Isoform-specific function and distribution of Na/K pumps in the frog lens epithelium.

Abstract

Epithelial cells from the anterior and equatorial surfaces of the frog lens were isolated and used the same day for studies of the Na/K ATPase. RNase protection assays showed that all cells express alpha(1)- and alpha(2)-isoforms of the Na/K pump but not the alpha(3)-isoform, however the alpha(2)-isoform dominates in anterior cells whereas the alpha(1)-isoform dominates in equatorial cells. The whole cell patch-clamp technique was used to record functional properties of the Na/K pump current (I(P)), defined as the current specifically inhibited by dihydro-ouabain (DHO). DHO-I(P) blockade data indicate the alpha(1)-isoform has a dissociation constant of 100 microm DHO whereas for the alpha(2)-isoform it is 0.75 microm DHO. Both alpha(1)- and alpha(2)-isoforms are half maximally activated at an intracellular Na(+)-concentration of 9 mm. The alpha(1)-isoform is half maximally activated at an extracellular K(+)-concentration of 3.9 mm whereas for the alpha(2)-isoform, half maximal activation occurs at 0.4 mm. Lastly, transport by the alpha(1)-isoform is inhibited by a drop in extracellular pH, which does not affect transport by the alpha(2)-isoform. Under normal physiological conditions, I(P) in equatorial cells is approximately 0.23 microA/microF, and in anterior cells it is about 0.14 microA/microF. These current densities refer to the area of cell membrane assuming a capacitance of around 1 microF/cm(2). Because cell size and geometry are different at the equatorial vs. anterior surface of the intact lens, we estimate Na/K pump current density per area of lens surface to be around 10 microA/cm(2) at the equator vs. 0.5 microA/cm(2) at the anterior pole.