Molecular determinants of hyperosmotically activated NKCC1-mediated K+/K+ exchange

Na(+)-K(+)-2Cl(-) cotransport (NKCC) mediates the movement of two Cl(-) ions for one Na(+) and one K(+) ion. Under isosmotic conditions or with activation of the kinases SPAK/WNK4, the NKCC1-mediated Cl(-) uptake in Xenopus laevis oocytes, as measured using (36)Cl, is twice the value of K(+) uptake, as determined using (86)Rb. Under hyperosmotic conditions, there is a significant activation of the bumetanide-sensitive K(+) uptake with only a minimal increase in bumetanide-sensitive Cl(-) uptake. This suggests that when stimulated by hypertonicity, the cotransporter mediates K(+)/K(+) and Cl(-)/Cl(-) exchange. Although significant stimulation of K(+)/K(+) exchange was observed with NKCC1, a significantly smaller hyperosmotic stimulatory effect was observed with NKCC2. In order to identify the molecular determinant(s) of this NKCC1-specific activation, we created chimeras of the mouse NKCC1 and the rat NKCC2. Swapping the regulatory amino termini of the cotransporters neither conferred activation to NKCC2 nor prevented activation of NKCC1. Using unique restrictions sites, we created additional chimeric molecules and determined that the first intracellular loop between membrane-spanning domains one and two and the second extracellular loop between membrane-spanning domains three and four of NKCC1 are necessary components of the hyperosmotic stimulation of K(+)/K(+) exchange.