Cl- channels in basolateral renal medullary membranes, VI. Cl- conductance expression in Xenopus oocytes

Cl- channels from basolateral membranes of the mammalian thick ascending limb of Henle differ significantly, in certain of their functional characteristics, from Cl- channels in apical membranes of secretory epithelia such as trachea or small intestine and from certain Cl- channels in the central nervous system. Yet there is no sequence information available about basolateral thick ascending limb Cl- channels. As an initial step in the isolation of a Cl- channel from the thick ascending limb of Henle's loop, we attempted a functional expression of a Cl- conductance in oocytes from Xenopus laevis. Oocytes injected with mRNA isolated from the outer medulla of rabbit kidney had a membrane conductance, measured using a two-electrode voltage clamp, which was sixfold greater than in water-injected oocytes (9.05 +/- 1.56 vs. 1.43 +/- 0.15 microS, respectively). Ion substitution experiments showed the conductance in the RNA-injected oocytes to be Cl- selective. In addition, the Cl(-)-channel blocker diphenylamine-2-carboxylate (1 mM) almost completely inhibited (79 +/- 6%) the increased conductance seen in the RNA-injected oocytes. Fractionation of the mRNA by sucrose gradient centrifugation revealed that peak Cl- channel activity was expressed using an mRNA fraction of 1.8-3.2 kb in size. These results demonstrate that a membrane Cl- conductance can be expressed in X. laevis oocytes injected with size-selected fractions of mRNA from rabbit outer renal medulla.