AJP - Renal Physiology, Vol 259, Issue 2 383-F388, Copyright © 1990 by American Physiological Society
Expression of the Na(+)-K(+)-2Cl- cotransporter in Xenopus oocytes
C. E. Burnham, J. Kidd and H. C. Palfrey
Department of Pharmacological and Physiological Sciences, University of Chicago, Illinois 60637.
The Na(+)-K(+)-2Cl- cotransporter (NKCCT) is important in mediating net
salt transport in a variety of cells. As a first step in obtaining
structural information on this transport system, we attempted to express it
in Xenopus laevis oocytes by injection of poly(A)+ mRNA from rat kidney.
Four days after injection, batches of oocytes were tested for 86Rb uptake
in presence and absence of 10 microM bumetanide to distinguish that
fraction of influx mediated by NKCCT. In the absence of bumetanide, the
oocytes formed a bimodal distribution with respect to 86Rb uptake, with
some oocytes accumulating significantly more 86Rb than others. In the
presence of bumetanide, or when Na was replaced with choline, that group of
oocytes taking up more 86Rb did not appear. Sham-injected oocytes did not
accumulate sufficient 86Rb to be distinguishable above background. Taken
together, these data suggest that some of the mRNA-injected oocytes
expressed the rat renal NKCCT. Crude poly(A)+ mRNA was separated by
acid-urea agarose gel electrophoresis, and fractions were injected into
oocytes. One fraction corresponding to messages of approximately 7
kilobases in length induced a bumetanide-sensitive 86Rb influx resembling
that seen with total mRNA. Poly(A)+ mRNA fractionated on sucrose density
gradients gave similar results. It is concluded that the rat kidney NKCCT
has been expressed in Xenopus oocytes from a high molecular weight fraction
of poly(A)+ mRNA.
