Nurses' perceptions of aids and obstacles to the provision of optimal end of life care in ICU

Contains fulltext : 172380.pdf (publisher's version ) (Open Access

Identification of an amiloride binding domain within the alpha-subunit of the epithelial Na+ channel.

Limited information is available regarding domains within the epithelial Na+ channel (ENaC) which participate in amiloride binding. We previously utilized the anti-amiloride antibody (BA7.1) as a surrogate amiloride receptor to delineate amino acid residues that contact amiloride, and identified a putative amiloride binding domain WYRFHY (residues 278-283) within the extracellular domain of alpharENaC. Mutations were generated to examine the role of this sequence in amiloride binding. Functional analyses of wild type (wt) and mutant alpharENaCs were performed by cRNA expression in Xenopus oocytes and by reconstitution into planar lipid bilayers. Wild type alpharENaC was inhibited by amiloride with a Ki of 169 nM. Deletion of the entire WYRFHY tract (alpharENaC Delta278-283) resulted in a loss of sensitivity of the channel to submicromolar concentrations of amiloride (Ki = 26.5 microM). Similar results were obtained when either alpharENaC or alpharENaC Delta278-283 were co-expressed with wt beta- and gammarENaC (Ki values of 155 nM and 22.8 microM, respectively). Moreover, alpharENaC H282D was insensitive to submicromolar concentrations of amiloride (Ki = 6.52 microM), whereas alpharENaC H282R was inhibited by amiloride with a Ki of 29 nM. These mutations do not alter ENaC Na+:K+ selectivity nor single-channel conductance. These data suggest that residues within the tract WYRFHY participate in amiloride binding. Our results, in conjunction with recent studies demonstrating that mutations within the membrane-spanning domains of alpharENaC and mutations preceding the second membrane-spanning domains of alpha-, beta-, and gammarENaC alters amiloride's Ki, suggest that selected regions of the extracellular loop of alpharENaC may be in close proximity to residues within the channel pore.Journal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, Non-P.H.S.Research Support, U.S. Gov't, P.H.S.info:eu-repo/semantics/publishe

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 α-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 αΔN-rENaC that displays accelerated kinetics (τo = 32 ± 13 ms, τc = 42 ± 11 ms), as compared with the WT channel (τc1 = 18 ± 8 ms, τc2 = 252 ± 31 ms, and τo = 157 ± 43 ms); and 2) WT and an amiloride binding mutant, αΔ278–283-rENaC. The channels that formed in a αWT:αΔN mixture fell into two groups: one with τo and τc that corresponded to those exhibited by the αΔN-rENaC alone, and another with a double-exponentially distributed closed time and a single-exponentially distributed open time that corresponded to the αWT-rENaC alone. Five channel subtypes with distinct sensitivities to amiloride were found in a 1αWT:1αΔ278–283 protein mixture. Statistical analyses of the distributions of channel phenotypes observed for either set of the WT:mutant combinations suggest a tetrameric organization of α-subunits as a minimal model for the core conduction element in ENaCs