Amino acid-permeable anion channels in early mouse embryos and their possible effects on cleavage

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 con centration-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- much greater than 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.