ω conotoxin gvia

For dissociated cell recordings, drugs were applied with a gravity-fed system that positioned a glass capillary tube 100 μm from the recording cell in the direction of superfusion flow. Solution changes were performed with a D.C. controlled microvalve system (Lee; Essex, CT, USA). This method allowed reversible drug applications [26 (link), 33 (link)]. For current clamp recordings drugs were administered into the bath saline. Substances used were the DA receptor D1-like selective agonist SKF 81297 (Cat# S143), DA receptor D1-like antagonist SCH 23390 (Cat# 125941-87-9), Ca²⁺ Ca_V1 antagonist nicardipine (Cat# N7510) all from Sigma-Aldrich-RBI (St Louis, MO, USA); Ca²⁺ Ca_V2.2 blocker ω-conotoxin GVIA (Cat# C-300), Ca²⁺ Ca_V3 blocker TTA-P2 (Cat# T-155), Ca²⁺ Ca_V2.3 blocker SNX-482 (Cat# RTS-500), Na⁺ blocker tetrodotoxin (TTX) (Cat# T-550) from Alomone Laboratories (Israel) and Ca²⁺ Ca_V2.1 blocker ω-agatoxin TK (Cat# 4294-s) from Peptides International (Louisville, KY).

The following drugs were used: bumetanide (200 μM, Sigma-Aldrich, Castle Hill, NSW, Australia), 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 100 μM, Sigma-Aldrich), ω-agatoxin IVA (200 nM, Alomone labs, Jerusalem, Israel), ω-conotoxin GVIA (5 μM, Alomone labs), mibefradil dihydrochloride (1 μM, Sigma-Aldrich,), nicardipine (2.5 μM, Sigma-Aldrich), tetraethylammonium (TEA, 2 mM, 10 mM or 30 mM, Sigma-Aldrich), 4-aminopyridine (4-AP, 0.1mM or 5 mM, Sigma-Aldrich), BDS-I (2.5 μM, Alomone labs) and linopirdine (10 μM, Sigma-Aldrich). The concentrations of the drugs used were based on previous publications [23 (link),31 (link),39 (link)–43 (link)]. With the exception of NPBB, BDS-I, TEA and 4-AP, all drugs were prepared as 1000-fold stock solutions of the highest final concentration used, and diluted to their final concentration in tissue culture medium (TCM: DMEM/F12 containing 10% fetal bovine serum, 6 mg/ml penicillin/streptomycin and 20 mM GlutaMAX (all from Invitrogen, Mulgrave, VIC, Australia). TEA was prepared as 80-fold, NPBB and BDS-I as 100-fold, and 4-AP as 400-fold stock solutions of the highest final concentration used.

The chemical reagents used in this study are as follows: 100 μM nifedipine (Sigma-Aldrich, St. Louis, MO; N7634); 500 μM gadolinium chloride (Sigma-Aldrich, G7532); 10 μM SKF96365 (Sigma-Aldrich, S7809); 1 M EGTA (Sigma-Aldrich, E4378); 500 μM lanthanum chloride (Sigma-Aldrich, L4131); 2 μM ω-Agatoxin IVA (Tocris, Bristol, UK; 2799); 2 μM SNX 482 (Tocris, 2799); 10 μM ω-Conotoxin GVIA (Alomone Labs, C-300); 10 μM U0126 (Sigma-Aldrich, U120); 50 mM potassium chloride (Sigma-Aldrich, P3911); 100 nM AP24534 (Tocris, 4274); 25 μM trifluoperazine dihydrochloride (Sigma Aldrich, T8516).
The following plasmids were purchased from Addgene: pGP-CMV-GCaMP6s was a gift from Douglas Kim (Addgene plasmid # 40753)^{33 (link)}; pGP-CMV-GCaMP6s-CAAX was a gift from Tobias Meyer (Addgene plasmid # 52228)^{34 (link)}; AAV-CAG-GFP was a gift from Karel Svoboda (Addgene plasmid # 28014)^{35 (link)}. The generation procedures for ERK1-dTomato construct were described previously^{36 (link)}. AAV-CAG-GCaMP6s-CAAX was cloned by replacing the GFP sequence in the AAV-CAG-GFP vector with GCaMP6s-CAAX PCR amplicon flanked BamHI/HindIII restriction sites. pHelper, and pAAV-RC1 plasmids were purchased from Cell Biolabs. RaichuEV-HRas FRET biosensor was kindly gifted from Dr. M. Matsuda (Kyoto University).

The apical and basolateral compartments of the Ussing chambers were filled with 10 ml carbogen aerated Krebs solution containing (in mM) 117 NaCl, 4.7 KCl, 1.2 MgCl₂, 1.2 NaH₂PO₄, 20 NaHCO₃, 2.5 CaCl₂ and 11 glucose (all from Sigma-Aldrich, Taufkirchen, Germany), maintained at 37°C. For experiments with Cl^- free buffer solution, Krebs solution was replaced by a carbogen aerated buffer solution containing (in mM) 58.5 NaSO₄, 2.4 KSO₄, 1.2 MgSO₄, 1.2 NaH₂PO₄, 20 NaHCO₃, 2.5 CaSO₄, 11 glucose and 85 mannitol. For experiments with Cl^- and HCO₃^- free buffer solution, Krebs solution was replaced by an oxygen aerated buffer solution containing (in mM) 58.5 NaSO₄, 2.4 KSO₄, 1.2 MgSO₄, 1.2 NaH₂PO₄, 20 HEPES, 2.5 CaSO₄, 11 glucose and 91 mannitol. Stock solutions of TTX (Carl Roth GmbH and Co. KG, Karlsruhe, Germany), ω-conotoxin GVIA (Alomone Labs, Jerusalem, Israel) dissolved in distilled water, as well as stock solutions of Piroxicam (P-5654, Sigma-Aldrich,) dissolved in chloroform and prostaglandin E₂ (PGE₂) (P5640, Sigma-Aldrich) dissolved in DMSO, were stored at -20°C until use at indicated final concentrations in Ussing chambers. Filters used for experiments to prevent distension had a pore size of 0.45 μM and a diameter of 2.5 cm (MF-Millipore, HABP02500, Merck Millipore Ltd., Ireland, Tullagreen, Carrigtwohill, County Cork, Ireland).