title: The role of Pre-H2 domains of alpha- and delta-epithelial Na+ channels in ion permeation, conductance, and amiloride sensitivity.
abstract: Epithelial Na(+) channels (ENaC) regulate salt and water re-absorption across the apical membrane of absorptive epithelia such as the kidney, colon, and lung. Structure-function studies have suggested that the second transmembrane domain (M2) and the adjacent pre- and post-M2 regions are involved in channel pore formation, cation selectivity, and amiloride sensitivity. Because Na(+) selectivity, unitary Na(+) conductance (gamma(Na)), and amiloride sensitivity of delta-ENaC are strikingly different from those of alpha-ENaC, the hypothesis that the pre-H2 domain may contribute to these characterizations has been examined by swapping the pre-H2, H2, and both (pre-H2+H2) domains of delta- and alpha-ENaCs. Whole-cell and single channel results showed that the permeation ratio of Li(+) and Na(+) (P(Li)/P(Na)) for the swap alpha chimeras co-expressed with betagamma-ENaC in Xenopus oocytes decreased significantly. In contrast, the ratio of P(Li)/P(Na) for the swap delta constructs was not significantly altered. Single channel studies confirmed that swapping of the H2 and the pre-H2+H2 domains increased the gamma(Na) of alpha-ENaC but decreased the gamma(Na) of delta-ENaC. A significant increment in the apparent inhibitory dissociation constant for amiloride (K(i)(amil)) was observed in the alpha chimeras by swapping the pre-H2, H2, and pre-H2+H2 domains. In contrast, a striking decline of K(i)(amil) was obtained in the chimeric delta constructs with substitution of the H2 and pre-H2+H2 domains. Our results demonstrate that the pre-H2 domain, combined with the H2 domain, contributes to the P(Li)/P(Na) ratio, single channel Na(+) conductance, and amiloride sensitivity of alpha- and delta-ENaCs.
authors.affiliation: Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005, USA. ji@physiology.uab.edu
authors.forename: Hong-Long
authors.initials: HL
authors.lastname: Ji
authors.forename: LaToya R
authors.initials: LR
authors.lastname: Bishop
authors.forename: Susan J
authors.initials: SJ
authors.lastname: Anderson
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Fuller
authors.forename: Dale J
authors.initials: DJ
authors.lastname: Benos
mesh_terms: D000584:Amiloride / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D002412:Cations / Q000378:metabolism*; D004553:Electric Conductivity; D053503:Epithelial Sodium Channels; D005260:Female; D015870:Gene Expression; D006801:Humans; D008094:Lithium / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D010539:Permeability; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D016415:Sequence Alignment; D012964:Sodium / Q000378:metabolism; D015222:Sodium Channels / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D013329:Structure-Activity Relationship; D014162:Transfection; D014981:Xenopus
chemical_list: D002412:Cations; D053503:Epithelial Sodium Channels; D011993:Recombinant Fusion Proteins; D015222:Sodium Channels; D000584:Amiloride; D008094:Lithium; D012964:Sodium