Intact cytoskeleton is required for small G protein dependent activation of the epithelial Na+ channel.
BACKGROUND
The Epithelial Na(+) Channel (ENaC) plays a central role in control of epithelial surface hydration and vascular volume. Similar to other ion channels, ENaC activity is regulated, in part, by cortical cytoskeleton. Besides, the cytoskeleton is an established target for small G proteins signaling. Here we studied whether ENaC activity is modulated by changes in the state of the cytoskeleton and whether cytoskeletal elements are involved in small G protein mediated increase of ENaC activity.


RESULTS
First, the functional importance of the cytoskeleton was established with whole-cell patch clamp experiments recording ENaC reconstituted in CHO cells. Pretreatment with Cytochalasin D (CytD; 10 microg/ml; 1-2 h) or colchicine (500 microM; 1-3 h) to disassembly F-actin and destroy microtubules, respectively, significantly decreased amiloride sensitive current. However, acute application of CytD induced rapid increase in macroscopic current. Single channel measurements under cell-attached conditions revealed similar observations. CytD rapidly increased ENaC activity in freshly isolated rat collecting duct, polarized epithelial mouse mpkCCD(c14) cells and HEK293 cells transiently transfected with ENaC subunits. In contrast, colchicine did not have an acute effect on ENaC activity. Small G proteins RhoA, Rac1 and Rab11a markedly increase ENaC activity. 1-2 h treatment with colchicine or CytD abolished effects of these GTPases. Interestingly, when cells were coexpressed with ENaC and RhoA, short-term treatment with CytD decreased ENaC activity.


CONCLUSIONS
We conclude that cytoskeleton is involved in regulation of ENaC and is necessary for small G protein mediated increase of ENaC activity.
Karpushev
Alexey V
AV
Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.
Ilatovskaya
Daria V
DV
Pavlov
Tengis S
TS
Negulyaev
Yuri A
YA
Staruschenko
Alexander
A
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D003078:Colchicine / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D015638:Cytochalasin D / Q000494:pharmacology; D053503:Epithelial Sodium Channels / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D051379:Mice; D018408:Patch-Clamp Techniques
D053503:Epithelial Sodium Channels; D015638:Cytochalasin D; D019204:GTP-Binding Proteins; D003078:Colchicine