Data describing the effects of potassium channels modulators on outward currents measured in human lymphoma cell lines

In the present work, applying the whole-cell patch-clamp technique in voltage clamp mode, we have investigated the effects of different drugs, such as riluzole, Psora-4 and Tram-34, on the potassium currents in four human lymphoma cell lines. We focused on outward currents mediated by two potassium channels (Kv1.3 and KCa3.1), which are known to play a key physiological role in lymphoid cells. The currents were evoked by voltage ramps ranging from -120 mV to +40 mV and the conductance of the two potassium channels was measured between +20 mV and +40 mV, both in the absence and in the presence of the specific blockers Psora-4 (Kv1.3; 1 µM) and Tram-34 (KCa3.1; 1 µM). The effect of the latter was tested after KCa3.1 channels were activated by riluzole 10 µM. Taken together, these data could be useful as an indication of the functional characteristics of the potassium channels in human lymphomas and represent a starting point for the study of potassium conductance in cellular models of these tumors.


a b s t r a c t
In the present work, applying the whole-cell patch-clamp technique in voltage clamp mode, we have investigated the effects of different drugs, such as riluzole, Psora-4 and Tram-34, on the potassium currents in four human lymphoma cell lines. We focused on outward currents mediated by two potassium channels (Kv1.3 and KCa3.1), which are known to play a key physiological role in lymphoid cells. The currents were evoked by voltage ramps ranging from -120 mV to + 40 mV and the conductance of the two potassium channels was measured between + 20 mV and + 40 mV, both in the absence and in the presence of the specific blockers Psora-4 (Kv1.3; 1 μM) and Tram-34 (KCa3.1; 1 μM). The effect of the latter was tested after KCa3.1 channels were activated by riluzole 10 μM. Taken together, these data could be useful as an indication of the functional characteristics of the potassium channels in human lymphomas and represent a starting point for the study of potassium conductance in cellular models of these tumors. © 2020 Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ) Table   Subject Cancer

Value of the Data
• A functional role of ion channels in different tumor types is increasingly recognized. The role of potassium channels has been widely studied in B lymphocyte physiology but not in lymphomas. Our dataset represents a start point for the study of potassium channels in cellular models of human lymphomas. • The shared data can be used by researchers as a comparative model for studying the conduction properties of potassium channels in tumor cell lines and especially for non-solid tumors (i.e. lymphocyte-derived cancer cells). • By providing a benchmark dataset the shared data are likely to be valuable in further development and refinement of ion channel conductance measurements on non-adhesive cell types.

Data Description
In SU-DHL4 cell line the application of 1 μM Psora-4 produced a significant reduction of the slope conductance from 11.6 ± 9.8 nS to 9.2 ± 8.6 nS and we measured an average net Psorasensitive conductance of 2.4 ± 2.5 nS ( p = 0.01, n = 10). On the other hand on SU-DHL-6 cells the  application of 1 μM Psora 4 produced a non significant reduction on the slope conductance (control: 18.1 ± 6.3 nS, treated: 17.2 ± 5.4 nS, n = 7; t -test). Similar results were obtained for WSU-DLCL2 (control: 1.3 ± 0.6 nS, treated: 0.7 ± 0.5 nS, n = 5) as well as EBV infected lymphocytes (control: 7.0 ± 6.3 nS, treated: 6.5 ± 6.7 nS, n = 8). Fig. 1 shows the time courses under control conditions and in the presence of Psora-4 (1 μM) representative of each cell line examined together with their relative current ramps.
-The raw data of the electrophysiological recordings are organized as follows in the Zenodo repository: -Each folder is named with the name of the cell line and contains the .csv files related to the electrophysiological recordings. Moreover, it contains an .xls file (eg. DHL-4_Kv1.3_raw.xls). This .xls file contains the measured conductance values in the various experimental conditions (ctrl, drugs etc.) relative to each single cell of that cell line. -Each .csv file corresponds to a cell of its relative cell line with the exception of files whose name ends with −1 or −2 (for example 191120_C1-1, 191120_C1-2). In these cases, the recording related to a cell has been divided in two files and −1 and −2 correspond to the first and second part of the recording, respectively. -For each .csv file: column 1 is an index column representing the sample number (not useful for analysis purposes), column 2 (Time) reports in ms each of the sample-by-sample timepoints, column 3 (Vcmd) is the potential value in mV imposed during the protocol, from column 4 (Sw…) onwards the single sweeps of that recording are reported (column by column) and each cell of a sweep column corresponds to the current (pA) measured sample by sample. -For the purposes of the analysis, the potential column (Vcmd) should be plotted on the x axis and one (or more) sweep column(s) on the y axis. So far it will be possible to display one (or more) ramps: from the Vcmd vs. Current plot it is then possible to perform a linear fit in the range between + 20 and + 40 mV. The slope of this fit corresponds to the conductance value for each ramp.

Experimental Design, Materials and Methods
Human lymphoma cell lines. Epstein-Barr virus-infected B lymphocytes were previously generated in our laboratory [1] . Epstein-Barr virus-infected B lymphocytes, the DLBCL (GC subtype) cell lines SU-DHL4 and SU-DHL6 were cultured in RPMI 1640 medium supplemented with 2 mM l -glutamine, 10% bovine calf serum (HyClone), 10 0 U/mL penicillin, and 10 0 μg/mL streptomycin. The DLBCL, SU-DHL4 and SU-DHL6 cell lines are derived from germinal center B-cell type diffuse large B-cell lymphoma, which is the most common type of B-cell lymphoma. These cell represent in vitro models to functionally study the ion channels expression pattern recently reported in clinical specimens [2] .
Electrophysiological recordings. Cells were plated on the experimental day directly on plastic. Electrophysiological recordings has been performed at room temperature ( ∼25 °C) in the wholecell configuration of the patch-clamp technique, after 1 hour in an incubator at 37 °C [3][4][5] . The patch pipettes were pulled from borosilicate glass capillary tubes, their resistance was 4-5 M and their capacitances were manually compensated up to 90-95% after the reaching of a stable gigaseal. Experimental protocols and data acquisition has been performed with the Multiclamp 700A amplifier and pCLAMP 9.2 software (Molecular Devices, Sunnyvale, California, USA) has been used for data analysis. All the outward potassium currents were measured with a 25KHz sampling rate and a 3 KHz low-pass filter. Cells' identification and patch has been performed at 40x magnification with a Nikon Eclipse TE300 microscope (Nikon Instruments Inc.), equipped with a Photometrics CoolSNAP CF camera (Teledyne Photometrics, Tucson AZ). Cells membrane potentials were held at −80 mV and outward potassium currents were elicited by 200 ms voltage ramps ( −120 to + 40 mV) with an intersweep interval of 10 s [6] . The internal pipette solution for Kv1.3 conductances measurement contained (in mM): 130 K + aspartate, 10 NaCl, 4 CaCl2, 2 MgCl2, 10 Hepes-NaOH, 10 EGTA, pH 7.3. The external solution, instead, contained (in mM): 130 NaCl, 5 KCl, 2 CaCl2, 2 MgCl2, 10 HEPES, 5 Glucose (EK = −80 mV), pH of 7.4. For KCa3.1 currents a different internal pipette solution was used (in mM): 145 K + aspartate, 8.55 CaCl2, 2 MgCl2, 10 HEPES, 10 EGTA, pH 7.2. In this case, the external solution was a Na + aspartate Ringer, containing (in mM): 160 Na + aspartate, 4.5 KCl, 2 CaCl2, 1 MgCl2, 5 Hepes-NaOH, pH 7.4. The GSlope values have been measured offline between + 20 and + 40 mV potentials range with the Clampfit software. The difference between the elicited currents recorded in control condition and the ones recorded in the presence of Psora-4 (1 μM) represented the Psora-4-sensitive current (Kv1.3 current). The effects of the KCa3.1 specific inhibitor TRAM-34 at 1 μM, instead, has been assessed on the maximal KCa3.1 activation, induced by Riluzole (10 μM). The 1 μM concentration for both Psora-4 and TRAM-34 was chosen because of its capability of evoking a maximal effect and drugs has been applied for at leat 5 min. Resting membrane potential (VREST) values were measured in I-0 mode.
Statistics. Parametric tests were used for statistical analysis, i.e., paired and unpaired t -test. Data are reported as mean ± SD and median ± interquartile range (IQR). The normality of data distribution was checked with Kolmogorov-Smirnov test. Statistical analysis was performed using Prism 6 software (GraphPad Software, San Diego, CA, USA). All statistical tests were two tailed with a significance level of 0.05.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.