For example, it appears that VRAC channels are hexamers composed of the essential LRRC8A subunit and at least one other subunit from the gene family (i

For example, it appears that VRAC channels are hexamers composed of the essential LRRC8A subunit and at least one other subunit from the gene family (i.e., LRRC8B-E). (Foster City, CA). qPCR primer pairs against human (cat. Pyridostatin hydrochloride no. “type”:”entrez-nucleotide”,”attrs”:”text”:”HP209551″,”term_id”:”306674741″,”term_text”:”HP209551″HP209551) and (cat. no. “type”:”entrez-nucleotide”,”attrs”:”text”:”HP205798″,”term_id”:”306668214″,”term_text”:”HP205798″HP205798) were purchased from Origene Technologies. The SsoAdvanced Universal Probes Supermix was used as a qPCR reagent for ACTB (Bio-Rad, Hercules, CA). The iTaq Universal SYBR Green One-Step Kit was used as a qPCR reagent for and (Bio-Rad, Hercules, CA). Reactions made up of 500 ng RNA were run from at least two biological replicates in duplicate in 10 L reactions according to the manufacturers specifications using a Bio-Rad CFX96 instrument (Bio-Rad, Hercules, CA). and were used as reference genes. Normalizations were done using the 2 2?CT method. Cell culture and Pyridostatin hydrochloride transient transfection. HEK293 cells were cultured in 75 cm2 flasks with Dulbeccos altered Eagles medium supplemented with 10% fetal bovine serum, and 1% penicillin-streptomycin. HEK293 cells stably transfected with the YFP(F46L/H148Q/I152L) fluorophore (i.e., HEK293-Ozzy cells) were cultured in the same medium supplemented with 700 g/mL G418 sulfate (Corning, Corning, NY). For patch-clamp experiments, HEK293 cells plated in 35-mm Nunclon dishes (Thermo Fisher Scientific, Rochester, NY) were co-transfected with 1 g CysLT1R plasmid DNA and 0.5 M green fluorescent protein (GFP; a transfection marker) using Lipofectamine LTX and studied the next day. For calcium imaging experiments, HEK293 cells were transfected with 8 g CysLT1R plasmid DNA in T-75 flasks (Thermo Fisher Scientific, Rochester, NY), plated in 384-well plates (Corning; see below) the next day, and allowed to recover for an additional 24 h before experiments. Patch-clamp electrophysiology. The day of experiments, parental or transfected (see above) HEK293 cells were rinsed with divalent-free Hanks balanced salt answer (HBSS), dissociated using 0.25% trypsin/1 mM EDTA for ~30-s, diluted with complete medium, plated on poly-l-lysine-coated round Pyridostatin hydrochloride glass coverslips, and allowed to recover at 37C in a 5% CO2 incubator for at least 1 h before experiments. Patch pipettes were pulled from Clark Custom 8520 Patch Glass (1.5 OD??1.16 ID) (Harvard Apparatus, Holliston, MA) using a P-1000 Flaming/Brown Microelectrode puller (Sutter Instruments) to a resistance of 2C4 M when filled with the following solution (in mM): 126 CsCl, 2 MgSO4, 20 HEPES, 1 EGTA, 2 Na2ATP, 0.5 GTP (pH 7.2 adjusted with CsOH, 275 mosM). The isotonic bath solution contained (in mM): 75 CsCl, 5 MgSO4, 1 calcium-d-gluconate, 12 HEPES, 8 Tris base, 5 glucose, 2 glutamine, and 95 sucrose (pH 7.4 adjusted with CsOH, 300 mosM). Hypotonic bath (250 mosM) was made by excluding sucrose from the isotonic bath. Macroscopic currents were recorded under voltage-clamp conditions using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA). Cells were voltage clamped at a holding potential of ?30 mV and whole cell currents were elicited by a voltage ramp or step protocol. For voltage ramps, membrane potential was first stepped to ?100 mV for 50 ms and then ramped over 1 s to +100 mV. This was followed by a step back to 0 mV for 4 s before this protocol was repeated. Step changes WBP4 in membrane voltage were induced by stepping membrane voltage to ?120 mV to +120 mV in 800 ms, 20 mV increments. Data were collected at 5 kHz and filtered at 1 kHz. Data acquisition and analysis were performed using Pyridostatin hydrochloride pClamp 9.2 software suite (Molecular Devices). Percent inhibition of compounds was determined by normalizing data to currents inhibited by 10 M DCPIB. Initial rate of swelling-induced VRAC activation was calculated by determining the linear slope of increasing current during the first 180 s following hypotonic answer addition. Fluorescence reporter assay of VRAC function. HEK293-Ozzy cells were dissociated using 0.25% trypsin-1 mM EDTA and plated at a density of 20,000 cells/well in clear bottomed, black-walled Corning PureCoat amine coated 384 well plates (Corning, Corning, NY) and cultured overnight at 37C in a 5% CO2 incubator. The following day Pyridostatin hydrochloride cells were washed with isotonic answer made up of (in mM) 140 NaCl, 5 KCl, and 20 HEPES (pH 7.4, 310 mosM). Compounds from the FDA library were dissolved in hypotonic answer made up of (in mM): 5 KCl, 20 HEPES, and 90 mannitol (pH 7.4, 120 mosM) and added simultaneously to all 384-wells at a final screening concentration of 10 M. After 5 min allowed for cell swelling and VRAC activation, 100 mM NaI was added to all 384-wells simultaneously and Ozzy fluorescence was measured at 1 Hz using a Panoptic kinetic imaging plate reader (WaveFront Bioscience, Franklin, TN). Fluorescence values from each well were normalized to baseline readings (i.e., common.


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