Hai M. Nguyen

Kv1.3 Deletion Biases T Cells toward an Immunoregulatory Phenotype and Renders Mice Resistant to Autoimmune Encephalomyelitis

Increasing evidence suggests ion channels have critical functions in the differentiation and plasticity of T cells. Kv1.3, a voltage-gated K+ channel, is a functional marker and a pharmacological target for activated effector memory T cells. Selective Kv1.3 blockers have been shown to inhibit proliferation and cytokine production by human and rat effector memory T cells. We used Kv1.3 knockout (KO) mice to investigate the mechanism by which Kv1.3 blockade affects CD4+ T cell differentiation during an inflammatory immune-mediated disease. Kv1.3 KO animals displayed significantly lower incidence and severity of myelin oligodendrocyte glycoprotein (MOG) peptide-induced experimental autoimmune encephalomyelitis. Kv1.3 was the only KV channel expressed in MOG 35–55-specific CD4+ T cell blasts, and no KV current was present in MOG-specific CD4+ T cell-blasts from Kv1.3 KO mice. Fewer CD4+ T cells migrated to the CNS in Kv1.3 KO mice following disease induction, and Ag-specific proliferation of CD4+ T cells from these mice was impaired with a corresponding cell-cycle delay. Kv1.3 was required for optimal expression of IFN-γ and IL-17, whereas its absence led to increased IL-10 production. Dendritic cells from Kv1.3 KO mice fully activated wild-type CD4+ T cells, indicating a T cell-intrinsic defect in Kv1.3 KO mice. The loss of Kv1.3 led to a suppressive phenotype, which may contribute to the mechanism by which deletion of Kv1.3 produces an immunotherapeutic effect. Skewing of CD4+ T cell differentiation toward Ag-specific regulatory T cells by pharmacological blockade or genetic suppression of Kv1.3 might be beneficial for therapy of immune-mediated diseases such as multiple sclerosis.

The potassium channel KCa3.1 constitutes a pharmacological target for neuroinflammation associated with ischemia/reperfusion stroke.

Activated microglia/macrophages significantly contribute to the secondary inflammatory damage in ischemic stroke. Cultured neonatal microglia express the K+ channels Kv1.3 and KCa3.1, both of which have been reported to be involved in microglia-mediated neuronal killing, oxidative burst and cytokine production. However, it is questionable whether neonatal cultures accurately reflect the K+ channel expression of activated microglia in the adult brain. We here subjected mice to middle cerebral artery occlusion with eight days of reperfusion and patch-clamped acutely isolated microglia/macrophages. Microglia from the infarcted area exhibited higher densities of K+ currents with the biophysical and pharmacological properties of Kv1.3, KCa3.1 and Kir2.1 than microglia from non-infarcted control brains. Similarly, immunohistochemistry on human infarcts showed strong Kv1.3 and KCa3.1 immunoreactivity on activated microglia/macrophages. We next investigated the effect of genetic deletion and pharmacological blockade of KCa3.1 in reversible middle cerebral artery occlusion. KCa3.1 −/− mice and wild-type mice treated with the KCa3.1 blocker TRAM-34 exhibited significantly smaller infarct areas on day-8 after middle cerebral artery occlusion and improved neurological deficit. Both manipulations reduced microglia/macrophage activation and brain cytokine levels. Our findings suggest KCa3.1 as a pharmacological target for ischemic stroke. Of potential, clinical relevance is that KCa3.1 blockade is still effective when initiated 12 h after the insult.

Kv1.3 channel-blocking immunomodulatory peptides from parasitic worms: implications for autoimmune diseases

The voltage‐gated potassium (Kv) 1.3 channel is widely regarded as a therapeutic target for immunomodulation in autoimmune diseases. ShK‐186, a selective inhibitor of Kv1.3 channels, ameliorates autoimmune diseases in rodent models, and human phase 1 trials of this agent in healthy volunteers have been completed. In this study, we identified and characterized a large family of Stichodactyla helianthus toxin (ShK)‐related peptides in parasitic worms. Based on phylogenetic analysis, 2 worm peptides were selected for study: AcK1, a 51‐residue peptide expressed in the anterior secretory glands of the dog‐infecting hookworm Ancylostoma caninum and the human‐infecting hookworm Ancylostoma ceylanicum, and BmK1, the C‐terminal domain of a metalloprotease from the filarial worm Brugia malayi. These peptides in solution adopt helical structures closely resembling that of ShK. At doses in the nanomolar‐micromolar range, they block native Kv1.3 in human T cells and cloned Kv1.3 stably expressed in L929 mouse fibroblasts. They preferentially suppress the proliferation of rat CCR7‐ effector memory T cells without affecting naive and central memory subsets and inhibit the delayed‐type hypersensitivity (DTH) response caused by skin‐homing effector memory T cells in rats. Further, they suppress IFNγ production by human T lymphocytes. ShK‐related peptides in parasitic worms may contribute to the potential beneficial effects of probiotic parasitic worm therapy in human autoimmune diseases.—Chhabra, S., Chang, S. C., Nguyen, H. M., Huq, R., Tanner, M. R., Londono, L. M., Estrada, R., Dhawan, V., Chauhan, S., Upadhyay, S. K., Gindin, M., Hotez, P. J., Valenzuela, J. G., Mohanty, B., Swarbrick, J. D., Wulff, H., Iadonato, S. P., Gutman, G. A., Beeton, C., Pennington, M. W., Norton, R. S., Chandy, K. G. Kv1.3 channel‐blocking immunomodulatory peptides from parasitic worms: implications for autoimmune diseases. FASEB J. 28, 3952‐3964 (2014). www.fasebj.org

Modulation of voltage-gated K+ channels by the sodium channel β1 subunit

Voltage-gated sodium (NaV) and potassium (KV) channels are critical components of neuronal action potential generation and propagation. Here, we report that NaVβ1 encoded by SCN1b, an integral subunit of NaV channels, coassembles with and modulates the biophysical properties of KV1 and KV7 channels, but not KV3 channels, in an isoform-specific manner. Distinct domains of NaVβ1 are involved in modulation of the different KV channels. Studies with channel chimeras demonstrate that NaVβ1-mediated changes in activation kinetics and voltage dependence of activation require interaction of NaVβ1 with the channel’s voltage-sensing domain, whereas changes in inactivation and deactivation require interaction with the channel’s pore domain. A molecular model based on docking studies shows NaVβ1 lying in the crevice between the voltage-sensing and pore domains of KV channels, making significant contacts with the S1 and S5 segments. Cross-modulation of NaV and KV channels by NaVβ1 may promote diversity and flexibility in the overall control of cellular excitability and signaling.

Differential Kv1.3, KCa3.1, and Kir2.1 expression in “classically” and “alternatively” activated microglia

Microglia are highly plastic cells that can assume different phenotypes in response to microenvironmental signals. Lipopolysaccharide (LPS) and interferon‐γ (IFN‐γ) promote differentiation into classically activated M1‐like microglia, which produce high levels of pro‐inflammatory cytokines and nitric oxide and are thought to contribute to neurological damage in ischemic stroke and Alzheimers disease. IL‐4 in contrast induces a phenotype associated with anti‐inflammatory effects and tissue repair. We here investigated whether these microglia subsets vary in their K+ channel expression by differentiating neonatal mouse microglia into M(LPS) and M(IL‐4) microglia and studying their K+ channel expression by whole‐cell patch‐clamp, quantitative PCR and immunohistochemistry. We identified three major types of K+ channels based on their biophysical and pharmacological fingerprints: a use‐dependent, outwardly rectifying current sensitive to the KV1.3 blockers PAP‐1 and ShK‐186, an inwardly rectifying Ba2+‐sensitive Kir2.1 current, and a Ca2+‐activated, TRAM‐34‐sensitive KCa3.1 current. Both KV1.3 and KCa3.1 blockers inhibited pro‐inflammatory cytokine production and iNOS and COX2 expression demonstrating that KV1.3 and KCa3.1 play important roles in microglia activation. Following differentiation with LPS or a combination of LPS and IFN‐γ microglia exhibited high KV1.3 current densities (∼50 pA/pF at 40 mV) and virtually no KCa3.1 and Kir currents, while microglia differentiated with IL‐4 exhibited large Kir2.1 currents (∼ 10 pA/pF at −120 mV). KCa3.1 currents were generally low but moderately increased following stimulation with IFN‐γ or ATP (∼10 pS/pF). This differential K+ channel expression pattern suggests that KV1.3 and KCa3.1 inhibitors could be used to inhibit detrimental neuroinflammatory microglia functions. GLIA 2016;65:106–121

Nanomolar Bifenthrin Alters Synchronous Ca2+ Oscillations and Cortical Neuron Development Independent of Sodium Channel Activity

Bifenthrin, a relatively stable type I pyrethroid that causes tremors and impairs motor activity in rodents, is broadly used. We investigated whether nanomolar bifenthrin alters synchronous Ca2+ oscillations (SCOs) necessary for activity-dependent dendritic development. Primary mouse cortical neurons were cultured 8 or 9 days in vitro (DIV), loaded with the Ca2+ indicator Fluo-4, and imaged using a Fluorescence Imaging Plate Reader Tetra. Acute exposure to bifenthrin rapidly increased the frequency of SCOs by 2.7-fold (EC50 = 58 nM) and decreased SCO amplitude by 36%. Changes in SCO properties were independent of modifications in voltage-gated sodium channels since 100 nM bifenthrin had no effect on the whole-cell Na+ current, nor did it influence neuronal resting membrane potential. The L-type Ca2+ channel blocker nifedipine failed to ameliorate bifenthrin-triggered SCO activity. By contrast, the metabotropic glutamate receptor (mGluR)5 antagonist MPEP [2-methyl-6-(phenylethynyl)pyridine] normalized bifenthrin-triggered increase in SCO frequency without altering baseline SCO activity, indicating that bifenthrin amplifies mGluR5 signaling independent of Na+ channel modification. Competitive [AP-5; (−)-2-amino-5-phosphonopentanoic acid] and noncompetitive (dizocilpine, or MK-801 [(5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate]) N-methyl-d-aspartate antagonists partially decreased both basal and bifenthrin-triggered SCO frequency increase. Bifenthrin-modified SCO rapidly enhanced the phosphorylation of cAMP response element–binding protein (CREB). Subacute (48 hours) exposure to bifenthrin commencing 2 DIV–enhanced neurite outgrowth and persistently increased SCO frequency and reduced SCO amplitude. Bifenthrin-stimulated neurite outgrowth and CREB phosphorylation were dependent on mGluR5 activity since MPEP normalized both responses. Collectively these data identify a new mechanism by which bifenthrin potently alters Ca2+ dynamics and Ca2+-dependent signaling in cortical neurons that have long term impacts on activity driven neuronal plasticity.

Sodium Channel Carboxyl-terminal Residue Regulates Fast Inactivation

The Nav1.2 and Nav1.3 voltage-gated sodium channel isoforms demonstrate distinct differences in their kinetics and voltage dependence of fast inactivation when expressed in Xenopus oocytes. Co-expression of the auxiliary β1 subunit accelerated inactivation of both the Nav1.2 and Nav1.3 isoforms, but it did not eliminate the differences, demonstrating that this property is inherent in the α subunit. By constructing chimeric channels between Nav1.2 and Nav1.3, we demonstrate that the carboxyl terminus is responsible for the differences. The Nav1.2 carboxyl terminus caused faster inactivation in the Nav1.3 backbone, and the Nav1.3 carboxyl terminus caused slower inactivation in the Nav1.2 channel. Through analysis of truncated channels, we identified a homologous 60-amino acid region within the carboxyl terminus of the Nav1.2 and the Nav1.3 channels that is responsible for this modulation of fast inactivation. Site-directed replacement of Nav1.3 lysine 1826 in this region to its Nav1.2 analogue glutamic acid 1880 (K1826E) shifted the voltage dependence of inactivation toward that of Nav1.2. The K1826E mutation also accelerated the inactivation kinetics to a level comparable with that of Nav1.2. The reverse Nav1.2 E1880K mutation exhibited much slower inactivation kinetics and depolarized inactivation voltage dependence. A complementary mutation located within the inactivation linker of Nav1.3 (K1453E) caused inactivation changes mirroring those caused by the K1826E mutation in Nav1.3. Therefore, we have identified a homologous carboxyl-terminal residue that regulates the kinetics and voltage dependence of fast inactivation in sodium channels, possibly via a charge-dependent interaction with the inactivation linker.

Domain structure and function of matrix metalloprotease 23 (MMP23): role in potassium channel trafficking

MMP23 is a member of the matrix metalloprotease family of zinc- and calcium-dependent endopeptidases, which are involved in a wide variety of cellular functions. Its catalytic domain displays a high degree of structural homology with those of other metalloproteases, but its atypical domain architecture suggests that it may possess unique functional properties. The N-terminal MMP23 pro-domain contains a type-II transmembrane domain that anchors the protein to the plasma membrane and lacks the cysteine-switch motif that is required to maintain other MMPs in a latent state during passage to the cell surface. Instead of the C-terminal hemopexin domain common to other MMPs, MMP23 contains a small toxin-like domain (TxD) and an immunoglobulin-like cell adhesion molecule (IgCAM) domain. The MMP23 pro-domain can trap Kv1.3 but not closely-related Kv1.2 channels in the endoplasmic reticulum, preventing their passage to the cell surface, while the TxD can bind to the channel pore and block the passage of potassium ions. The MMP23 C-terminal IgCAM domain displays some similarity to Ig-like C2-type domains found in IgCAMs of the immunoglobulin superfamily, which are known to mediate protein–protein and protein–lipid interactions. MMP23 and Kv1.3 are co-expressed in a variety of tissues and together are implicated in diseases including cancer and inflammatory disorders. Further studies are required to elucidate the mechanism of action of this unique member of the MMP family.

Intracellular Trafficking of the KV1.3 Potassium Channel Is Regulated by the Prodomain of a Matrix Metalloprotease

Background: Noncanonical functions of matrix metalloproteases are poorly characterized. Results: The prodomain of MMP23 co-localizes with and traps KV1.3 channels intracellularly, thereby suppressing KV1.3 currents. Conclusion: A novel metalloprotease-independent channel-modulating function of the MMP23 prodomain has been identified. Significance: The topological similarity of the prodomain of MMP23 and KCNE proteins suggests a shared mechanism of channel modulation. Matrix metalloproteases (MMPs) are endopeptidases that regulate diverse biological processes. Synthesized as zymogens, MMPs become active after removal of their prodomains. Much is known about the metalloprotease activity of these enzymes, but noncanonical functions are poorly defined, and functions of the prodomains have been largely ignored. Here we report a novel metalloprotease-independent, channel-modulating function for the prodomain of MMP23 (MMP23-PD). Whole-cell patch clamping and confocal microscopy, coupled with deletion analysis, demonstrate that MMP23-PD suppresses the voltage-gated potassium channel KV1.3, but not the closely related KV1.2 channel, by trapping the channel intracellularly. Studies with KV1.2-1.3 chimeras suggest that MMP23-PD requires the presence of the KV1.3 region from the S5 trans-membrane segment to the C terminus to modulate KV1.3 channel function. NMR studies of MMP23-PD reveal a single, kinked trans-membrane α-helix, joined by a short linker to a juxtamembrane α-helix, which is associated with the surface of the membrane and protected from exchange with the solvent. The topological similarity of MMP23-PD to KCNE1, KCNE2, and KCNE4 proteins that trap KV1.3, KV1.4, KV3.3, and KV3.4 channels early in the secretory pathway suggests a shared mechanism of channel regulation. MMP23 and KV1.3 expression is enhanced and overlapping in colorectal cancers where the interaction of the two proteins could affect cell function.

Structural Insights into the Atomistic Mechanisms of Action of Small Molecule Inhibitors Targeting the KCa3.1 Channel Pore

The intermediate-conductance Ca2+-activated K+ channel (KCa3.1) constitutes an attractive pharmacological target for immunosuppression, fibroproliferative disorders, atherosclerosis, and stroke. However, there currently is no available crystal structure of this medically relevant channel that could be used for structure-assisted drug design. Using the Rosetta molecular modeling suite we generated a molecular model of the KCa3.1 pore and tested the model by first confirming previously mapped binding sites and visualizing the mechanism of TRAM-34 (1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole), senicapoc (2,2-bis-(4-fluorophenyl)-2-phenylacetamide), and NS6180 (4-[[3-(trifluoromethyl)phenyl]methyl]-2H-1,4-benzothiazin-3(4H)-one) inhibition at the atomistic level. All three compounds block ion conduction directly by fully or partially occupying the site that would normally be occupied by K+ before it enters the selectivity filter. We then challenged the model to predict the receptor sites and mechanisms of action of the dihydropyridine nifedipine and an isosteric 4-phenyl-pyran. Rosetta predicted receptor sites for nifedipine in the fenestration region and for the 4-phenyl-pyran in the pore lumen, which could both be confirmed by site-directed mutagenesis and electrophysiology. While nifedipine is thus not a pore blocker and might be stabilizing the channel in a nonconducting conformation or interfere with gating, the 4-phenyl-pyran was found to be a classical pore blocker that directly inhibits ion conduction similar to the triarylmethanes TRAM-34 and senicapoc. The Rosetta KCa3.1 pore model explains the mechanism of action of several KCa3.1 blockers at the molecular level and could be used for structure-assisted drug design.