Choline and NMDG directly reduce outward currents: reduced outward current when these substances replace Na⁺is alone not evidence of Na⁺-activated K⁺currents

Abstract

Choline chloride is often, and N-methyl-d-glucamine (NMDG) sometimes, used to replace sodium chloride in studies of sodium-activated potassium channels. Given the high concentrations used in sodium replacement protocols, it is essential to test that it is not the replacement substances themselves, as opposed to the lack of sodium, that cause any observed effects. We therefore compared, in lobster stomatogastric neurons and leech Retzius cells, the effects of applying salines in which choline chloride replaced sodium chloride, and in which choline hydroxide or sucrose was added to normal saline. We also tested, in stomatogastric neurons, the effect of adding NMDG to normal saline. These protocols allowed us to measure the direct effects (i.e., effects not due to changes in sodium concentration or saline osmolarity or ionic strength) of choline on stomatogastric and leech currents, and of NMDG on stomatogastric currents. Choline directly reduced transient and sustained depolarization-activated outward currents in both species, and NMDG directly reduced transient depolarization-activated outward currents in stomatogastric neurons. Experiments with lower choline concentrations showed that adding as little as 150 mM (stomatogastric) or 5 mM (leech) choline reduced at least some depolarization-activated outward currents. Reductions in outward current with choline chloride or NMDG replacement alone are thus not evidence of sodium-activated potassium currents. NEW & NOTEWORTHY We show that choline or N-methyl-d-glucamine (NMDG) directly (i.e., not due to changes in extracellular sodium) decrease outward currents. Prior work studying sodium-activated potassium channels in which sodium was replaced with choline or NMDG without an addition control may therefore be artifactual.

Acid‐sensing ion channel 1 contributes to pulmonary arterial smooth muscle cell depolarization following hypoxic pulmonary hypertension

“…3B). Although the effect of NMDG to blunt outward currents in PASMC from CH rats is unclear, it may be due to a direct effect of NMDG to inhibit outward currents, as demonstrated by others (Thuma & Hooper, 2018). On the other hand, I NSCC was minimally affected by the Clchannel blockers, 5-nitro-2-(3-phenylpropylamino) benzoic acid (5-NPPB, 100 μM) and N-phenylanthranilic acid (NPA, 1 mM) added to bath and pipette (Fig.…”

Sodium action potentials in placozoa: Insights into behavioral integration and evolution of nerveless animals

“…To reduce movements, placozoans were immobilized in 1% agarose made either artificial seawater (ASW: NaCl-450mM, KCl-13.4mM, MgCl 2 -24mM, CaCl 2 -9.5mM, MgSO 4 -5mM; pH = 8.0) or sodium-free seawater, where NaCl was replaced by N-methyl-dglucamine (NMDG, 390 mM, pH = 8.0) [38]. Initially, 1% agarose and seawater were heated to 40 °C, and then we placed a small (<0.1-0.2 mL) drop of the agarose solution in a dish at room temperature.…”

Sodium regulates PLC and IP3R-mediated calcium signalling in invasive breast cancer cells

“…These findings also present a cautionary tale for cell physiologists using the methods employed in this study for exploring the relationship between Na + conductance, Ca 2+ signalling and wider cell behaviour. Application of 0Na + /NMDG or 0Na + /ChoCl is widely utilised for isolating cell responses from the influence of extracellular Na + ; however, these manoeuvres are not inert (Thuma & Hooper, 2018). In addition to assessing for reverse mode NCX activity (Chovancova et al ., 2020), Na + regulation of GPCRs (and resultant effects on Ca 2+ dynamics) should be taken into careful consideration and controlled for when using 0Na + /NMDG, 0Na + /ChoCl or similar manoeuvres to probe Na + conductance mechanisms.…”