Study of the effect of hypotonic stimuli on mouse epithelial sodium channel (ENaC) activity in Xenopus laevis oocytes

GALIZIA L; PALMA AG; MARINO GI; KOTSIAS BA

Carlos Paz (Córdoba)

Congreso; XLII Reunión anual de la Sociedad Argentina de Biofísica; 2013

SAB

The regulation of the epithelial Na+ channel (ENaC) during cell swelling is relevant in cellular processes in which cell volume changes occurs. Its sensitivity to hypotonic induced swelling was investigated in the Xenopus oocyte expression system with the injection of the three subunits of mouse ENaC (mENaC). We used the voltage clamp technique to study the amiloride-sensitive Na+ currents (INa(amil)) and video microscopy methodologies to assess oocyte volume changes. Under conditions of mild swelling (25 % reduced hypotonicity) inward current amplitude decreased rapidly during 1.5 minutes. INa(amil) kinetics analysis revealed a decrease in slower inactivation time constant during the hypotonic stimuli. We evaluate the role of external Cl- in the inhibition of INa(amil) by hypotonicity. The reduction of extracellular Cl- concentration did not affect the observed response. To study the role of actin cytoskeleton in this response we evaluate short (20 minutes) and long term (2-5hs) effects of cytochalasin D pre-treatment on hypotonic mediated decrease in ENaC activity. Neither short nor long term cytochalasin D treatment affected the observed response. Oocytes expressing a DEG mutant β-ENaC subunit (β-S518K) with an open probability of 1 showed a reduced INa(amil) hypotonic response in comparison to the oocytes injected with the wild type ENaC subunits. During the hypotonic response the ENaC injected oocytes did not show cell volume difference compared with water injected oocytes. On this basis we suggest that hypotonicity-dependent ENaC inhibition is principally mediated through an effect on open probability of channels in the membrane and is independent of actin cytoskeleton.