Sodium reabsorbing epithelia, such as renal distal and collecting tubules, have as their major function the regulation of whole-body sodium balance. These epithelia contain sodium channels localized in their luminal or apical membranes that are inhibited by the diuretic drug amiloride. It is at the level of these channels that the primary feedback control mechanisms necessary for the maintenance for sodium homeostasis occur. Recently, an epithelial sodium channel (termed ENaC) has been cloned. This channel, at a minimum, consists of at least three structurally related subunits (termed alpha-ENaC, beta-ENaC, and gamma-ENaC). The primary and predicted secondary structures of these ENaCs have been described, but the assignment of specific functional roles to domains within each ENaC subunit has just begun. The long term goal of this project is to understand the functional roles of specific amino acid domains within ENaC. We hypothesize that the cation selectivity filter and channel pore regions of ENaC are spatially distinct. Moreover, we hypothesize that there are specific glutamic acid residues in the pre-H2M2 region of each ENaC subunit that participates in calcium binding/inhibition of the channel. We also hypothesize that there are specific negatively charged amino acids (aspartic acid and glutamic acid) located within the M2 region of each ENaC subunit that constitute part of the conduction pathway, or pore of the channel. This application has two specific aims: (1) to test the hypothesis that two specific negatively charged amino acids, located 4 and 12 amino acids upstream from the beginning of the second large hydrophobic domain (H2M2) within the selectivity filter of alpha, beta, and gamma-rENaC, are the sites involved in Ca2+ block of both alpha and alphabetagamma-rENaC; and (2) to test the hypothesis that specific hydrophilic residues (E568, E571, and D575) within the M2 region of each ENaC subunit are essential for the conduction properties of the pore. Important aspects of this study are that unique biochemical, physiological, and molecular biological characteristics of ENaCs will be elucidated; regions of protein sequence that participate in specific channel functions will be identified; and new insights into the gross molecular architecture of a functional ENaC will be developed. These results will offer new insights into the nature of amiloride-sensitive sodium channels, in that important structural regions of the channel involved in cation selectivity, calcium interactions with the channel, and conductions through the pore will be defined.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK056095-03
Application #
6381577
Study Section
General Medicine B Study Section (GMB)
Program Officer
Scherbenske, M James
Project Start
1999-09-01
Project End
2003-08-31
Budget Start
2001-09-01
Budget End
2002-08-31
Support Year
3
Fiscal Year
2001
Total Cost
$197,744
Indirect Cost
Name
University of Alabama Birmingham
Department
Physiology
Type
Schools of Medicine
DUNS #
004514360
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Ji, Hong-Long; Bishop, LaToya R; Anderson, Susan J et al. (2004) The role of Pre-H2 domains of alpha- and delta-epithelial Na+ channels in ion permeation, conductance, and amiloride sensitivity. J Biol Chem 279:8428-40
Ji, Hong-Long; Benos, Dale J (2004) Degenerin sites mediate proton activation of deltabetagamma-epithelial sodium channel. J Biol Chem 279:26939-47
Ji, Hong-Long; Jovov, Biljana; Fu, Jian et al. (2002) Up-regulation of acid-gated Na(+) channels (ASICs) by cystic fibrosis transmembrane conductance regulator co-expression in Xenopus oocytes. J Biol Chem 277:8395-405
Ji, H L; Parker, S; Langloh, A L et al. (2001) Point mutations in the post-M2 region of human alpha-ENaC regulate cation selectivity. Am J Physiol Cell Physiol 281:C64-74
Berdiev, B K; Latorre, R; Benos, D J et al. (2001) Actin modifies Ca2+ block of epithelial Na+ channels in planar lipid bilayers. Biophys J 80:2176-86
Berdiev, B K; Mapstone, T B; Markert, J M et al. (2001) pH alterations ""reset"" Ca2+ sensitivity of brain Na+ channel 2, a degenerin/epithelial Na+ ion channel, in planar lipid bilayers. J Biol Chem 276:38755-61
Markert, J M; Fuller, C M; Gillespie, G Y et al. (2001) Differential gene expression profiling in human brain tumors. Physiol Genomics 5:21-33
Langloh, A L; Berdiev, B; Ji, H L et al. (2000) Charged residues in the M2 region of alpha-hENaC play a role in channel conductance. Am J Physiol Cell Physiol 278:C277-91
Benos, D J; Stanton, B A (1999) Functional domains within the degenerin/epithelial sodium channel (Deg/ENaC) superfamily of ion channels. J Physiol 520 Pt 3:631-44