Characterization and imaging of A6 epithelial cell clones expressing fluorescently labeled ENaC subunits

被引:22
作者
Blazer-Yost, BL
Butterworth, M
Hartman, AD
Parker, GE
Faletti, CJ
Els, WJ
Rhodes, SJ
机构
[1] Indiana Univ Purdue Univ, Dept Biol, Indianapolis, IN 46202 USA
[2] Univ Cape Town, Sch Med, Dept Anat & Cell Biol, ZA-7925 Cape Town, South Africa
来源
AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY | 2001年 / 281卷 / 02期
关键词
sodium transport; amiloride; aldosterone; insulin; channel trafficking; adenosine; 3; 5 '-cyclic monophosphate; signal transduction; epithelial sodium channel; green fluorescent protein;
D O I
10.1152/ajpcell.2001.281.2.C624
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
A6 model renal epithelial cells were stably transfected with enhanced green fluorescent protein (EGFP)-tagged alpha- or beta -subunits of the epithelial Na+ channel (ENaC). Transfected RNA and proteins were both expressed in low abundance, similar to the endogenous levels of ENaC in native cells. In living cells, laser scanning confocal microscopy revealed a predominately subapical distribution of EGFP-labeled subunits, suggesting a readily accessible pool of subunits available to participate in Na+ transport. The basal level of Na+ transport in the clonal lines was enhanced two- to fourfold relative to the parent line. Natriferic responses to insulin or aldosterone were similar in magnitude to the parent line, while forskolin-stimulated Na+ transport was 64% greater than control in both the alpha- and beta -transfected lines. In response to forskolin, EGFP-labeled channel subunits traffic to the apical membrane. These data suggest that channel regulators, not the channel per se, form the rate-limiting step in response to insulin or aldosterone stimulation, while the number of channel subunits is important for basal as well as cAMP-stimulated Na+ transport.
引用
收藏
页码:C624 / C632
页数:9
相关论文
共 35 条
[1]   Feedback inhibition of rat amiloride-sensitive epithelial sodium channels expressed in Xenopus laevis oocytes [J].
Abriel, H ;
Horisberger, JD .
JOURNAL OF PHYSIOLOGY-LONDON, 1999, 516 (01) :31-43
[2]   Interactions between subunits of the human epithelial sodium channel [J].
Adams, CM ;
Snyder, PM ;
Welsh, MJ .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1997, 272 (43) :27295-27300
[3]   Aldosterone-induced increase in the abundance of Na+ channel subunits [J].
Asher, C ;
Wald, H ;
Rossier, BC ;
Garty, H .
AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY, 1996, 271 (02) :C605-C611
[4]  
Ausubel FM., 1994, Curr. Protoc. Mol. Biol
[5]  
Barbry P, 1996, Ion Channels, V4, P115
[6]   Imaging exocytosis and endocytosis [J].
Betz, WJ ;
Mao, F ;
Smith, CB .
CURRENT OPINION IN NEUROBIOLOGY, 1996, 6 (03) :365-371
[7]   Hormonal regulation of ENaCs: insulin and aldosterone [J].
Blazer-Yost, BL ;
Liu, XH ;
Helman, SI .
AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY, 1998, 274 (05) :C1373-C1379
[8]   INSULIN-LIKE GROWTH FACTOR-I STIMULATES RENAL EPITHELIAL NA+-TRANSPORT [J].
BLAZERYOST, BL ;
COX, M .
AMERICAN JOURNAL OF PHYSIOLOGY, 1988, 255 (03) :C413-C417
[9]  
BLAZERYOST BL, 1992, BIOCHEM INT, V26, P887
[10]   The amiloride-sensitive epithelial Na+ channel: Binding sites and channel densities [J].
BlazerYost, BL ;
Helman, SI .
AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY, 1997, 272 (03) :C761-C769