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: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.
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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 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: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:Ion channels gated by cyclic nucleotides have crucial roles in neuronal excitability and signal transduction of sensory neurons. Here, we studied ligand binding of a cyclic nucleotide-activated K(+) channel from Mesorhizobium loti and its isolated cyclic nucleotide-binding domain. The channel and the binding domain alone bind cyclic AMP with similar affinity in a non-cooperative manner. The cAMP sensitivities of binding and activation coincide. Thus, each subunit in the tetrameric channel acts independently of the others. The binding and gating properties of the bacterial channel are distinctively different from those of eukaryotic cyclic nucleotide-gated channels.
| S-EPMC1978089 | biostudies-other
Project description:Human mutations of the Na+-activated K+ channel Slack (KCNT1) are associated with epilepsy and intellectual disability. Accordingly, Slack knockout mice (Slack-/-) exhibit cognitive flexibility deficits in distinct behavioral tasks. So far, however, the underlying causes as well as the role of Slack in hippocampus-dependent memory functions remain enigmatic. We now report that infant (P6-P14) Slack-/- lack both hippocampal LTD and LTP, likely due to impaired NMDA receptor (NMDAR) signaling. Postsynaptic GluN2B levels are reduced in infant Slack-/-, evidenced by lower amplitudes of NMDAR-meditated excitatory postsynaptic potentials. Low GluN2B affected NMDAR-mediated Ca2+-influx, rendering cultured hippocampal Slack-/-neurons highly insensitive to the GluN2B-specific inhibitor Ro 25-6981. Furthermore, dephosphorylation of the AMPA receptor (AMPAR) subunit GluA1 at S845, which is involved in AMPAR endocytosis during homeostatic and neuromodulator-regulated plasticity, is reduced after chemical LTD (cLTD) in infant Slack-/-. We additionally detect a lack of mGluR-induced LTD in infant Slack-/-, possibly caused by upregulation of the recycling endosome-associated small GTPase Rab4 which might accelerate AMPAR recycling from early endosomes. Interestingly, LTP and mGluR LTD, but not LTD and S845 dephosphorylation after cLTD are restored in adult Slack-/-. This together with normalized expression levels of GluN2B and Rab4 hints to developmental "restoration" of LTP expression despite Slack ablation, whereas in infant and adult brain, NMDAR-dependent LTD induction depends on this channel. Based on the present findings, NMDAR and vesicular transport might represent novel targets for the therapy of intellectual disability associated with Slack mutations. Consequently, careful modulation of hippocampal Slack activity should also improve learning abilities.
| S-EPMC8629810 | biostudies-literature
Project description:The epithelial Na(+) channel (ENaC) regulates Na(+) absorption in epithelial tissues including the lung, colon and sweat gland, and in the distal nephrons of the kidney. When Na(+)-channel function is disrupted, salt and water homoeostasis is affected. The cytoplasmic regions of the Na(+)-channel subunits provide binding sites for other proteins to interact with and potentially regulate Na(+)-channel activity. Previously we showed that a proline-rich region of the alpha subunit of the Na(+) channel bound to a protein of 116 kDa from human lung cells. Here we report the identification of this protein as human Nedd4, a ubiquitin-protein ligase that binds to the Na(+)-channel subunits via its WW domains. Further, we show that WW domains 2, 3 and 4 of human Nedd4 bind to the alpha, beta and gamma Na(+)-channel subunits but not to a mutated beta subunit. In addition, when co-expressed in Xenopus oocytes, human Nedd4 down-regulates Na(+)-channel activity.
| S-EPMC1220784 | biostudies-other
Project description:Specific small interfering RNA (siRNA) constructs were used to test for the functional relation of subunits alpha, beta, and gamma of the epithelial Na(+) channel (ENaC) to the hypertonicity-induced cation channel (HICC) in confluent rat hepatocytes. In current-clamp recordings, hypertonic stress (300 --> 400 mosM) increased membrane conductance from 75.4 +/- 9.4 to 91.1 +/- 11.2 pS (p < 0.001). The effect was completely blocked by 100 microM amiloride and reduced to 46, 30, and 45% of the control value by anti-alpha-, anti-beta-, and anti-gamma-rENaC siRNA, respectively. Scanning acoustic microscopy revealed an initial shrinkage of cells from 6.98 +/- 0.45 to 6.03 +/- 0.43 pl within 2 min. This passive response was then followed by a regulatory volume increase (RVI) by 0.42 +/- 0.05 pl (p < 0.001). With anti-alpha-, anti-beta-, and anti-gamma-rENaC siRNA, the volume response was reduced to 31, 31, and 36% of the reference level, respectively. It is concluded that all three subunits of the ENaC are functionally related to RVI and HICC activation in rat hepatocytes.
| S-EPMC2756400 | biostudies-other
Project description:The Shaker-like family of voltage-gated K+ channels comprises four functionally independent gene subfamilies, Shaker (Kv1), Shab (Kv2), Shaw (Kv3), and Shal (Kv4), each of which regulates distinct aspects of neuronal excitability. Subfamily-specific assembly of tetrameric channels is mediated by the N-terminal T1 domain and segregates Kv1-4, allowing multiple channel types to function independently in the same cell. Typical Shaker-like Kv subunits can form functional channels as homotetramers, but a group of mammalian Kv2-related genes (Kv5.1, Kv6s, Kv8s, and Kv9s) encodes subunits that have a "silent" or "regulatory" phenotype characterized by T1 self-incompatibility. These channels are unable to form homotetramers, but instead heteromerize with Kv2.1 or Kv2.2 to diversify the functional properties of these delayed rectifiers. While T1 self-incompatibility predicts that these heterotetramers could contain up to two regulatory (R) subunits, experiments show a predominance of 3:1R stoichiometry in which heteromeric channels contain a single regulatory subunit. Substitution of the self-compatible Kv2.1 T1 domain into the regulatory subunit Kv6.4 does not alter the stoichiometry of Kv2.1:Kv6.4 heteromers. Here, to identify other channel structures that might be responsible for favoring the 3:1R stoichiometry, we compare the sequences of mammalian regulatory subunits to independently evolved regulatory subunits from cnidarians. The most widespread feature of regulatory subunits is the presence of atypical substitutions in the highly conserved consensus sequence of the intracellular S6 activation gate of the pore. We show that two amino acid substitutions in the S6 gate of the regulatory subunit Kv6.4 restrict the functional stoichiometry of Kv2.1:Kv6.4 to 3:1R by limiting the formation and function of 2:2R heteromers. We propose a two-step model for the evolution of the asymmetric 3:1R stoichiometry, which begins with evolution of self-incompatibility to establish the regulatory phenotype, followed by drift of the activation gate consensus sequence under relaxed selection to limit stoichiometry to 3:1R.
| S-EPMC6279357 | biostudies-literature