Subunit Composition Determines the Single Channel Kinetics of the Epithelial Sodium Channel (original) (raw)
Related papers
Diversity of Channels Generated by Different Combinations of Epithelial Sodium Channel Subunits
The Journal of General Physiology, 1997
The epithelial sodium channel is a multimeric protein formed by three homologous subunits: α, β, and γ; each subunit contains only two transmembrane domains. The level of expression of each of the subunits is markedly different in various Na+absorbing epithelia raising the possibility that channels with different subunit composition can function in vivo. We have examined the functional properties of channels formed by the association of α with β and of α with γ in theXenopusoocyte expression system using two-microelectrode voltage clamp and patch-clamp techniques. We found that αβ channels differ from αγ channels in the following functional properties: (a) αβ channels expressed larger Na+than Li+currents (INa+/ILi+1.2) whereas αγ channels expressed smaller Na+than Li+currents (INa+/ILi+0.55); (b) the Michaelis Menten constants (Km) of activation of current by increasing concentrations of external Na+and Li+of αβ channels were larger (Km> 180 mM) than those of αγ channels (Kmof 35...
The Second Hydrophobic Domain Contributes to the Kinetic Properties of Epithelial Sodium Channels
Journal of Biological Chemistry, 1999
The epithelial sodium channel (ENaC) is the prototype of a new class of ion channels known as the ENaC/Deg family. The hallmarks of ENaC are a high selectivity for Na ؉ , block by amiloride, small conductance, and slow kinetics that are voltage-independent. We have investigated the contribution of the second hydrophobic domain of each of the homologous subunits ␣, , and ␥ to the kinetic properties of ENaC. Chimeric subunits were constructed between ␣ and  subunits (␣-) and between ␥ and  subunits (␥-). Chimeric and wild-type subunits were expressed in various combinations in Xenopus oocytes. Analysis of whole-cell and unitary currents made it possible to correlate functional properties with specific sequences in the subunits. Functional channels were generated without the second transmembrane domain from ␣ subunits, indicating that it is not essential to form functional pores. The open probability and kinetics varied with the different channels and were influenced by the second hydrophobic domains. Amiloride affinity, Li ؉ /Na ؉ selectivity, and single channel conductance were also affected by this segment.
Subunit Stoichiometry of the Epithelial Sodium Channel
Journal of Biological Chemistry, 1998
The epithelial Na ؉ Channel (ENaC) mediates Na ؉ reabsorption in a variety of epithelial tissues. ENaC is composed of three homologous subunits, termed ␣, , and ␥. All three subunits participate in channel formation as the absence of any one subunit results in a significant reduction or complete abrogation of Na ؉ current expression in Xenopus oocytes. To determine the subunit stoichiometry, a biophysical assay was employed utilizing mutant subunits that display significant differences in sensitivity to channel blockers from the wild type channel. Our results indicate that ENaC is a tetrameric channel with an ␣ 2 ␥ stoichiometry, similar to that reported for other cation selective channels, such as K v , K ir , as well as voltage-gated Na ؉ and Ca 2؉ channels that have 4-fold internal symmetry.
Journal of Biological Chemistry, 2008
Maturation of the epithelial sodium channel (ENaC) involves furin-dependent cleavage at two extracellular sites within the ␣ subunit and at a single extracellular site within the ␥ subunit. Channels lacking furin processing of the ␣ subunit have very low activity. We recently identified a prostasin-dependent cleavage site (RKRK 186) in the ␥ subunit. We also demonstrated that the tract ␣ D206-R231, between the two furin cleavage sites in the ␣ subunit, as well as the tract ␥ E144-K186, between the furin and prostasin cleavage sites in the ␥ subunit, are inhibitory domains. ENaC cleavage by furin, and subsequently by prostasin, leads to a stepwise increase in the open probability of the channel as a result of release of the ␣ and ␥ subunit inhibitory tracts, respectively. We examined whether release of either the ␣ or ␥ inhibitory tract has a dominant role in activating the channel. Co-expression of prostasin and either wild type channels or mutant channels lacking furin cleavage of the ␣ subunit (␣R205A,R208A,R231A␥) in Xenopus laevis oocytes led to increases in whole cell currents to similar levels. In an analogous manner and independent of the proteolytic processing of the ␣ subunit, amiloride-sensitive currents in oocytes expressing channels carrying ␥ subunits with both a mutation in the furin cleavage site and a deletion of the inhibitory tract (␣␥R143A,⌬E144-K186 and ␣R205A,R208A,R231A␥R143A, ⌬E144-K186) were significantly higher than those from oocytes expressing wild type ENaC. When channels lacked the ␣ and ␥ subunit inhibitory tracts, ␣ subunit cleavage was required for channels to be fully active. Channels lacking both furin cleavage and the inhibitory tract in the ␥ subunit (␣␥R143A,⌬E144-K186) showed a significant reduction in the efficacy of block by the synthetic ␣-26 inhibitory peptide representing the tract ␣D206-R231. Our data indicate that removal of the inhibitory tract from the ␥ subunit, in the absence of ␣ subunit cleavage, results in nearly full activation of the channel. Epithelial sodium channels (ENaCs) 2 mediate Na ϩ transport across apical membranes of cells lining the distal nephron, air-* This work was supported, in whole or in part, by National Institutes of Health Grants R01/R56 DK065161 and P30 DK079307. This work was also supported by the Cystic Fibrosis Foundation (R883CR02 and Kleyma08P0). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. We dedicate this report to the memory of James B. Bruns.
The epithelial sodium channel -subunit: new notes for an old song
AJP: Renal Physiology, 2012
old song -subunit: new notes for an δ The epithelial sodium channel You might find this additional info useful... 101 articles, 64 of which you can access for free at: This article cites http://ajprenal.physiology.org/content/303/3/F328.full#ref-list-1 including high resolution figures, can be found at: Updated information and services http://ajprenal.physiology.org/content/303/3/F328.full found at: can be American Journal of Physiology -Renal Physiology about Additional material and information The epithelial sodium channel ␦-subunit: new notes for an old song.
The epithelial sodium channel δ-subunit: new notes for an old song
American Journal of Physiology-Renal Physiology, 2012
Amiloride-sensitive epithelial Na+ channels (ENaCs) can be formed by different combinations of four homologous subunits, named α, β, γ, and δ. In addition to providing an apical entry pathway for transepithelial Na+ reabsorption in tight epithelia such as the kidney distal tubule and collecting duct, ENaCs are also expressed in nonepithelial cells, where they may play different functional roles. The δ-subunit of ENaC was originally identified in humans and is able to form amiloride-sensitive Na+ channels alone or in combination with β and γ, generally resembling the canonical kidney ENaC formed by α, β, and γ. However, δ differs from α in its tissue distribution and channel properties. Despite the low sequence conservation between α and δ (37% identity), their similar functional characteristics provide an excellent model for exploring structural correlates of specific ENaC biophysical and pharmacological properties. Moreover, the study of cellular mechanisms modulating the activity ...
Cell surface expression and turnover of the alpha-subunit of the epithelial sodium channel
American journal of physiology. Renal physiology, 2001
The renal epithelial cell line A6, derived from Xenopus laevis, expresses epithelial Na(+) channels (ENaCs) and serves as a model system to study hormonal regulation and turnover of ENaCs. Our previous studies suggest that the alpha-subunit of Xenopus ENaC (alpha-xENaC) is detectable as 150- and 180-kDa polypeptides, putative immature and mature alpha-subunit heterodimers. The 150- and 180-kDa alpha-xENaC were present in distinct fractions after sedimentation of A6 cell lysate through a sucrose density gradient. Two anti-alpha-xENaC antibodies directed against distinct domains demonstrated that only 180-kDa alpha-xENaC was expressed at the apical cell surface. The half-life of cell surface-expressed alpha-xENaC was 24-30 h, suggesting that once ENaC matures and is expressed at the plasma membrane, its turnover is similar to that reported for mature cystic fibrosis transmembrane conductance regulator. No significant changes in apical surface expression of alpha-xENaC were observed af...
Defining an inhibitory domain in the -subunit of the epithelial sodium channel
AJP: Renal Physiology, 2007
Epithelial sodium channels (ENaC) are processed by proteases as they transit the biosynthetic pathway. We recently observed that furin-dependent processing of the α-subunit of ENaC at two sites within its extracellular domain is required for channel activation due to release of a 26-residue inhibitory domain. While channels with α-subunits lacking the furin sites are not cleaved and have very low activity, channels lacking the furin consensus sites as well as the tract between these sites (αD206–R231) are active. We analyzed channels with a series of deletions in the tract αD206–R231 and lacking the α-subunit furin consensus sites in Xenopus laevis oocytes. We found an eight-residue tract that, when deleted, restored channel activity to the level found in oocytes expressing wild-type ENaC. A synthetic peptide, LPHPLQRL, representing the tract αL211–L218, inhibited wild-type ENaC expressed in oocytes with an IC50 of 0.9 μM, and inhibited channels expressed in collecting duct cells an...
2006
Native amiloride-sensitive Na+ channels exhibit a variety of biophysical properties, including variable sensitivities to amiloride, different ion selectivities, and diverse unitary conductances. The molecular basis of these differences has not been elucidated. We tested the hypothesis that co-expression of δ-epithelial sodium channel (ENaC) underlies, at least in part, the multiplicity of amiloride-sensitive Na+ conductances in epithelial cells. For example, the δ-subunit may form multimeric channels with αβγ-ENaC. Reverse transcription-PCR revealed that δ-ENaC is co-expressed with αβγ-subunits in cultured human lung (H441 and A549), pancreatic (CFPAC), and colonic epithelial cells (Caco-2). Indirect immunofluorescence microscopy revealed that δ-ENaC is co-expressed with α-, β-, and γ-ENaC in H441 cells at the protein level. Measurement of current-voltage that cation selectivity ratios for the revealed relationships Na+/Li+/K+/Cs+/Ca2+/Mg2+, the apparent dissociation constant (Ki) f...