Activation by cleavage of the epithelial Na+ channel α and γ subunits independently coevolved with the vertebrate terrestrial migration.
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ABSTRACT:
SUBMITTER: Wang XP
PROVIDER: S-EPMC8791634 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The epithelial Na+ channel (ENaC) possesses a large extracellular domain formed by a β-strand core enclosed by three peripheral α-helical subdomains, which have been dubbed thumb, finger, and knuckle. Here we asked whether the ENaC thumb domains play specific roles in channel function. To this end, we examined the characteristics of channels lacking a thumb domain in an individual ENaC subunit (α, β, or γ). Removing the γ subunit thumb domain had no effect on Na+ currents when expressed in Xenopus oocytes, but moderately reduced channel surface expression. In contrast, ENaCs lacking the α or β subunit thumb domain exhibited significantly reduced Na+ currents along with a large reduction in channel surface expression. Moreover, channels lacking an α or γ thumb domain exhibited a diminished Na+ self-inhibition response, whereas this response was retained in channels lacking a β thumb domain. In turn, deletion of the α thumb domain had no effect on the degradation rate of the immature α subunit as assessed by cycloheximide chase analysis. However, accelerated degradation of the immature β subunit and mature γ subunit was observed when the β or γ thumb domain was deleted, respectively. Our results suggest that the thumb domains in each ENaC subunit are required for optimal surface expression in oocytes and that the α and γ thumb domains both have important roles in the channel's inhibitory response to external Na+ Our findings support the notion that the extracellular helical domains serve as functional modules that regulate ENaC biogenesis and activity.
| S-EPMC6231141 | biostudies-literature
Project description:The epithelial Na+ channel (ENaC) is a member of the ENaC/degenerin family of ion channels. In the structure of a related family member, the "thumb" domain's base interacts with the pore, and its tip interacts with the divergent "finger" domain. Between the base and tip, the thumb domain is characterized by a conserved five-rung disulfide ladder holding together two anti-parallel α helices. The ENaC α and γ subunits' finger domains harbor autoinhibitory tracts that can be proteolytically liberated to activate the channel and also host an ENaC-specific pair of cysteines. Using a crosslinking approach, we show that one of the finger domain cysteines in the α subunit (αCys-263) and both of the finger domain cysteines in the γ subunit (γCys-213 and γCys-220) lie near the dynamic finger-thumb domain interface. Our data suggest that the αCys-256/αCys-263 pair is not disulfide-bonded. In contrast, we found that the γCys-213/γCys-220 pair is disulfide-bonded. Our data also suggest that the γ subunit lacks the terminal rung in the thumb domain disulfide ladder, suggesting asymmetry between the subunits. We also observed functional asymmetry between the α and γ subunit finger-thumb domain interfaces; crosslinks bridging the α subunit finger-thumb interface only inhibited ENaC currents, whereas crosslinks bridging the γ subunit finger-thumb interface activated or inhibited currents dependent on the length of the crosslinker. Our data suggest that reactive cysteines lie at the dynamic finger-thumb interfaces of the α and γ subunits and may play a yet undefined role in channel regulation.
| S-EPMC5880152 | biostudies-literature
Project description:Voltage-gated Na(+) channels (VGSCs) in mammals contain a pore-forming ? subunit and one or more ? subunits. There are five mammalian ? subunits in total: ?1, ?1B, ?2, ?3, and ?4, encoded by four genes: SCN1B-SCN4B. With the exception of the SCN1B splice variant, ?1B, the ? subunits are type I topology transmembrane proteins. In contrast, ?1B lacks a transmembrane domain and is a secreted protein. A growing body of work shows that VGSC ? subunits are multifunctional. While they do not form the ion channel pore, ? subunits alter gating, voltage-dependence, and kinetics of VGSC? subunits and thus regulate cellular excitability in vivo. In addition to their roles in channel modulation, ? subunits are members of the immunoglobulin superfamily of cell adhesion molecules and regulate cell adhesion and migration. ? subunits are also substrates for sequential proteolytic cleavage by secretases. An example of the multifunctional nature of ? subunits is ?1, encoded by SCN1B, that plays a critical role in neuronal migration and pathfinding during brain development, and whose function is dependent on Na(+) current and ?-secretase activity. Functional deletion of SCN1B results in Dravet Syndrome, a severe and intractable pediatric epileptic encephalopathy. ? subunits are emerging as key players in a wide variety of physiopathologies, including epilepsy, cardiac arrhythmia, multiple sclerosis, Huntington's disease, neuropsychiatric disorders, neuropathic and inflammatory pain, and cancer. ? subunits mediate multiple signaling pathways on different timescales, regulating electrical excitability, adhesion, migration, pathfinding, and transcription. Importantly, some ? subunit functions may operate independently of ? subunits. Thus, ? subunits perform critical roles during development and disease. As such, they may prove useful in disease diagnosis and therapy.
| S-EPMC3181431 | biostudies-literature
Project description:BackgroundThe epithelial Na+ channel (ENaC) is intrinsically linked to fluid volume homeostasis and blood pressure. Specific rare mutations in SCNN1A, SCNN1B, and SCNN1G, genes encoding the α, β, and γ subunits of ENaC, respectively, are associated with extreme blood pressure phenotypes. No associations between blood pressure and SCNN1D, which encodes the δ subunit of ENaC, have been reported. A small number of sequence variants in ENaC subunits have been reported to affect functional transport in vitro or blood pressure. The effects of the vast majority of rare and low-frequency ENaC variants on blood pressure are not known.MethodsWe explored the association of low frequency and rare variants in the genes encoding ENaC subunits, with systolic blood pressure, diastolic blood pressure, mean arterial pressure, and pulse pressure. Using whole-genome sequencing data from 14 studies participating in the Trans-Omics in Precision Medicine Whole-Genome Sequencing Program, and sequence kernel association tests.ResultsWe found that variants in SCNN1A and SCNN1B were associated with diastolic blood pressure and mean arterial pressure (P<0.00625). Although SCNN1D is poorly expressed in human kidney tissue, SCNN1D variants were associated with systolic blood pressure, diastolic blood pressure, mean arterial pressure, and pulse pressure (P<0.00625). ENaC variants in 2 of the 4 subunits (SCNN1B and SCNN1D) were also associated with estimated glomerular filtration rate (P<0.00625), but not with stroke.ConclusionsOur results suggest that variants in extrarenal ENaCs, in addition to ENaCs expressed in kidneys, influence blood pressure and kidney function.
| S-EPMC9669116 | biostudies-literature
Project description:Ion selectivity of the potassium channel is crucial for regulating electrical activity in living cells; however, the mechanism underlying the potassium channel selectivity that favors large K+ over small Na+ remains unclear. Generally, Na+ is not completely excluded from permeation through potassium channels. Herein, the distinct nature of Na+ conduction through the prototypical KcsA potassium channel was examined. Single-channel current recordings revealed that, at a high Na+ concentration (200 mM), the channel was blocked by Na+, and this blocking was relieved at high membrane potentials, suggesting the passage of Na+ across the channel. At a 2,000 mM Na+ concentration, single-channel Na+ conductance was measured as one-eightieth of the K+ conductance, indicating that the selectivity filter allows substantial conduit of Na+ Molecular dynamics simulations revealed unprecedented atomic trajectories of Na+ permeation. In the selectivity filter having a series of carbonyl oxygen rings, a smaller Na+ was distributed off-center in eight carbonyl oxygen-coordinated sites as well as on-center in four carbonyl oxygen-coordinated sites. This amphipathic nature of Na+ coordination yielded a continuous but tortuous path along the filter. Trapping of Na+ in many deep free energy wells in the filter caused slow elution. Conversely, K+ is conducted via a straight path, and as the number of occupied K+ ions increased to three, the concerted conduction was accelerated dramatically, generating the conductance selectivity ratio of up to 80. The selectivity filter allows accommodation of different ion species, but the ion coordination and interactions between ions render contrast conduction rates, constituting the potassium channel conductance selectivity.
| S-EPMC8000460 | biostudies-literature
Project description:The affinity for K+ of silkworm Na+/K+-ATPase, which is composed of α and β subunits, is remarkably lower than that of mammalian Na+/K+-ATPase, with a slightly higher affinity for Na+. Because the α subunit had more than 70% identity to the mammalian α subunit in the amino acid sequence, whereas the β subunit, a glycosylated protein, had less than 30% identity to the mammalian β subunit, it was suggested that the β subunit was involved in the affinities for Na+ and K+ of Na+/K+-ATPase. To confirm this hypothesis, we examined whether replacing the silkworm β subunit with the mammalian β subunit affected the affinities for Na+ and K+ of Na+/K+-ATPase. Cloned silkworm α and cloned rat β1 were co-expressed in BM-N cells, a cultured silkworm ovary-derived cell lacking endogenous Na+/K+-ATPase, to construct a hybrid Na+/K+-ATPase, in which the silkworm β subunit was replaced with the rat β1 subunit. The hybrid Na+/K+-ATPase increased the affinity for K+ by 4.1-fold and for Na+ by 0.65-fold compared to the wild-type one. Deglycosylation of the silkworm β subunit did not affect the K+ affinity. These results support the involvement of the β subunit in the Na+ and K+ affinities of Na+/K+-ATPase.
| S-EPMC9483571 | biostudies-literature
Project description:The epithelial Na+ channel (ENaC) emerged early in vertebrates and has played a role in Na+ and fluid homeostasis throughout vertebrate evolution. We previously showed that proteolytic activation of the channel evolved at the water-to-land transition of vertebrates. Sensitivity to extracellular Na+, known as Na+ self-inhibition, reduces ENaC function when Na+ concentrations are high and is a distinctive feature of the channel. A fourth ENaC subunit, δ, emerged in jawed fishes from an α subunit gene duplication. Here, we analyzed 849 α and δ subunit sequences and found that a key Asp in a postulated Na+ binding site was nearly always present in the α subunit, but frequently lost in the δ subunit (e.g. human). Analysis of site evolution and codon substitution rates provide evidence that the ancestral α subunit had the site and that purifying selection for the site relaxed in the δ subunit after its divergence from the α subunit, coinciding with a loss of δ subunit expression in renal tissues. We also show that the proposed Na+ binding site in the α subunit is a bona fide site by conferring novel function to channels comprising human δ subunits. Together, our findings provide evidence that ENaC Na+ self-inhibition improves fitness through its role in Na+ homeostasis in vertebrates.
| S-EPMC11331422 | biostudies-literature
Project description:The cardiac voltage-gated Ca(2+) channel, Ca(v)1.2, mediates excitation-contraction coupling in the heart. The molecular composition of the channel includes the pore-forming ?1 subunit and auxiliary ?2/?-1 and ? subunits. Ca(2+) channel ? subunits, of which there are 8 isoforms, consist of 4 transmembrane domains with intracellular N- and C-terminal ends. The ?1 subunit was initially detected in the skeletal muscle Ca(v)1.1 channel complex, modulating current amplitude and activation and inactivation properties. The ?1 subunit also shifts the steady-state inactivation to more negative membrane potentials, accelerates current inactivation, and increases peak currents, when coexpressed with the cardiac ?1c subunit in Xenopus oocytes and human embryonic kidney (HEK) 293 cells. The ?1 subunit is not expressed, however, in cardiac muscle. We sought to determine whether ? subunits that are expressed in cardiac tissue physically associate with and modulate Ca(v)1.2 function. We now demonstrate that ?4, ?6, ?7, and ?8 subunits physically interact with the Ca(v)1.2 complex. The ? subunits differentially modulate Ca(2+) channel function when coexpressed with the ?1b and ?2/?-1 subunits in HEK cells, altering both activation and inactivation properties. The effects of ? on Ca(v)1.2 function are dependent on the subtype of ? subunit. Our results identify new members of the cardiac Ca(v)1.2 macromolecular complex and identify a mechanism by which to increase the functional diversity of Ca(v)1.2 channels.
| S-EPMC3042847 | biostudies-literature
Project description:The stable structural conformations that occur along the complete reaction coordinate for ion channel opening have never been observed. In this study, we describe the equilibrium ensemble of structures of Slo2.2, a neuronal Na+-activated K+ channel, as a function of the Na+ concentration. We find that Slo2.2 exists in multiple closed conformations whose relative occupancies are independent of Na+ concentration. An open conformation emerges from an ensemble of closed conformations in a highly Na+-dependent manner, without evidence of Na+-dependent intermediates. In other words, channel opening is a highly concerted, switch-like process. The midpoint of the structural titration matches that of the functional titration. A maximum open conformation probability approaching 1.0 and maximum functional open probability approaching 0.7 imply that, within the class of open channels, there is a subclass that is not permeable to ions.
| S-EPMC5382815 | biostudies-literature
Project description:Skeletal muscle contraction depends on activation of clustered acetylcholine receptors (AchRs) and muscle-specific Na+ channels (Nav1.4). Some Nav1.4 channels are highly enriched at the neuromuscular junction (NMJ), and their clustering is thought to be essential for effective muscle excitation. However, this has not been experimentally tested, and how NMJ Na+ channels are clustered is unknown. Here, using muscle-specific ankyrinR, ankyrinB, and ankyrinG single, double, and triple-conditional knockout mice, we show that Nav1.4 channels fail to cluster only after deletion of all three ankyrins. Remarkably, ankyrin-deficient muscles have normal NMJ morphology, AchR clustering, sarcolemmal levels of Nav1.4, and muscle force, and they show no indication of degeneration. However, mice lacking clustered NMJ Na+ channels have significantly reduced levels of motor activity and their NMJs rapidly fatigue after repeated nerve-dependent stimulation. Thus, the triple redundancy of ankyrins facilitates NMJ Na+ channel clustering to prevent neuromuscular synapse fatigue.
| S-EPMC8440394 | biostudies-literature