Ferenc Papp

Vm24, a natural immunosuppressive peptide, potently and selectively blocks Kv1.3 potassium channels of human T cells.

Blockade of Kv1.3 K+ channels in T cells is a promising therapeutic approach for the treatment of autoimmune diseases such as multiple sclerosis and type 1 diabetes mellitus. Vm24 (α-KTx 23.1) is a novel 36-residue Kv1.3-specific peptide isolated from the venom of the scorpion Vaejovis mexicanus smithi. Vm24 inhibits Kv1.3 channels of human lymphocytes with high affinity (Kd = 2.9 pM) and exhibits >1500-fold selectivity over other ion channels assayed. It inhibits the proliferation and Ca2+ signaling of human T cells in vitro and reduces delayed-type hypersensitivity reactions in rats in vivo. Our results indicate that Vm24 has exceptional pharmacological properties that make it an excellent candidate for treatment of certain autoimmune diseases.

Voltage-Gated Sodium Channel Nav1.7 Maintains the Membrane Potential and Regulates the Activation and Chemokine-Induced Migration of a Monocyte-Derived Dendritic Cell Subset

Expression of CD1a protein defines a human dendritic cell (DC) subset with unique functional activities. We aimed to study the expression of the Nav1.7 sodium channel and the functional consequences of its activity in CD1a− and CD1a+ DC. Single-cell electrophysiology (patch-clamp) and quantitative PCR experiments performed on sorted CD1a− and CD1a+ immature DC (IDC) showed that the frequency of cells expressing Na+ current, current density, and the relative expression of the SCN9A gene encoding Nav1.7 were significantly higher in CD1a+ cells than in their CD1a− counterparts. The activity of Nav1.7 results in a depolarized resting membrane potential (−8.7 ± 1.5 mV) in CD1a+ IDC as compared with CD1a− cells lacking Nav1.7 (−47 ± 6.2 mV). Stimulation of DC by inflammatory signals or by increased intracellular Ca2+ levels resulted in reduced Nav1.7 expression. Silencing of the SCN9A gene shifted the membrane potential to a hyperpolarizing direction in CD1a+ IDC, resulting in decreased cell migration, whereas pharmacological inhibition of Nav1.7 by tetrodotoxin sensitized the cells for activation signals. Fine-tuning of IDC functions by a voltage-gated sodium channel emerges as a new regulatory mechanism modulating the migration and cytokine responses of these DC subsets.

A selective blocker of Kv1.2 and Kv1.3 potassium channels from the venom of the scorpion Centruroides suffusus suffusus

A novel potassium channel blocker peptide was purified from the venom of the scorpion Centruroides suffusus suffusus by high-performance liquid chromatography and its amino acid sequence was completed by Edman degradation and mass spectrometry analysis. It contains 38 amino acid residues with a molecular weight of 4000.3Da, tightly folded by three disulfide bridges. This peptide, named Css20, was shown to block preferentially the currents of the voltage-dependent K+-channels Kv1.2 and Kv1.3. It did not affect several other ion channels tested at 10 nM concentration. Concentration-response curves of Css20 yielded an IC50 of 1.3 and 7.2 nM for Kv1.2- and Kv1.3-channels, respectively. Interestingly, despite the similar affinities for the two channels the association and dissociation rates of the toxin were much slower for Kv1.2, implying that different interactions may be involved in binding to the two channel types; an implication further supported by in silico docking analyses. Based on the primary structure of Css20, the systematic nomenclature proposed for this toxin is alpha-KTx 2.13.

Transient receptor potential vanilloid-2 mediates the effects of transient heat shock on endocytosis of human monocyte-derived dendritic cells

Our goal was to investigate the effect of heat shock on human monocyte‐derived dendritic cells (DCs) and to dissect the role of thermosensitive transient receptor potential (TRP) channels in the process. We provide evidence that a short heat shock challenge (43 °C) decreased the endocytotic activity of the DCs and that this effect could be alleviated by the RNAi‐mediated knockdown of TRPV2 but, importantly, not by the pharmacological (antagonists) or molecular (RNAi) suppression of TRPV1 and TRPV4 activities/levels. Likewise, the heat shock‐induced robust membrane currents were selectively and markedly inhibited by TRPV2 “silencing” whereas modulation of TRPV1 and TRPV4 activities, again, had no effect. These intriguing data introduce TRPV2‐coupled signaling as a key player in mediating the cellular actions of heat shock on DCs.

A new mechanism of voltage-dependent gating exposed by KV10.1 channels interrupted between voltage sensor and pore

Voltage-gated ion channels couple transmembrane potential changes to ion flow. Conformational changes in the voltage-sensing domain (VSD) of the channel are thought to be transmitted to the pore domain (PD) through an &agr;-helical linker between them (S4–S5 linker). However, our recent work on channels disrupted in the S4–S5 linker has challenged this interpretation for the KCNH family. Furthermore, a recent single-particle cryo-electron microscopy structure of KV10.1 revealed that the S4–S5 linker is a short loop in this KCNH family member, confirming the need for an alternative gating model. Here we use “split” channels made by expression of VSD and PD as separate fragments to investigate the mechanism of gating in KV10.1. We find that disruption of the covalent connection within the S4 helix compromises the ability of channels to close at negative voltage, whereas disconnecting the S4–S5 linker from S5 slows down activation and deactivation kinetics. Surprisingly, voltage-clamp fluorometry and MTS accessibility assays show that the motion of the S4 voltage sensor is virtually unaffected when VSD and PD are not covalently bound. Finally, experiments using constitutively open PD mutants suggest that the presence of the VSD is structurally important for the conducting conformation of the pore. Collectively, our observations offer partial support to the gating model that assumes that an inward motion of the C-terminal S4 helix, rather than the S4–S5 linker, closes the channel gate, while also suggesting that control of the pore by the voltage sensor involves more than one mechanism.

OcyKTx2, a new K+-channel toxin characterized from the venom of the scorpion Opisthacanthus cayaporum

Opisthacanthus cayaporum belongs to the Liochelidae family, and the scorpions from this genus occur in southern Africa, Central America and South America and, therefore, can be considered a true Gondwana heritage. In this communication, the isolation, primary structure characterization, and K⁺-channel blocking activity of new peptide from this scorpion venom are reported. OcyKTx2 is a 34 amino acid long peptide with four disulfide bridges and molecular mass of 3807 Da. Electrophysiological assays conducted with pure OcyKTx2 showed that this toxin reversibly blocks Shaker B K⁺-channels with a Kd of 82 nM, and presents an even better affinity toward hKv1.3, blocking it with a Kd of ∼18 nM. OcyKTx2 shares high sequence identity with peptides belonging to subfamily 6 of α-KTxs that clustered very closely in the phylogenetic tree included here. Sequence comparison, chain length and number of disulfide bridges analysis classify OcyKTx2 into subfamily 6 of the α-KTx scorpion toxins (systematic name, α-KTx6.17).

Investigating the Function of a Novel Voltage-Sensing Protein

We have identified a protein coded by the C15orf27 gene that we named NVS (Novel Voltage Sensor). NVS contains 531 residues, and contains an S1-S4 domain, a 90 residue N-terminus and a 307 residue C-terminus, both of which are predicted to be intracellular. The most critical residues found in S1-S4 domains of other voltage sensors are conserved in NVS, including 3 Arg and a Lys in the S4 helix, 4 conserved acidic residues in S1-S3 and the charge-transfer Phe in S2. In addition, the C-terminus is predicted to contain a coiled-coil domain, similar to voltage-activated proton (Hv1) channels. Our hypothesis is that NVS functions as a voltage sensor that couples to intracellular signaling pathways or interacts with Hv1 to form heteroligomers through the C-terminal coiled-coil domain. We used site-specific voltage-clamp fluorometry and identified several positions at the outer ends of S3 and S4 where labeled Cys residues produced changes in fluorescence as a function of membrane potential. Several positions give complex fluorescence responses, starting with a rapid increase in fluorescence followed by slower decrease in fluorescence. We also investigated whether NVS can oligomerize with Hv1, but observe no change in the gating properties of Hv1 when coexpressed with NVS, and NVS was not capable of interacting with Hv1 in pull down assays. Having no apparent interaction with Hv1, we set out to determine whether NVS forms oligomers. Using chemical crosslinking and pull-down assays, we see formation of oligomers that are consistent with dimers and dependent on the C-terminus. Taken together, our results support the hypothesis that NVS is a voltage sensing protein capable of dimerization, with a function independent of Hv1. We are currently using these findings and approaches to investigate the structural assembly of NVS and to identify interaction partners.

Voltage-Gated Sodium Channel Nav1.7 Maintains the Membrane Potential and Regulates Chemokine-Induced Migration of a Subpopulation of Monocyte-Derived Dendritic Cells

Expression of CD1a protein defines two human dendritic cell (DC) subsets with distinct functions. We aimed to study the expression of the Nav1.7 sodium channel and the functional consequences of its activity in CD1a- and CD1a+ DC. Single-cell electrophysiology (patch-clamp) and Q-PCR experiments performed on immature sorted CD1a- and CD1a+ DC populations showed that the frequency of cells expressing Na+ current, current density and the relative expression of the SCN9A gene encoding Nav1.7 were significantly higher in CD1a+ cells than in their CD1a- counterparts. Down-regulation of Nav 1.7 expression accompanying DC maturation is abolished by increasing cytosolic Ca2+ concentration using ionomycin and thapsigargin or inhibiting the NF-κB-pathway. The activity of Nav1.7 results in a depolarized resting potential (−8.7 +/- 1.5 mV) in CD1a+ IDCs as compared to CD1a- cells lacking Nav1.7 (−47 +/- 6.2 mV) or mature DCs used as ¯controls¯ with reduced Nav1.7 expression. Silencing of the SCN9A gene shifted the membrane potential to a hyperpolarizing direction in CD1a+ immature DC resulting in decreased cell migration, similarly to the pharmacological inhibition of Nav1.7 by TTX. The control of IDC function by a voltage-gated sodium channel emerges as a new regulatory mechanism modulating the migration and other responses of these DC subsets.

Molecular Rearrangements During Slow Inactivation of the Shaker-Ir Potassium Channel

Crosstalk between the activation and slow inactivation gates in Shaker potassium channels is now well-established. The activation gate perceives the conformation of the inactivation gate (Panyi and Deutsch, 2006, 2007). Closure of the inactivation gate speeds opening and slows closing of the activation gate, i.e., stabilizing the gate in the open configuration. If this coupling involves movement of the S6 transmembrane segment, then we predict state-dependent changes in accessibility of residues lining the channel cavity. We engineered cysteines, one at a time, at positions 470, 471, 472, 473, and 474 in a T449A Shaker-IR background and determined modification rates for the cysteine modifying reagents, MTSET and MTSEA, in the open, closed, and inactivated state of the channel. Neither reagent, applied from the intracellular side, modifies cysteines at 470-474 in the closed state. Both 470C and 474C are rapidly modified in the open state and at approximately one-tenth this rate in the inactivated state. In contrast, 471C is not modified in the open state but can be modified by MTSEA but not MTSET in the inactivated state. Residue 472C cannot be modified in any of the three states. Mutant 473C did not express current. Our findings are consistent with a rotation of S6 in the inactivated state, which increases the accessibility of residue 471 while simultaneously decreasing accessibility of residues 470 and 474. Any model of C-type inactivation in the Shaker Kv channel must conform to these experimental observations.[Supported by NIH grant GM 069837 (CD) and OTKA K 75904 (GP)].