-expression, 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 cover slip. 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 h after transfection solution containing (in mM): 145 mM NaCl, 4 mM KCl, 1 mM MgCl2, 2 mM CaCl2 and 10 mM HEPES. Glass pipettes of resistance between 3 and 6 MΩ were filled with intracellular solution containing (in mM): 140 KCl, 10 Hepes, 3 Na2ATP, 0.2 Na2GTP, 5 EGTA, 3 MgCl2, pH 7.4. Cells were voltage clamped using an Axopatch 200A (Molecular Devices) amplifier in the whole cell mode.
Hippocampal neuron whole cell patch clamp electrophysiology was performed 3–6 days after transfection (DIV 12–15 for cultured neurons; DIV 6–8 for slices). For voltage and current clamp experiments in cultured neurons, extracellular solution contained (in mM): 138 NaCl, 1.5 KCl, 1.2 MgCl2, 2.5 CaCl2, 10 glucose, 5 Hepes, (plus 10 CNQX, 10 Bicucculine only for voltage clamp experiments), pH 7.4. In slices ACSF contained (in mM) 19 NaCl, 2.5 KCl, 1.3 MgSO4, 1 NaH2PO4-H2O, 26.2 NaHCO3, 11 glucose and 2.5 CaCl2 and was continuously perfused and bubbled with 95% O2/5% CO2. For all experiments, intracellular solution contained (in mM): 140 K-Gluconate, 10 NaCl, 5 EGTA, 2 MgCl2, 1 CaCl2, 10 Hepes, 2 MgATP, 0.3 Na2GTP, pH 7.2. For slice experiments MAQ was diluted in NMDG-labeling solution containing (in mM): 150 NMDG-HCl, 3 KCl, 0.5 CaCl2, 5 MgCl2, 10 HEPES and 5 glucose, pH 7.4. Only cells with a resting potential <−45 mV were analyzed. All pharmacological compounds for voltage clamp recording were dissolved in appropriate extracellular buffers before application using a gravity-driven perfusion system.
Illumination was controlled using a Polychrome V monochromator (TILL Photonics) through a 20× objective or with a Lambda DG4 high speed wavelength switcher (Sutter) with 380 nm and 500 nm filters through a 40× objective. pClamp software was used for both data acquisition and control of illumination. To conjugate MAQ, cells were incubated in 50–100 µM MAQ for 60 minutes in the dark at room temperature in standard extracellular cell buffer for either HEK293 cells or hippocampal neurons. The percentage of block was calculated from the current induced by a voltage-ramp at −20 mV as (I500-I380/I500)*100.
Preparation of cultured hippocampal slices
Hippocampi were obtained from postnatal Sprague-Dawley rats (postnatal days 6 and 7), cut into 400-µm slices and cultured on 0.4-µm Millicell culture inserts (Millipore) in Neurobasal-A medium (Gibco) supplemented with 20% horse serum (vol/vol), insulin, ascorbic acid, GlutaMAX (Gibco), penicillin/streptomycin, HEPES and Ara-C. Slices were transfected 2–3 d after isolation by Biolistic gene transfer using a BioRad Helios Gene Gun and gold microcarriers coated with both DNA encoding TREK1-PCS in Pires2EGFP and cytosolic tdTomato (to aid in the visualization of the transfected cells).