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Tsiferova Nargiza A., Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan, Tashkent, Uzbekistan
Merzlyak Petr G., DSc., Institute of Biophysics and Biochemistry, National University of Uzbekistan, Tashkent, Uzbekistan
Sabirov Ravshan Z., Acad., Institute of Biophysics and Biochemistry, National University of Uzbekistan Center for Advanced Technologies Department of Biophysics, National University of Uzbekistan, Tashkent, Uzbekistan E-mail: [email protected]
VSOR CHANNEL PROPERTIES IN MELANOMA CELLS CULTURED IN DIFFERENT MEDIUMS
Abstract. Effect of DMEM and RPMI-1640 mediums on VSOR anion channel properties in a melanoma cell line (KML) was studied. Selectivity and single channel amplitude exhibited significant differences in KML cells cultured under different conditions.
Keywords: Melanoma, B16, ion channel, VSOR, VRAC.
Introduction
Malignant melanoma is one of the most aggressive skin cancers. The percentage of the illness in Caucasian populations has extremely increased comparing to other tumors over the last 30 years [1]. Mortality risks in malignant melanomas are higher due to its resistance to conventional treatment [2]. Ion channels may emerge as new targets for melanoma treatment. Volume-sensitive outwardly rectifying (VSOR) anion channel plays a major role in cell volume regulation system, also involved in different physiological and pathophysiological processes [3]. However, the role of VSOR channel in malignant melanoma tumorigenesis and melanogenesis remains poorly understood. B16 mouse melanoma cell line is widely used as an appropriate model for metastasis, melanogenesis and tumor formation studies. Modification of B16 cells culture conditions are known to induce morphological changes (differentiation), as well as alterations in malignancy, melanogenesis and unusual metastasis locations [4; 5]. In the present study, we found that cell culture conditions significantly affect the biophysical properties (selectivity and single-channel amplitude) of the VSOR anion channel in KML melanoma cells.
Materials and methods. KML melanoma cell line (patent UZ IAP 02729) was originally generated by continuous cultur-ing of excised primary lung tumors from mice intravenously in-j ected with B16 melanoma. KML were cultured in RPMI-1640 (HiMEDIA, India) or in DMEM (GIBCO, Japan) supplemented with 100 U/ml penicillin plus 100 ^g/ml streptomycin as
well as with 10% fetal bovine serum. Cells were kept at 37 °C at 5% CO2. For patch-clamp experiments, cells were cultured in suspension under gentle stirring during 4-5 hours.
Standard Ringer solution contained (in mM): 135 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 11 HEPES, and 5 glucose (pH 7.4 adjusted with NaOH, 290 mosmol/kg-H2O). The standard pipette solution for whole-cell experiments contained (in mM): 125 CsCl, 2 CaCl2, 1 MgCl2, 3 Na2ATP, 5 HEPES (pH 7.4 adjusted with CsOH), 10 EGTA, and 50 mannitol (pCa 7.65; 320 mosmol/kg-H2O). For measurements of glutamate permeability, the low-Cl bath solution was prepared by replacing 135 mM NaCl in standard Ringer solution with 135 mM Na-glutamate. Hypotonic solution for single-channel measurements contained (in mM): 100 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES (220 mosmol/kg-H2O, pH-7.4, adjusted with KOH). Pipette solution for on-cell experiments contained (in mM): 100 CsCl, 2 CaCl2, 1 MgCl2, 5 HEPES (210 mosmol/kg-H2O, pH-7.4, adjusted with CsOH). Osmolality of experimental solutions was measured with a vapor pressure osmometer VAPOR5600 (WESCOR, South Logan, UT).
Patch electrodes were fabricated from borosilicate glass capillaries (outer diameter 1.4 mm, inner diameter 1.0 mm) with a micropipette puller (model PP-830; Narishige, Japan) and had a tip resistance of 2-5 MH when filled with pipette solutions. Fast and slow capacitative transients were routinely compensated for. For whole-cell recordings, the access resistance did not exceed 6 MH and was always compensated
Section 1. Biology
for by 70-80%. Membrane currents were measured with an EPC-9 patch-clamp system (Heka-Electronics, Lambrecht/ Pfalz, Germany). Data acquisition and analysis were done using Pulse+PulseFit (Heka-Electronics). Currents were filtered at 1 kHz and sampled at 5-10 kHz. When appropriate, off-line correction was made for changes in liquid junction potentials calculated using pCLAMP 8.1 (Axon Instruments, Foster, CA) algorithms. Whenever the bath Cl- concentration was altered, a salt bridge containing 3 M KCl in 2% agarose was used to minimize variations of the bath electrode potential. All experiments were performed at room temperature (23-25 °C).
Data were analyzed by OriginPro 8 (MicroCal Software, Northampton, MA). Pooled data are given as means ± SEM of n observations. Statistical differences of the data were evalu-
ated by the unpaired Student's t test and considered significant at P < 0.05.
Results and Discussion. Under whole-cell patch-clamp configuration, hypertonic pipette solution (osmolality 320) induced visual swelling of the KML cells, which was accompanied by an increase of whole-cell current (Figure 1. A, B). Time of half- activation of swelling-activated conductance was app. 9.5 min (Figure 2. A). The activated current exhibited outward rectification and time-dependent inactivation at large positive potentials higher than +80mV The macroscopic current density at +100 mV was app. 240 pA/pF. The current asymmetry (ratio of the outward to inward current) was around 6.5 (Figure 2. B). The activation time, current density and asymmetry did not show any significant difference in KML cells for both culturing mediums.
Figure 1. Activation and selectivity of the VSOR channel in melanoma cells cultured in RPMI-1640 and DMEM:
(A) Activation of the VSOR currents in KML/RPMI-1640 at ± 25 mV upon osmotic swelling. (B) Activation of the VSOR currents in KML/DMEM at ± 25 mV upon osmotic swelling. (C) Demonstration of the VSOR channel selectivity measurements in KML/DMEM. Ramp pulses ±100 mV were applied every 10 s to the pre-swollen cell to elucidate the currents. (D) Voltage-current relationship of the macroscopic current in standard Ringer solution (146 mM Cl-; E = -3,1 mV), and low Cl- solution (135 mM glutamate+11 mM Cl-; E = 29.4 mV) in KML/DMEM
In order to elucidate changes in the selectivity properties of VSOR in KML cells, which were influenced by alteration of culture medium, the low-chloride bath solution was used. When activated current reached the steady state level, the standard Ringer solution (146 mM Cl-) was replaced by Na-gluta-mate solution (135 mM glutamate + 11 mM Cl-) (Figure 1. C, D). As illustrated in Figure 1C, this maneuver resulted in a decrease of the whole-cell current and shift of the reversal poten-
tial (Fig. 1D) for KML/DMEM to Erev = 30.6 ± 0.9 mV (n = 9) and for KML/ RPMI-1640 to E = 36.9 ± 2.6 mV (n = 6)
rev v '
(Figure 2. D). The shift of the reversal potentials to positive (more depolarized) values confirmed anion selectivity of the recorded currents. As shown in (Figure 2. D), comparison of the mean values of the reversal potentials in KML cells cultured in different mediums showed that the differences were subtle but significant. Permeability ratio PGlu/PCl was 0.213 ±
0.01 (n = 9) and 0.17 ± 0.03 (n = 6) for KML/DMEM and KML/RPMI-1640 conditions, respectively (Figure 2. E).
For single-channel experiments, bath hypotonic solution with high [K+] was used. High [K+] in bath and presence of Cs+ ions in pipette nullified potassium potential on the membrane and decreased cationic K+ - flux through the membrane. Before forming a gigaseal contact, cells were incubated for 5-10 min in hypotonic solution to induce sustained swelling. Single channel events were recorded in on-cell mode at +140 mV. The mean single-channel amplitude was 3.6±0.1 pA (n = 87 from 9 patches) for KML/DMEM and
4.5 ± 0.9 pA for KML/RPMI-1640 (n = 113 from 9 patches). As shown in (Figure 2. C), the difference in single-channel amplitude was statistically significant for KML/DMEM vs KML/RPMI-1640.
In conclusion, culturing the KML melanoma cells in different culture mediums causes significant changes in VSOR anion channel biophysical properties, such as selectivity and single channel conductance.
This work was supported by the grant FA-F5-014 from the Ministry of Innovational Development of the Republic of Uzbekistan.
Figure 2. The summary graphs representing half-maximal activation time (A), current asymmetry determined as
a ratio of the whole-cell current at +100 mV to that at -100 mV (B), mean single-channel amplitudes at +140 mV (C), reversal potential in the presence of Na-glutamate bath solution (D), and permeability ratio of glutamate over chloride calculated using Goldman-Hodgkin-Katz equation (E). * P < 0.05 DMEM vs. RPMI-1640
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