Subunit stoichiometry of a core conduction element in a cloned epithelial amiloride-sensitive Na+ channel.
The molecular composition of a core conduction element formed by the alpha-subunit of cloned epithelial Na+ channels (ENaC) was studied in planar lipid bilayers. Two pairs of in vitro translated proteins were employed in combinatorial experiments: 1) wild-type (WT) and an N-terminally truncated alphaDeltaN-rENaC that displays accelerated kinetics (tauo = 32 +/- 13 ms, tauc = 42 +/- 11 ms), as compared with the WT channel (tauc1 = 18 +/- 8 ms, tauc2 = 252 +/- 31 ms, and tauo = 157 +/- 43 ms); and 2) WT and an amiloride binding mutant, alphaDelta278-283-rENaC. The channels that formed in a alphaWT:alphaDeltaN mixture fell into two groups: one with tauo and tauc that corresponded to those exhibited by the alphaDeltaN-rENaC alone, and another with a double-exponentially distributed closed time and a single-exponentially distributed open time that corresponded to the alphaWT-rENaC alone. Five channel subtypes with distinct sensitivities to amiloride were found in a 1alphaWT:1alphaDelta278-283 protein mixture. Statistical analyses of the distributions of channel phenotypes observed for either set of the WT:mutant combinations suggest a tetrameric organization of alpha-subunits as a minimal model for the core conduction element in ENaCs.
Berdiev
B K
BK
Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005, USA.
Karlson
K H
KH
Jovov
B
B
Ripoll
P J
PJ
Morris
R
R
Loffing-Cueni
D
D
Halpin
P
P
Stanton
B A
BA
Kleyman
T R
TR
Ismailov
I I
II
D000584:Amiloride / Q000494:pharmacology*; D000818:Animals; D003001:Cloning, Molecular; D004594:Electrophysiology; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D011510:Proteolipids / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D015222:Sodium Channels / Q000737:chemistry* / Q000235:genetics; D014981:Xenopus
D008051:Lipid Bilayers; D011510:Proteolipids; D011994:Recombinant Proteins; D015222:Sodium Channels; C020485:proteoliposomes; D000584:Amiloride