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.
Zimniak
L
L
Department of Internal Medicine, University of Arkansas College of Medicine, Little Rock.
Reeves
W B
WB
Andreoli
T E
TE
D000818:Animals; D018118:Chloride Channels; D004553:Electric Conductivity; D005260:Female; D007267:Injections; D007679:Kidney Medulla / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism* / Q000502:physiology; D008566:Membranes / Q000378:metabolism; D009865:Oocytes / Q000502:physiology*; D011061:Poly A / Q000378:metabolism; D012333:RNA, Messenger / Q000008:administration & dosage / Q000378:metabolism; D011817:Rabbits; D014982:Xenopus laevis / Q000502:physiology*; D062367:ortho-Aminobenzoates / Q000494:pharmacology
D018118:Chloride Channels; D008565:Membrane Proteins; D012333:RNA, Messenger; D062367:ortho-Aminobenzoates; D011061:Poly A; C001209:fenamic acid