Retigabine

We used several pharmacological agents including Hm1a (Alomone Labs STH-601), linopirdine (Sigma L134), Retigabine (Alomone D-23129), the cholinomimetic carbamoylcholine chloride (carbachol; Sigma C4382), muscarine (Sigma M6532), and the nicotinic agonist 4-Acetyl-1,1-dimethylpiperazinium iodide (Tocris 0352). Hm1a, muscarine, and 4-Acetyl-1,1-dimethylpiperazinium iodide were dissolved in deionized water, aliquoted, and frozen at −20° C. carbachol was stored as a powder at RT, and both linopirdine and Retigabine were stored as 100 mM stock solutions in DMSO (Sigma) aliquoted in −20°C. For pharmacological experiments in which drug was dissolved in DMSO, the same concentration of DMSO was present in control external solution. After a baseline recording, drugs were perfused in at 3 mL/min while continuously recording membrane potential; repeat measurements were performed 10 min after wash-in. A subset of cells were recorded for up to 1 hr following washout of each drug, as needed in some cases to observe complete or near-complete washout. For experiments using carbachol, repeat measurements were made 5 min after wash-in.

NNC 55-0396 and edelfosine were from Tocris Bioscience (UK); XE991, retigabine, nifedipine, AVP, 2-Aminoethoxydiphenyl borate (2-APB), tetracaine and all other chemicals were obtained from Sigma-Aldrich (UK) (Supplemental Table III).

Phenylephrine hydrochloride, sodium nitroprusside (SNP), ANP (127–150 rat), XE991 (10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride), carbachol chloride, isoprenaline hydrochloride, forskolin, mefenamic acid, diclofenac sodium, levcromakalim and retigabine were sourced from Sigma-Aldrich. Linopirdine hydrochloride, HMR1556 (N-[(3R,4S)-3,4-Dihydro-3-hydroxy-2,2-dimethyl-6-(4,4,4-trifluorobutoxy)-2H-1-benzopyran-4-yl]-N-methylmetanesulfonamide), treprostinil, R-L3 (5-(2-Fluorophenyl)-1,3-dihydro-3-(1H-indol-3-ylmethyl)-1-methyl-2H-1,4-benzodiazepin-2-one), ML277 ((2R)-N-[4-(4-Methoxyphenyl)-2-thiazolyl]-1-[(4-methylphenyl)sulfonyl]-2-piperidinecarboxamide), ICA-069673 (N-(2-Chloro-5-pyrimidinyl)-3,4-difluorobenzamide), GW0742, SC-51322 and iberiotoxin were obtained from Tocris Bioscience (Bristol, UK). Sildenafil citrate was supplied by Cambridge Bioscience (Cambridge, UK), A23187 was from Calbiochem (Beeston, Nottinghamshire, UK) and glyceryl trinitrate (GTN) from Aspire Pharma Ltd. (Petersfield, UK). Depending on solubility, stock solutions were prepared as 100 or 10 mM drug in water or dimethyl sulphoxide (DMSO) and stored as frozen aliquots. DMSO was present at <0.1% in the myograph chamber.