EXTERNAL-ANION-DEPENDENT ANIONIC CURRENT IN BLASTODERM CELLS OF EARLYMEDAKA FISH EMBRYOS

1. Anionic current was studied by a whole-cell variation of the patch- clamp technique in blastoderm cells dissociated from medaka (Oryzias l atipes) embryos at the early blastula stage. The blastoderm cells were mechanically dissociated without using proteolytic enzymes. 2. The an ionic current was deactivated by hyperpolarizing steps. The steady-sta te current-voltage (I-V) relationship of the anion current was accurat ely represented by the Boltzmann relation with z = 1.01 +/- 0.02 (+/- S.E.M., n = 4) for voltage-dependent activation when internal calcium ions were buffered at 100 nM by BAPTA-Ca2+ buffer. 3. When the interna l calcium concentration was reduced to 10 nM, this anionic current bec ame an external-calcium-dependent current and was remarkably decreased by removal of external calcium ions. Furthermore, this anionic curren t was almost abolished when the internal calcium concentration was gre atly reduced by chelating: Ca2+ ions with 10 mM BAPTA. 4. This current was not affected by replacing external Na+ with TEA(+) or N-methyl-D- glucamine. The reversal potentials shifted by +58.6 mV for a 10-fold d ecrease in the external Cl- concentration. External Cl- ions were subs tituted with various anions and respective current-reversal potentials were measured. The order of permeability was I- > Br- > Cl- > F-. Bot h the outward and inward currents almost disappeared in external Cl--f ree solutions. 5. The voltage dependency of the anionic current shifte d in a positive direction with the reduction in the external Cl- conce ntration, the potentials at which half the channels were activated (V- 1/2) being -59.8 +/- 1.2 mV (n = 4), -40.7 +/- 0.8 mV (n = 4), -14/9 /- 0.7 mV (n = 4), and -0.7 +/- 0.6 mV (n = 4) for 135, 90, 67, and 45 mM Cl-, respectively. This implies that the inward current, brought b y efflux of internal anions, decreases with reductions in the external Cl- concentration. In the presence of external 135 mM Br-, the voltag e dependency of the anionic current shifted in the negative direction (V-1/2 = -84.5 +/- 1.0 mV and z = 0.91 +/- 0.02 (n = 3)) in comparison with that in the external Cl- medium. It further shifted in the negat ive direction in the presence of 135 mM I- (V-1/2 = -93.8 +/- 1.5 mV a nd z = 0.85 +/- 0.03 (n = 3)). The inward current of the anion channel is considered to be affected by both the type and the concentration o f external halide ions. 6. At the beginning of the blastula stage, the blastocoel cavity is formed for the first time. A mechanism is necess ary to transport ions to the cavity without losing them to pond water outside the embryos. Since this anion channel is external-anion depend ent, the current cannot be activated in the membrane facing the pond w ater, where concentrations of anions are much lower than those of the intracavernous solution. Only after blastocoel formation are blastoder m cells first exposed to the extracellular fluid with high ionic conce ntrations. Then, the anion channel would become effective for maintain ing the Cl- equilibrium potential.