Gc150f 10

Hippocampal slices (300-µm thick) were prepared from P10 and older mice, and incubated at 34 °C in sucrose-containing artificial cerebrospinal fluid (sucrose-ACSF) (85 mM NaCl, 75 mM sucrose, 2.5 mM KCl, 25 mM glucose, 1.25 mM NaH₂PO₄, 4 mM MgCl₂, 0.5 mM CaCl₂, and 24 mM NaHCO₃) for 0.5 h, and then held at room temperature until recording. Cells were visualized by infrared differential interference contrast optics in an upright microscope (Olympus; BX-51WI) using a Hamamatsu Orca-Flash 4.0 CMOS camera. Recordings were performed using borosilicate glass pipettes with filament (Harvard Apparatus; GC150F-10; o.d., 1.5 mm; i.d., 0.86 mm; 10-cm length) at 33 °C in ACSF (126 mM NaCl, 2.5 mM KCl, 10 mM glucose, 1.25 mM NaH₂PO₄, 2 mM MgCl₂, 2 mM CaCl₂, and 26 mM NaHCO₃) with a standard intracellular solution (95 mM K-gluconate, 50 mM KCl, 10 mM HEPES, 4 mM Mg-ATP, 0.5 Na-GTP, 10 mM phosphocreatine; pH 7.2, KOH adjusted, 300 mOsm). All recordings were made using MultiClamp700B amplifier (Molecular Devices), and signals are filtered at 10 kHz (Bessel filter) and digitized (50 kHz) with a Digidata1440A and pClamp10 (Molecular Devices).

Both control and salt-treated seedlings were immobilized in a measuring chamber as described earlier (Bose et al., 2014a) and were kept for pre-conditioning in BSM (with/without NaCl) for 50–60min. A conventional microelectrode (GC 150F-10, Harvard Apparatus Ltd, Kent, UK) with a tip diameter of ~0.5 µm was filled with 0.5M KCl and connected to a MIFE electrometer (Shabala et al., 1997 (link)) via an Ag–AgCl half-cell. The mounted electrode was then impaled in the external cortex cells of intact roots using a manually operated hydraulic micromanipulator (MHW-4-1; Narishige, Tokyo, Japan). The steady-state MP measurements were conducted from at least six individual roots (from both the mature and elongation zones as described above) with no more than four impalements per root undertaken. Each measurement was averaged for at least a 30s interval (Bose et al., 2014a).

Late wandering third instar larvae were fillet dissected in standard HL3 buffer (adapted from Stewart et al., 1994 (link)), 70 mM NaCl, 5 mM KCl, 20 mM MgCl₂, 10 mM NaHCO₃, 115 mM Sucrose, 5 mM HEPES, 1.5 mM CaCl, pH 7.25) ventral surface down with a lateral incision in order preserve both the ventral and dorsal midlines. Suction (GC150F-10 Harvard Apparatus) and sharp (GC100F-10 Harvard Apparatus) electrodes were pulled using a P-97 pipette puller (Sutter Instrument Company). Sharp electrode muscle impalement (DA1 muscle, at the dorsal midline) and inter-segmental nerve suction (ventral midline) were performed using an Olympus BX50WI compound microscope with 10X air (Olympus 10x/0.25 N.A.) and 20X dipping (Olympus UMPlanFL 20x/0.5 N.A.) objective lenses. Recordings were made at 21°C in HL3 using an Axopatch-1D amplifier (Axon Instruments), a 1322A DigiData (Axon Instruments), a DS2A-MkII Constant Voltage Isolated Stimulator (Digitimer Ltd.) and pCLAMP 10.4 acquisition software (Molecular Devices). mEJP (2–3 min) and eEJP (3 rounds of 20 stimulations, 5V) recordings were made in current-clamp mode from muscle cells with an input resistance above 8MOhm and a stable resting membrane potential between −40 mV and −70 mV. Analysis of eEJPs was performed using Clampfit10.6 (Molecular Devices) and mEJPs using Mini-Analysis6.0.7 (Synaptosoft).

BEL-7404 cell lines overexpressing human ether-a-go-go-related gene (hERG) were used for the experiment (69 (link)). The cultured cells were placed on 35-mm diameter Petri dishes in extracellular solution and incubated at 37°C for 30 min to adhere. A whole-cell patch-clamp method was performed to record hERG currents at room temperature (22°C) using WPC-100 resistive feedback amplifiers (ESF electronic). When filled with internal pipette solution, the resistance of the patch pipettes (GC150F-10; Harvard Apparatus) averaged 2–3 MΩ. Patch pipettes were pulled from borosilicate glass by a P-97 horizontal puller (Sutter Instrument Co.). For drug application, PIX or cisapride (positive control) was applied using a piezoelectric-driven micromanipulator (P-287.70, Physik Instrumente) at a flow rate of 100–200 μL/min, as described previously (70 (link)).

HEK-293 were transiently co-transfected with 0.3 μg of pCR3.1-CD8 and 1.5 μg of plasmid (pCR3.1) containing either Kir7.1wt or Kir7.1-HA cDNA using Lipofectamine 3000 (Invitrogen #L3000). At 24 h post-transfection, the cells were visualized applying Dynabeads-CD8 (Invitrogen #11147D) and recordings were performed from isolated cells using standard whole-cell patch-clamp technique as described elsewhere (Díaz and Sepúlveda, 1995 (link)). Currents were recorded using an Axopatch 200B amplifier (Axon Instruments). Acquisition was at 2 kHz with filter at 1kHz employing pClamp10 software (Axon Instruments). For experiments, borosilicate capillaries (Harvard Apparatus GC150F-10, 1.5 mm OD × 1.17 mm ID) were fire polished to 2–4 Mω and covered in beeswax. The bath solution contained in mM: 135 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 1 Glucose, 10 HEPES/Tris, pH7.5 and sucrose to obtain 310 mOsm/L. In alternative bathing solutions NaCl was omitted and KCl or RbCl increased to 140 mM. BaCl₂ at 1 mM was added to the last solution as indicated. The pipette solution contained in mM: 140 KCl, 1 MgCl₂, 1 Glucose, 10 EGTA and 10 HEPES/Tris, pH7.5 (300 mOsm/L).