COMPARISON OF NA-K-CL COTRANSPORTERS - NKCC1, NKCC2, AND THE HEK CELLNA-K-CL COTRANSPORTER

The Na-K-Cl cotransporter (NKCC) mediates the coupled movement of ions into most animal cells, playing important roles in maintenance of cel l volume and in epithelial Cl transport. Two forms of NKCC have been d escribed: NKCC1, the ''housekeeping'' isoform that is also responsible for C1 accumulation in secretory epithelial cells, and NKCC2, which m ediates apical Na+K+Cl entry into renal epithelial cells. Here we exam ine the kinetic properties of NKCC1, NKCC2, and the endogenous HEK-293 cell cotransporter, Stable expression of rabbit NKCC2A was obtained i n HEK-293 cells utilizing a chimera (h(1)r(2A)0.7) in which the 5'-unt ranslated region and cDNA encoding 104 amino acids of the N terminus a re replaced by the corresponding sequence of NKCC1. h(1)r(2A)0.7 exhib its Na and C1 affinities near those of NKCC1, but it has a 4-fold lowe r Rb affinity, and a 3-fold higher affinity for the inhibitor bumetani de, The activity of h(1)r(2A)0.7 is increased on incubation in low [C1 ] media as is NKCC1, but the resting level of activity is higher in h( 1)r(2A)0.7 and activation is more rapid. h(1)r(2A)0.7 exhibits an appr opriate volume response, unlike NKCC1 for which concomitant changes in [Cl](i) appear to be the overriding factor. These results support a m odel in which apical NKCC2 activity is matched to basolateral C1 exit through changes in [C1],, Reverse transcriptase-polymerase chain react ion of HEK-293 cell mRNA is positive with NKCC1 primers and negative w ith NKCC2 primers, Surprisingly, we found that the behavior of the end ogenous HEK cell Na-K-Cl cotransporter is unlike either of the two for ms which have been described: compared with NKCC1, HEK cell cotranspor ter has a 2.5-fold lower Na affinity, an 8-fold lower Rb affinity, and a 4-fold higher bumetanide affinity. These results suggest the presen ce of a novel isoform of NKCC in HEK-293 cells.