The expression of Kv1.3 channels in D10 cells was confirmed from the inhibition of the current ITK Inhibitor by margatoxin (MgTx), a high affinity inhibitor of Kv1.3 channels applied at its known blocking concentration  (Number 1B), determining the midpoint of the voltage dependence of steady-state activation (V1/2 = ?25 2 mV, = 13, Figure A1, panel B) and the time constant of inactivation kinetics ( = 364 26 ms, = 17, Figure 1A). set up of channels and the constrained space between the interacting cells creates a favorable environment for these oscillations, which may enhance the signaling process leading to T cell activation. = 18). The manifestation of Kv1.3 channels in D10 cells was confirmed from the inhibition of the current by margatoxin (MgTx), a high affinity inhibitor of Kv1.3 channels applied at its known blocking concentration  (Number 1B), determining the midpoint of the voltage dependence of steady-state activation (V1/2 = ?25 2 mV, = 13, Figure A1, panel B) and the time constant of inactivation kinetics ( = 364 26 ms, = 17, Figure 1A). A pipette remedy having 1 M free Ca2+ concentration was used to ITK Inhibitor measure the manifestation of KCa3.1 channels in D10 cells in response to voltage ramps from ?120 mV to +50 mV . The slope of the current below the activation threshold of Kv1.3 is characteristic for the KCa3.1 conductance of the membrane. The current magnitude was 209 33 pA at ?20 mV membrane potential (= 12). The presence of KCa3.1 channels was confirmed from the inhibition of the wholeCcell current by TRAM-34 , a selective small molecule inhibitor of these channels (Number 1C). The formation of cell conjugates between APCs and T cells was initiated by co-centrifugation of ITK Inhibitor conalbumin antigen-pulsed CH12 cells and D10 cells at a percentage of 1 1:1 . The formation of the Is definitely was confirmed from the characteristic recruitment of the GFP-tagged PKC into the synapse using confocal microscopy (Number 2) [22,34]. In subsequent experiments the specific recruitment of PKC-GFP into the Is definitely was used to identify appropriate cell conjugates for electrophysiological experiments. Open in a separate window Number 1 Kv1.3 and KCa3.1 currents are expressed in D10 cells. (A): Representative Kv1.3 K+ current in one D10 cell recorded during a 1.5-s-long test pulse to +50 mV from a holding potential of C120 mV. The superimposed dashed collection indicates the best match solitary exponential with =252 ms. (B): Representative Kv1.3 K+ currents from a single D10 cell in control solution, and after the equilibration of the prevent in the presence of 15 pM MgTx (test pulse: +50 mV). (C): Voltage ramps from C120 mV to +50 mV (period: 150 ms) evoked KCa3.1 currents from a single D10 cell. Traces display the current in control remedy, after the equilibration of the block in the presence of 250 nM TRAM-34, and after wash-out. The voltage range below the activation threshold of Kv1.3 channels is shown only. Open in a separate windowpane Number 2 Recruitment of PKC-GFP and Kv1.3 into the IS. Representative confocal images of a D10 cell only (ACD) or conjugated to a CH12 cell (E-H). Panels from remaining T to right display: (A,E): GFP transmission of PKC (green), (B,F): Cy3 fluorescence of Kv1.3 signal (reddish), (C,G): merge of the PKC-GFP and Kv1.3 signs. (D,H): bright field image of the cells. Slice ITK Inhibitor thickness was arranged to 1 1 m. The image was taken 20 min after combining and centrifuging collectively the two cell types. Scale bar is definitely 10 m. 2.2. The Membrane Potential Oscillates More Frequently in Conjugated T Cells Than in Lone T Cells We recorded the membrane potential of D10 cells not conjugated (lone) or conjugated with specific antigen showing CH12 cells using the patch-clamp technique  in I = 0.