Rat ENaC expressed in Xenopus laevis oocytes is activated by cAMP and blocked by Ni(2+).
We used oocytes of the South African clawed toad Xenopus laevis to express the three subunits of the epithelial Na(+) channel from rat distal colon (rENaC). We combined conventional dual-microelectrode voltage-clamp with continuous capacitance (C(m)) measurements and noise analysis to evaluate the effects of cAMP and Ni(2+) on rENaC. Control oocytes or rENaC-expressing oocytes exhibited no spontaneous fluctuations in current. However, in rENaC-expressing oocytes amiloride induced a marked plateau-shaped rise of the power density spectra. Recordings using four different concentrations of amiloride revealed that the blocker-channel interactions were of the first order. A cocktail of the membrane permeant cAMP analogue chlorophenylthio-cAMP and IBMX (cAMP cocktail) increased amiloride-sensitive current (I(ami)) and conductance (G(ami)). Furthermore, C(m) was also increased following cAMP application, indicating an increase in plasma membrane surface area. Noise analysis showed that cAMP increased the number of active channels in the oocyte membrane while single-channel current decreased. From these data we conclude that cAMP triggered exocytotic delivery of preformed rENaCs to the plasma membrane. Ni(2+) (2.5 mM) inhibited about 60% of the rENaC current and conductance while C(m) remained unaffected. Noise analysis revealed that this inhibition could be attributed to a decrease in the apparent channel density, while single-channel current did not change significantly. These observations argue for direct effects of Ni(2+) on channel activity rather than induction of endocytotic removal of active channels from the plasma membrane.
Segal
Andrei
A
Laboratory of Physiology, KU Leuven, Campus Gasthuisberg, B-3000, Leuven, Belgium.
Cucu
Dana
D
Van Driessche
Willy
W
Weber
Wolf Michael
WM
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D025802:Electric Capacitance; D004791:Enzyme Inhibitors / Q000494:pharmacology; D053503:Epithelial Sodium Channels; D005089:Exocytosis / Q000187:drug effects; D007424:Intracellular Fluid / Q000378:metabolism; D008845:Microinjections; D009532:Nickel / Q000494:pharmacology*; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D051381:Rats; D012815:Signal Processing, Computer-Assisted; D012964:Sodium / Q000378:metabolism; D015222:Sodium Channels / Q000096:biosynthesis* / Q000187:drug effects* / Q000235:genetics; D014982:Xenopus laevis
D004791:Enzyme Inhibitors; D053503:Epithelial Sodium Channels; D015222:Sodium Channels; D000584:Amiloride; D009532:Nickel; D012964:Sodium; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases