Browsing by Subject "Post-translational modification (PTM)"

Now showing 1 - 4 of 4
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    CaMKII enhances voltage-gated sodium channel Nav1.6 activity and neuronal excitability
    (American Society for Biochemistry and Molecular Biology, 2020-08-14) Zybura, Agnes S.; Baucum, Anthony J., II.; Rush, Anthony M.; Cummins, Theodore R.; Hudmon, Andy; Biology, School of Science
    Nav1.6 is the primary voltage-gated sodium channel isoform expressed in mature axon initial segments and nodes, making it critical for initiation and propagation of neuronal impulses. Thus, Nav1.6 modulation and dysfunction may have profound effects on input-output properties of neurons in normal and pathological conditions. Phosphorylation is a powerful and reversible mechanism regulating ion channel function. Because Nav1.6 and the multifunctional Ca2+/CaM-dependent protein kinase II (CaMKII) are independently linked to excitability disorders, we sought to investigate modulation of Nav1.6 function by CaMKII signaling. We show that inhibition of CaMKII, a Ser/Thr protein kinase associated with excitability, synaptic plasticity, and excitability disorders, with the CaMKII-specific peptide inhibitor CN21 reduces transient and persistent currents in Nav1.6-expressing Purkinje neurons by 87%. Using whole-cell voltage clamp of Nav1.6, we show that CaMKII inhibition in ND7/23 and HEK293 cells significantly reduces transient and persistent currents by 72% and produces a 5.8-mV depolarizing shift in the voltage dependence of activation. Immobilized peptide arrays and nanoflow LC-electrospray ionization/MS of Nav1.6 reveal potential sites of CaMKII phosphorylation, specifically Ser-561 and Ser-641/Thr-642 within the first intracellular loop of the channel. Using site-directed mutagenesis to test multiple potential sites of phosphorylation, we show that Ala substitutions of Ser-561 and Ser-641/Thr-642 recapitulate the depolarizing shift in activation and reduction in current density. Computational simulations to model effects of CaMKII inhibition on Nav1.6 function demonstrate dramatic reductions in spontaneous and evoked action potentials in a Purkinje cell model, suggesting that CaMKII modulation of Nav1.6 may be a powerful mechanism to regulate neuronal excitability.
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    CaMKII Inhibition Attenuates Distinct Gain-of-Function Effects Produced by Mutant Nav1.6 Channels and Reduces Neuronal Excitability
    (MDPI, 2022-07-04) Zybura, Agnes S.; Sahoo, Firoj K.; Hudmon, Andy; Cummins, Theodore R.; Biology, School of Science
    Aberrant Nav1.6 activity can induce hyperexcitability associated with epilepsy. Gain-of-function mutations in the SCN8A gene encoding Nav1.6 are linked to epilepsy development; however, the molecular mechanisms mediating these changes are remarkably heterogeneous and may involve post-translational regulation of Nav1.6. Because calcium/calmodulin-dependent protein kinase II (CaMKII) is a powerful modulator of Nav1.6 channels, we investigated whether CaMKII modulates disease-linked Nav1.6 mutants. Whole-cell voltage clamp recordings in ND7/23 cells show that CaMKII inhibition of the epilepsy-related mutation R850Q largely recapitulates the effects previously observed for WT Nav1.6. We also characterized a rare missense variant, R639C, located within a regulatory hotspot for CaMKII modulation of Nav1.6. Prediction software algorithms and electrophysiological recordings revealed gain-of-function effects for R639C mutant channel activity, including increased sodium currents and hyperpolarized activation compared to WT Nav1.6. Importantly, the R639C mutation ablates CaMKII phosphorylation at a key regulatory site, T642, and, in contrast to WT and R850Q channels, displays a distinct response to CaMKII inhibition. Computational simulations demonstrate that modeled neurons harboring the R639C or R850Q mutations are hyperexcitable, and simulating the effects of CaMKII inhibition on Nav1.6 activity in modeled neurons differentially reduced hyperexcitability. Acute CaMKII inhibition may represent a promising mechanism to attenuate gain-of-function effects produced by Nav1.6 mutations.
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    Role of Novel Serine 316 Phosphorylation of the p65 Subunit of NF-κB in Differential Gene Regulation
    (American Society for Biochemistry & Molecular Biology, 2015-06-16) Wang, Benlian; Prabhu, Lakshmi; Zhao, Wei; Martin, Matthew; Hartley, Antja-Voy; Lu, Tao; Wei, Han; Department of Pharmacology and Toxicology, IU School of Medicine
    Nuclear factor κB (NF-κB) is a central coordinator in immune and inflammatory responses. Constitutive NF-κB is often found in some types of cancers, contributing to oncogenesis and tumor progression. Therefore, knowing how NF-κB is regulated is important for its therapeutic control. Post-translational modification of the p65 subunit of NF-κB is a well known approach for its regulation. Here, we reported that in response to interleukin 1β, the p65 subunit of NF-κB is phosphorylated on the novel serine 316. Overexpression of S316A (serine 316 → alanine) mutant exhibited significantly reduced ability to activate NF-κB and decreased cell growth as compared with wtp65 (wild type p65). Moreover, conditioned media from cells expressing the S316A-p65 mutant had a considerably lower ability to induce NF-κB than that of wtp65. Our data suggested that phosphorylation of p65 on Ser-316 controls the activity and function of NF-κB. Importantly, we found that phosphorylation at the novel Ser-316 site and other two known phosphorylation sites, Ser-529 and Ser-536, either individually or cooperatively, regulated distinct groups of NF-κB-dependent genes, suggesting the unique role of each individual phosphorylation site on NF-κB-dependent gene regulation. Our novel findings provide an important piece of evidence regarding differential regulation of NF-κB-dependent genes through phosphorylation of different p65 serine residues, thus shedding light on novel mechanisms for the pathway-specific control of NF-κB. This knowledge is key to develop strategies for prevention and treatment of constitutive NF-κB-driven inflammatory diseases and cancers.
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    S-Palmitoylation of the sodium channel Nav1.6 regulates its activity and neuronal excitability
    (Elsevier, 2020-05) Pan, Yanling; Xiao, Yucheng; Pei, Zifan; Cummins, Theodore R.; Biology, School of Science
    S-Palmitoylation is a reversible post-translational lipid modification that dynamically regulates protein functions. Voltage-gated sodium channels are subjected to S-palmitoylation and exhibit altered functions in different S-palmitoylation states. Our aim was to investigate whether and how S-palmitoylation regulates Nav1.6 channel function and to identify S-palmitoylation sites that can potentially be pharmacologically targeted. Acyl-biotin exchange assay showed that Nav1.6 is modified by S-palmitoylation in the mouse brain and in a Nav1.6 stable HEK 293 cell line. Using whole-cell voltage clamp, we discovered that enhancing S-palmitoylation with palmitic acid increases Nav1.6 current, whereas blocking S-palmitoylation with 2-bromopalmitate reduces Nav1.6 current and shifts the steady-state inactivation in the hyperpolarizing direction. Three S-palmitoylation sites (Cys1169, Cys1170, and Cys1978) were identified. These sites differentially modulate distinct Nav1.6 properties. Interestingly, Cys1978 is exclusive to Nav1.6 among all Nav isoforms and is evolutionally conserved in Nav1.6 among most species. Cys1978S-palmitoylation regulates current amplitude uniquely in Nav1.6. Furthermore, we showed that eliminating S-palmitoylation at specific sites alters Nav1.6-mediated excitability in dorsal root ganglion neurons. Therefore, our study reveals S-palmitoylation as a potential isoform-specific mechanism to modulate Nav activity and neuronal excitability in physiological and diseased conditions.