Effects of several Cl− channel blockers on ionic currents in mouse embryos were studied using whole-cell patch-clamp and microelectrode methods. Microelectrode measurements showed that the resting membrane potential of early embryonic cells (1-cell stage) was −23 mV and that reduction of extracellular Cl− concentration depolarized the membrane, suggesting that Cl− conductance is a major contributor for establishing the resting membrane potential. Membrane currents recorded by whole-cell voltage clamp showed outward rectification and confirmed that a major component of these embryonic currents are carried by Cl− ions. A Cl− channel blocker, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), suppressed the outward rectifier current in a voltage- and concentration-dependent manner. Other Cl− channel blockers (5-nitro-2-[3-phenylpropyl-amino] benzoic acid and 2-[3-(trifluoromethyl)-anilino] nicotinic acid [niflumic acid]) similarly inhibited this current. Simultaneous application of niflumic acid with DIDS further suppressed the outward rectifier current. Under high osmotic condition, niflumic acid, but not DIDS, inhibited the Cl−current, suggesting the presence of two types of Cl− channels: a DIDS-sensitive (swelling-activated) channel, and a DIDS-insensitive (niflumic acid-sensitive) Cl− channel. Anion permeability of the DIDS-insensitive Cl− current differed from that of the compound Cl− current: Rank order of anion permeability of the DIDS-sensitive Cl− channels was I− = Br− > Cl− > gluconate−, whereas that of the DIDS-insensitive Cl− channel was I− = Br− > Cl− ≫ gluconate−. These results indicate that early mouse embryos have a Cl− channel that is highly permeable to amino acids, which may regulate intracellular amino acid concentration.
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