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.