The NH2 terminus of the epithelial sodium channel contains an endocytic motif

被引:39
作者
Chalfant, ML
Denton, JS
Langloh, AL
Karlson, KH
Loffing, J
Benos, DJ
Stanton, BA
机构
[1] Dartmouth Med Sch, Dept Physiol, Hanover, NH 03755 USA
[2] Univ Alabama, Dept Physiol & Biophys, Birmingham, AL 35233 USA
[3] Univ Zurich, Inst Anat, CH-8057 Zurich, Switzerland
关键词
D O I
10.1074/jbc.274.46.32889
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
An epithelial sodium channel (ENaC) is composed of three homologous subunits: alpha, beta, and gamma. To elucidate the function of the cytoplasmic, NH2 terminus of rat ENaC (rENaC) subunits, a series of mutant cDNAs was constructed and the cRNAs for all three subunits were expressed in Xenopus oocytes. Amiloride-sensitive Na+ currents (I-Na) were measured by the two-electrode voltage clamp technique. Deletion of the cytoplasmic, NH2 terminus of alpha (Delta 2-109), beta (Delta 2-49), or gamma-rENaC (Delta 2-53) dramatically reduced I-Na. A series of progressive, NH2 terminal deletions of alpha-rENaC were constructed to identify motifs that regulate I-Na. Deletion of amino acids 2-46 had no effect on I-Na: however, deletion of amino acids 2-51, 2-55, 2-58, and 2-67 increased I-Na by similar to 4-fold. By contrast, deletion of amino acids 2-79, 2-89, 2-100, and 2-109 eliminated I-Na. To evaluate the mechanism whereby Delta 2-67-alpha-rENaC increased I-Na, single channels were evaluated by patch clamp. The single-channel conductance and open probability of alpha,beta,gamma-rENaC and Delta 2-67-alpha,beta,gamma-rENaC were similar. However, the number of active channels in the membrane increased from 6 +/- 1 channels per patch with alpha,beta,gamma-rENaC to 11 +/- 1 channels per patch with Delta 2-67-alpha,beta,gamma-rENaC. Laser scanning confocal microscopy confirmed that there were more Delta 2-67-alpha,beta,gamma-rENaC channels in the plasma membrane than alpha,beta,gamma-rENaC channels. Deletion of amino acids 2-67 in alpha-rENaC reduced the endocytic retrieval of channels from the plasma membrane and increased the half-life of the channel in the membrane from 1.1 +/- 0.2 to 3.5 +/- 1.1 h. We conclude that the cytoplasmic, NH2 terminus of alpha-, beta-, and gamma-rENaC is required for channel activity, The cytoplasmic, NH2 terminus of alpha-rENaC contains two key motifs. One motif regulates the endocytic retrieval of the channel from the plasma membrane. The second motif is required for channel activity.
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页码:32889 / 32896
页数:8
相关论文
共 40 条
[1]   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
[2]   Regulation of a cloned epithelial Na+ channel by its β- and γ-subunits [J].
Awayda, MS ;
Tousson, A ;
Benos, DJ .
AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY, 1997, 273 (06) :C1889-C1899
[3]   Diversity and regulation of amiloride-sensitive Na+ channels [J].
Benos, DJ ;
Awayda, MS ;
Berdiev, BK ;
Bradford, AL ;
Fuller, CM ;
Senyk, O ;
Ismailov, II .
KIDNEY INTERNATIONAL, 1996, 49 (06) :1632-1637
[4]  
BENOS DJ, 1999, IN PRESS J PHYSL
[5]   Subunit stoichiometry of a core conduction element in a cloned epithelial amiloride-sensitive Na+ channel [J].
Berdiev, BK ;
Karlson, KH ;
Jovov, B ;
Ripoll, PJ ;
Morris, R ;
Loffing-Cueni, D ;
Halpin, P ;
Stanton, BA ;
Kleyman, TR ;
Ismailov, II .
BIOPHYSICAL JOURNAL, 1998, 75 (05) :2292-2301
[6]   AMILORIDE-SENSITIVE EPITHELIAL NA+ CHANNEL IS MADE OF 3 HOMOLOGOUS SUBUNITS [J].
CANESSA, CM ;
SCHILD, L ;
BUELL, G ;
THORENS, B ;
GAUTSCHI, I ;
HORISBERGER, JD ;
ROSSIER, BC .
NATURE, 1994, 367 (6462) :463-467
[7]  
Canessa CM, 1996, NEWS PHYSIOL SCI, V11, P195
[8]   EPITHELIAL SODIUM-CHANNEL RELATED TO PROTEINS INVOLVED IN NEURODEGENERATION [J].
CANESSA, CM ;
HORISBERGER, JD ;
ROSSIER, BC .
NATURE, 1993, 361 (6411) :467-470
[9]   Intracellular H+ regulates the α-subunit of ENaC, the epithelial Na+ channel [J].
Chalfant, ML ;
Denton, JS ;
Berdiev, BK ;
Ismailov, II ;
Benos, DJ ;
Stanton, BA .
AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY, 1999, 276 (02) :C477-C486
[10]   THE USE OF XENOPUS OOCYTES FOR THE STUDY OF ION CHANNELS [J].
DASCAL, N .
CRC CRITICAL REVIEWS IN BIOCHEMISTRY, 1987, 22 (04) :317-387