Singlet oxygen modification abolishes voltage-dependent inactivation of the sea urchin spHCN channel.
Ontology highlight
ABSTRACT:
SUBMITTER: Idikuda V
PROVIDER: S-EPMC6122923 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:The voltage-gated potassium (Kv) channel Kv1.5 mediates the I(Kur) repolarizing current in human atrial myocytes and regulates vascular tone in multiple peripheral vascular beds. Understanding the complex regulation of Kv1.5 function is of substantial interest because it represents a promising pharmacological target for the treatment of atrial fibrillation and hypoxic pulmonary hypertension. Herein we demonstrate that posttranslational modification of Kv1.5 by small ubiquitin-like modifier (SUMO) proteins modulates Kv1.5 function. We have identified two membrane-proximal and highly conserved cytoplasmic sequences in Kv1.5 that conform to established SUMO modification sites in transcription factors. We find that Kv1.5 interacts specifically with the SUMO-conjugating enzyme Ubc9 and is a target for modification by SUMO-1, -2, and -3 in vivo. In addition, purified recombinant Kv1.5 serves as a substrate in a minimal in vitro reconstituted SUMOylation reaction. The SUMO-specific proteases SENP2 and Ulp1 efficiently deconjugate SUMO from Kv1.5 in vivo and in vitro, and disruption of the two identified target motifs results in a loss of the major SUMO-conjugated forms of Kv1.5. In whole-cell patch-clamp electrophysiological studies, loss of Kv1.5 SUMOylation, by either disruption of the conjugation sites or expression of the SUMO protease SENP2, leads to a selective approximately 15-mV hyperpolarizing shift in the voltage dependence of steady-state inactivation. Reversible control of voltage-sensitive channels through SUMOylation constitutes a unique and likely widespread mechanism for adaptive tuning of the electrical excitability of cells.
| S-EPMC1794304 | biostudies-literature
Project description:The transient receptor potential melastatin 5 (TRPM5) channel is a monovalent-permeable cation channel that is activated by intracellular Ca2+. Expression of TRPM5 has been shown in taste cells, pancreas, brainstem and olfactory epithelium, and this channel is thought to be involved in controlling membrane potentials. In whole-cell patch-clamp recordings, TRPM5 exhibited voltage-dependent inactivation at negative membrane potentials and time constant of voltage-dependent inactivation of TRPM5 did not depend on the intracellular Ca2+ concentrations between 100 and 500 nM. Alanine substitution at Y913 and I916 in the pore helix of TRPM5 increased time constant of voltage-dependent inactivation. Meanwhile, voltage-dependent inactivation was reduced in TRPM5 mutants having glycine substitution at L901, Y913, Q915 and I916 in the pore helix. From these results, we conclude that the pore helix in the outer pore loop might play a role in voltage-dependent inactivation of TRPM5.
| S-EPMC7848652 | biostudies-literature
Project description:Singlet oxygen ((1)O2), which is generated through metabolic reactions and oxidizes numerous biological molecules, has been a useful tool in basic research and clinical practice. However, its role as a signaling factor, as well as a mechanistic understanding of the oxidation process, remains poorly understood. Here, we show that hyperpolarization-activated, cAMP-gated (HCN) channels--which conduct the hyperpolarization-activated current (Ih) and the voltage-insensitive instantaneous current (Iinst), and contribute to diverse physiological functions including learning and memory, cardiac pacemaking, and the sensation of pain--are subject to modification by (1)O2. To increase the site specificity of (1)O2 generation, we used fluorescein-conjugated cAMP, which specifically binds to HCN channels, or a chimeric channel in which an in-frame (1)O2 generator (SOG) protein was fused to the HCN C terminus. Millisecond laser pulses reduced Ih current amplitude, slowed channel deactivation, and enhanced Iinst current. The modification of HCN channel function is a photodynamic process that involves (1)O2, as supported by the dependence on dissolved oxygen in solutions, the inhibitory effect by a (1)O2 scavenger, and the results with the HCN2-SOG fusion protein. Intriguingly, (1)O2 modification of the HCN2 channel is state dependent: laser pulses applied to open channels mainly slow down deactivation and increase Iinst, whereas for the closed channels, (1)O2 modification mainly reduced Ih amplitude. We identified a histidine residue (H434 in S6) near the activation gate in the pore critical for (1)O2 modulation of HCN function. Alanine replacement of H434 abolished the delay in channel deactivation and the generation of Iinst induced by photodynamic modification. Our study provides new insights into the instantaneous current conducted by HCN channels, showing that modifications to the region close to the intracellular gate underlie the expression of Iinst, and establishes a well-defined model for studying (1)O2 modifications at the molecular level.
| S-EPMC4003188 | biostudies-literature
Project description:Calcium-dependent inactivation (CDI) is a fundamental autoregulatory mechanism in CaV1 and CaV2 voltage-gated calcium channels. Although CDI initiates with the cytoplasmic calcium sensor, how this event causes CDI has been elusive. Here, we show that a conserved selectivity filter (SF) domain II (DII) aspartate is essential for CDI. Mutation of this residue essentially eliminates CDI and leaves key channel biophysical characteristics untouched. DII mutants regain CDI by placing an aspartate at the analogous SF site in DIII or DIV, but not DI, indicating that CaV SF asymmetry is key to CDI. Together, our data establish that the CaV SF is the CDI endpoint. Discovery of this SF CDI gate recasts the CaV inactivation paradigm, placing it squarely in the framework of voltage-gated ion channel (VGIC) superfamily members in which SF-based gating is important. This commonality suggests that SF inactivation is an ancient process arising from the shared VGIC pore architecture.
| S-EPMC8878153 | biostudies-literature
Project description:Singlet oxygen ((1)O(2)) is a reactive oxygen species that may be generated in biological systems. Photodynamic therapy generates (1)O(2) by photoexcitation of sensitizers resulting in intracellular oxidative stress and induction of apoptosis. (1)O(2) oxidizes amino acid side chains of proteins and inactivates enzymes when generated in vitro. Among proteogenic amino acids, His, Tyr, Met, Cys, and Trp are known to be oxidized by (1)O(2) at physiological pH. However, there is a lack of direct evidence of oxidation of proteins by (1)O(2). Because (1)O(2) is difficult to detect in cells, identifying oxidized cellular products uniquely derived from (1)O(2) could serve as a marker of its presence. In the present study, (1)O(2) reactions with model peptides analyzed by tandem mass spectrometry provide insight into the mass of prominent adducts formed with the reactive amino acids. Analysis by MALDI-TOF and tandem mass spectrometry of peptides of cytochrome c exposed to (1)O(2) generated by photoexcitation of the phthalocyanine Pc 4 showed unique oxidation products, which might be used as markers of the presence of (1)O(2) in the mitochondrial intermembrane space. Differences in the elemental composition of the oxidized amino acid residues observed with cytochrome c and the model peptides suggest that the protein environment can affect the oxidation pathway.
| S-EPMC2424268 | biostudies-literature
Project description:Sperm guidance is controlled by chemical and physical cues. In many species, Ca(2+) bursts in the flagellum govern navigation to the egg. In Arbacia punctulata, a model system of sperm chemotaxis, a cGMP signaling pathway controls these Ca(2+) bursts. The underlying Ca(2+) channel and its mechanisms of activation are unknown. Here, we identify CatSper Ca(2+) channels in the flagellum of A. punctulata sperm. We show that CatSper mediates the chemoattractant-evoked Ca(2+) influx and controls chemotactic steering; a concomitant alkalization serves as a highly cooperative mechanism that enables CatSper to transduce periodic voltage changes into Ca(2+) bursts. Our results reveal intriguing phylogenetic commonalities but also variations between marine invertebrates and mammals regarding the function and control of CatSper. The variations probably reflect functional and mechanistic adaptations that evolved during the transition from external to internal fertilization.
| S-EPMC4339123 | biostudies-literature
Project description:Direct regulation of N-type calcium channels by G-proteins is essential to control neuronal excitability and neurotransmitter release. Binding of the G(betagamma) dimer directly onto the channel is characterized by a marked current inhibition ("ON" effect), whereas the pore opening- and time-dependent dissociation of this complex from the channel produce a characteristic set of biophysical modifications ("OFF" effects). Although G-protein dissociation is linked to channel opening, the contribution of channel inactivation to G-protein regulation has been poorly studied. Here, the role of channel inactivation was assessed by examining time-dependent G-protein de-inhibition of Ca(v)2.2 channels in the presence of various inactivation-altering beta subunit constructs. G-protein activation was produced via mu-opioid receptor activation using the DAMGO agonist. Whereas the "ON" effect of G-protein regulation is independent of the type of beta subunit, the "OFF" effects were critically affected by channel inactivation. Channel inactivation acts as a synergistic factor to channel activation for the speed of G-protein dissociation. However, fast inactivating channels also reduce the temporal window of opportunity for G-protein dissociation, resulting in a reduced extent of current recovery, whereas slow inactivating channels undergo a far more complete recovery from inhibition. Taken together, these results provide novel insights on the role of channel inactivation in N-type channel regulation by G-proteins and contribute to the understanding of the physiological consequence of channel inactivation in the modulation of synaptic activity by G-protein coupled receptors.
| S-EPMC2703660 | biostudies-literature
Project description:Intracellular singlet oxygen generation in photofrin-loaded cells caused cell death without discrimination between nonmalignant and malignant cells. In contrast, extracellular singlet oxygen generation caused apoptosis induction selectively in tumor cells through singlet oxygen-mediated inactivation of tumor cell protective catalase and subsequent reactivation of intercellular ROS-mediated apoptosis signaling through the HOCl and the NO/peroxynitrite signaling pathway. Singlet oxygen generation by extracellular photofrin alone was, however, not sufficient for optimal direct inactivation of catalase, but needed to trigger the generation of cell-derived extracellular singlet oxygen through the interaction between H2O2 and peroxynitrite. Thereby, formation of peroxynitrous acid, generation of hydroxyl radicals and formation of perhydroxyl radicals (HO2(.)) through hydroxyl radical/H2O2 interaction seemed to be required as intermediate steps. This amplificatory mechanism led to the formation of singlet oxygen at a sufficiently high concentration for optimal inactivation of membrane-associated catalase. At low initial concentrations of singlet oxygen, an additional amplification step needed to be activated. It depended on singlet oxygen-dependent activation of the FAS receptor and caspase-8, followed by caspase-8-mediated enhancement of NOX activity. The biochemical mechanisms described here might be considered as promising principle for the development of novel approaches in tumor therapy that specifically direct membrane-associated catalase of tumor cells and thus utilize tumor cell-specific apoptosis-inducing ROS signaling.
| S-EPMC4532730 | biostudies-literature
Project description:Mutations in CACNA1C that increase current through the CaV1.2 L-type Ca2+ channel underlie rare forms of long QT syndrome (LQTS), and Timothy syndrome (TS). We identified a variant in CACNA1C in a male child of Filipino descent with arrhythmias and extracardiac features by candidate gene sequencing and performed functional expression studies to electrophysiologically characterize the effects of the variant on CaV1.2 channels. As a baby, the subject developed seizures and displayed developmental delays at 30 months of age. At age 5 years, he displayed a QTc of 520 ms and experienced recurrent VT. Physical exam at 17 years of age was notable for microcephaly, short stature, lower extremity weakness and atrophy with hyperreflexia, spastic diplegia, multiple dental caries and episodes of rhabdomyolysis. Candidate gene sequencing identified a G>C transversion at position 5731 of CACNA1C (rs374528680) predicting a glycine>arginine substitution at residue 1911 (p.G1911R) of CaV1.2. The allele frequency of this variant is 0.01 in Malays, but absent in 984 Caucasian alleles and in the 1000 genomes project. In electrophysiological analyses, the variant decreased voltage-dependent inactivation, thus causing a gain of function of CaV1.2. We also observed a negative shift of V1/2 of activation and positive shift of V1/2 of channel inactivation, resulting in an increase of the window current. Together, these suggest a gain-of-function effect on CaV1.2 and suggest increased susceptibility for arrhythmias in certain clinical settings. The p.G1911R variant was also identified in a case of sudden unexplained infant death (SUID), for which an increasing number of clinical observations have demonstrated can be associated with arrhythmogenic mutations in cardiac ion channels. In summary, the combined effects of the CACNA1C variant to diminish voltage-dependent inactivation of CaV1.2 and increase window current expand our appreciation of mechanisms by which a gain of function of CaV1.2 can contribute to QT prolongation.
| S-EPMC4153713 | biostudies-literature
Project description: Not available
| S-EPMC4187349 | biostudies-literature