Orsolya Szilagyi

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.

Functional consequences of Kv1.3 ion channel rearrangement into the immunological synapse.

Formation of immunological synapse (IS), the interface between T cells and antigen presenting cells, is a crucial step in T cell activation. This conjugation formation results in the rearrangement and segregation of a set of membrane bound and cytosolic proteins, including that of the T cell receptor, into membrane domains. It was showed earlier that Kv1.3, the dominant voltage-gated potassium channel of T cells redistributes into the IS on interaction with its specific APC. In the present experiments we investigated the functional consequences of the translocation of Kv1.3 channels into the IS formed between mouse helper T (T(h)2) and B cells. Biophysical characteristics of whole-cell Kv1.3 current in standalone cells (c) or ones in IS (IS) were determined using voltage-clamp configuration of standard whole-cell patch-clamp technique. Patch-clamp recordings showed that the activation of Kv1.3 current slowed (tau(a,IS)=2.36+/-0.13 ms (n=7); tau(a,c)=1.36+/-0.06 ms (n=18)) whereas the inactivation rate increased (tau(i,IS)=263+/-29 ms (n=7); tau(i,c)=365+/-27 ms (n=17)) in cells being in IS compared to the standalone cells. The equilibrium distribution between the open and the closed states of Kv1.3 (voltage-dependence of steady-state activation) was shifted toward the depolarizing potentials in T cells engaged into IS (V(1/2,IS)=-20.9+/-2 mV (n=7), V(1/2,c)=-26.4+/-1.5 mV (n=12)). Thus, segregation of Kv1.3 channels into the IS modifies the gating properties of the channels. Application of protein kinase (PK) inhibitors (PKC: GF109203X, PKA: H89, p56Lck: damnacanthal) demonstrated that increase in the inactivation rate can be explained by the dephosphorylation of the channel protein. However, the slower activation kinetics of Kv1.3 in IS is likely to be the consequence of the redistribution of the channels into distinct membrane domains.

Membrane microdomain organization, calcium signal, and NFAT activation as an important axis in polarized Th cell function.

T helper lymphocytes become polarized upon antigen and cytokine stimuli received after their maturation in the thymus. Since the balance of Th1 and Th2 responses is critical in healthy and pathological immune responses, understanding the molecular base of T cell polarization still remained an important question. Using our Th0/Th1/Th2 hybridoma model system, we performed a comparative study on polarized Th1 and Th2 cells in terms of their membrane raft expression/composition, their TCR mediated activation signaling, and sensitivity to activation‐induced cell death (AICD) using flow and image cytometric methods. We show here that the TCR stimulation induced more intense and sustained Ca2+‐response in Th1 cells compared to Th2 ones correlates well with a shorter nuclear residence time of the Ca2+‐dependent NFAT transcription factor in Th2 cells. In addition, NFAT translocation directly depended on lipid raft integrity/membrane cholesterol level. Expression pattern of raftophilic accessory proteins (CD4, CD59, and CD48) and lipids (GM1, cholesterol) were also different in the Th1 and Th2 hybridomas, similarly to differentiated spleen Th cells. The activation‐induced, remarkably clustered and polarized membrane distribution of TCR/CD3 complex in Th1, but not in Th2 cells, together with an increased raft localization of Kv1.3 ion channels regulating the Ca2+‐response, are consistent with the above properties of NFAT. Finally, the polarized Th cells, especially Th1, were more sensitive to AICD than their unpolarized Th0 precursor. These results suggest that the membrane microdomain organization—Ca2+‐signaling—NFAT activation axis is an important determinant of polarized Th cell effector function and fate.

Analysis of the K+ current in human CD4+ T lymphocytes in hypercholesterolemic state

Atherosclerosis involves immune mechanisms: T lymphocytes are found in atherosclerotic plaques, suggesting their activation during atherogenesis. The predominant voltage-gated potassium channel of T cells, Kv1.3 is a key regulator of the Ca(2+)-dependent activation pathway. In the present experiments we studied the proliferation capacity and functional changes of Kv1.3 channels in T cells from healthy and hypercholestaeremic patients. By means of CFSE-assay (carboxyfluorescein succinimidyl ester) we showed that spontaneous activation rate of lymphocytes in hypercholesterolemia was elevated and the antiCD3/antiCD28 co-stimulation was less effective as compared to the healthy group. Using whole-cell patch-clamping we obtained that the activation and deactivation kinetics of Kv1.3 channels were faster in hypercholesterolemic state but no change in other parameters of Kv1.3 were found (inactivation kinetics, steady-state activation, expression level). We suppose that incorporation of oxLDL species via its raft-rupturing effect can modify proliferative rate of T cells as well as the gating of Kv1.3 channels.

The anti-proliferative effect of cation channel blockers in T lymphocytes depends on the strength of mitogenic stimulation

Ion channels are crucially important for the activation and proliferation of T lymphocytes, and thus, for the function of the immune system. Previous studies on the effects of channel blockers on T cell proliferation reported variable effectiveness due to differing experimental systems. Therefore our aim was to investigate how the strength of the mitogenic stimulation influences the efficiency of cation channel blockers in inhibiting activation, cytokine secretion and proliferation of T cells under standardized conditions. Human peripheral blood lymphocytes were activated via monoclonal antibodies targeting the TCR-CD3 complex and the co-stimulator CD28. We applied the blockers of Kv1.3 (Anuroctoxin), KCa3.1 (TRAM-34) and CRAC (2-Apb) channels of T cells either alone or in combination with rapamycin, the inhibitor of the mammalian target of rapamycin (mTOR). Five days after the stimulation ELISA and flow cytometric measurements were performed to determine IL-10 and IFN-γ secretion, cellular viability and proliferation. Our results showed that ion channel blockers and rapamycin inhibit IL-10 and IFN-γ secretion and cell division in a dose-dependent manner. Simultaneous application of the blockers for each channel along with rapamycin was the most effective, indicating synergy among the various activation pathways. Upon increasing the extent of mitogenic stimulation the anti-proliferative effect of the ion channel blockers diminished. This phenomenon may be important in understanding the fine-tuning of T cell activation.

The C-terminal HRET sequence of Kv1.3 regulates gating rather than targeting of Kv1.3 to the plasma membrane

Kv1.3 channels are expressed in several cell types including immune cells, such as T lymphocytes. The targeting of Kv1.3 to the plasma membrane is essential for T cell clonal expansion and assumed to be guided by the C-terminus of the channel. Using two point mutants of Kv1.3 with remarkably different features compared to the wild-type Kv1.3 (A413V and H399K having fast inactivation kinetics and tetraethylammonium-insensitivity, respectively) we showed that both Kv1.3 channel variants target to the membrane when the C-terminus was truncated right after the conserved HRET sequence and produce currents identical to those with a full-length C-terminus. The truncation before the HRET sequence (NOHRET channels) resulted in reduced membrane-targeting but non-functional phenotypes. NOHRET channels did not display gating currents, and coexpression with wild-type Kv1.3 did not rescue the NOHRET-A413V phenotype, no heteromeric current was observed. Interestingly, mutants of wild-type Kv1.3 lacking HRET(E) (deletion) or substituted with five alanines for the HRET(E) motif expressed current indistinguishable from the wild-type. These results demonstrate that the C-terminal region of Kv1.3 immediately proximal to the S6 helix is required for the activation gating and conduction, whereas the presence of the distal region of the C-terminus is not exclusively required for trafficking of Kv1.3 to the plasma membrane.

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.

Answer to the "Comment on functional consequences of Kv1.3 ion channel rearrangement into the immunological synapse" by Stefan Bittner et al. [Immunol. Lett. 125 (Aug 15 (2)) (2009) 156-157]

A critical comment to our paper on Kv1.3 channels in the immunological synapse (IS) was published, which emphasized the role of two-pore potassium channels (TASK-1 and 3) in the physiology of T lymphocytes [1]. Here we provide several lines of evidence that presence of TASK channels in the T cell line (a murine Th2 Cell line, D10) used in our experimental model cannot explain the changes in the biophysical parameters of the whole-cell current upon formation of an IS [2].