Store-operated cationic channels in human myeloid leukaemia K562 cells

Using the whole-cell patch clamp technique, single channels operated by intracellular Ca(2+)-store depletion were first revealed in human myeloid leukaemia cells K562. A single store-operated channel could be detected in divalent-free extracellular solutions with Na+ as a permeant ion, and intracellular solutions with strong Ca(2+)-helating agent with some delay after whole-cell formation. Addition of inositol-1,4,5-triphosphate to the pipette solution resulted in a significant decrease of this latency. These channels had a conductance of 29 pS, and were inhibited by low concentration of external Ca2+. Our results enable us to assume that the revealed channels are calcium release-activated calcium channels, operated by Ca2+ depletion of endoplasmic reticulum.     

Activation of mechanosensitive ion channels in plasma membrane of K562 cells

Patch clamp method in cell-attached configuration was used to search for mechanogated ion channels in plasma membrane of human myeloid leukemia K562 cells. A reversible activation of transmembrane currents in response to negative pressure applied to membrane patch was observed. Four types of mechanosensitive channels were identified in K562 cells: two main types were characterized with conductance values of 16 and 25 pS; while two others, showing higher conductance values (about 35 and 50 pS), were rarely met. In terms of gating, all channels described here could be assigned to the stretch-activated type. No inactivation of mechanosensitive channels at the sustained stimulation was observed. The activation of mechanosensitive channels in K562 cells was not dependent upon the presence of bivalent cations in the extracellular solution.     

Inhibiting and stimulating effect of amiloride on potential-dependent cation channels in K562 cells

Non-voltage-gated ion channels play an essential role in cellular signalling and ionic homeostasis in nonexcitable cells. The patch clamp method in cell-attached configuration was used to search for the effects of amiloride and gadolinium (Gd3+) exerted on two types of voltage-insensitive cationic channels in plasma membrane of human leukemia K562 cells: Na-selective channels activated by actin disassembly, and mechanosensitive channels. Here we demonstrate that amiloride in high concentrations (1 mM) caused a full inhibition of mechanosensitive channels in K562 cells similarly to Gd3+ effect in micromolecular concentration range. Na-selective channels controlled by actin dynamics were shown to be unaffected by Gd3+ similarly as by amiloride. We also found that application of amiloride to the extracellular surface of membrane patch resulted in a significant increase in the activity of sodium channels. This unexpected stimulatory effect of amiloride may represent an unknown mechanism of activation of non-voltage-gated sodium channels. The data show an essential difference of the activation and blockage of these types of cation-selective channels.     

Change in the selectivity of the sodium channels of a nerve fiber membrane under the action of veratrine]

The ionic currents of the nodal membrane were measured under voltage clamp conditions. The membrane being +40 mv. The replacing of the external Na+-ions to K+- and NH4+-ions have showed that the relative pearmeabilities of the veratrine-modified channels calculated from the constant field theory are arranged in the following row: PNa:PK:PNH4 = 1:0.29:0.61, which differs from the same row for the normal channels. The decreasing of the slope of current-voltage relations of the modified channels with the replacing of Na+-ions to K+- and NH4+-ions is the evidence of a more strong binding of these ions to external mouth of the modified channel compared to the binding of Na+-ions.     

Comparative study of the action of procaine and benzocaine on normal and aconitine-modified sodium channels]

Ionic currents of normal and aconitine modified sodium channels of the Ranvier node membrane were measured under voltage clamp conditions. The experiments with local anesthetics in the external Ringer solution have showed that dissociation constant (Kdis) of normal channel-anesthetic complex for procaine is 0.27 + 0.03 mM, and for benzocaine is 0.68 +/- 0.04 mM. With aconitine modified channels, Kdis increases and becomes 1.32 +/- 0.5 mM and 1.52 +/- 0.3 mM for procaine and benzocaine, respectively. It is ascertained that the development of aconitine effect is inhibited by neutral benzocaine to a lesser extent than by procaine. It is shown that the aconitine effect cannot be reversed by a high concentration of anesthetic. Hence, it appears that aconitine and anesthetic receptors do not coincide.     

Mechanism of epithelial sodium channel (ENaC) regulation by cortactin: involvement of dynamin

We have recently shown that epithelial sodium channels (ENaC) are regulated by the actin-binding protein cortactin via the Arp2/3 protein complex. However, it has been also demonstrated that GTPase, dynamin, which is known to regulate clathrin-mediated endocytosis, can as well initiate signaling cascades regulated by cortactin. This study was designed to investigate the involvement of dynamin into cortactin-mediated regulation of ENaC. Initially, a recently described inhibitor of dynamin, dynasore, was used. However, use of this inhibitor seemed to be inappropriate due to discovered side effects. F. i., treatment of mpkCCD(c14) cells monolayers with dynasore (in concentrations of 10 and 100 microM) resulted in a decrease in ENaC-mediated transepithelial currents. Besides, the same concentrations of dynasore caused reduced currents in CHO cells transfected with ENaC subunits. Therefore, the data demonstrated that dynasore down regulates both native and overexpressed channel's activity and is not suitable for studies of a role of dynamin in the clathrin-mediated endocytosis of ENaC. This effect is most likely caused either by dynasore's toxic effect upon the cells or by enhanced endocytosis of ENaC-activating proteins. In the following experiments designed to study the role of dynamin different plasmids encoding mutant forms of dynamin and cortactin were used. Dominant negative dynamin K44A transfected into CHO cells together with ENaC subunits significantly increased amiloride-sensitive current density compared to cells transfected with ENaC subunits only (control); additional transfection of cortactin in this system resulted in current density restitution back to the control level. Moreover, ENaC overexpression with the SH3 domain of cortactin, which is responsible for dynamin binding, caused a decrease if ENaC current. Thus, we have shown in this study that cortactin can mediate ENaC activity not only via the Arp2/3 complex, but apart from that dynamin and related processes also might be involved into ENaC regulation by cortactin.     

The average cell size as a factor reflecting the interaction of the CHO cells during process of proliferation

Changes in the cell area during cultivation of the CHO line cells were studied using time-lapse technique (start of registration in one day after cell plating). It was established that the size of the daughter cells after mitosis remains lees than the size of the mother cell for a long time (up to 6 h). Nevertheless, the average cell area of the whole population is constant throughout the observation period (up to 18 h). We assume that this phenomenon could be a result of interaction among dividing and not-dividing cells. The experimental data confirming this conclusion are presented.     

The estimation of similarity in characteristics of the post-mitotic daughter L-929 cells during their migration along the substrate

Using time-lapse microscopy, spreading of the post-mitotic daughter cells has been studied. The work was performed on non-synchronized cells of established L-929 cell line. The study was aimed to characterize the morphology of the cells as they move along the substrate and to determine whether the area of the migrating cells changes nonrandom. Two new parameters have been proposed for comparison of cell morphology: the identity indicator (II) and the synchronism indicator (SI). Time-dependent changes in the area in pairs of cells were measured to calculate these parameters. The first indicator shows the degree of coincidence between the absolute values of the area in the pair of the cells, whereas the second indicator shows synchronism of the changes in the cell areas and does not depend on their absolute values. The lower are the indicators, the higher is the similarity in the time-dependent changes in the areas of cell pairs studied. The indicators were shown to be approximately 1.5-fold lower for the pairs of the post-mitotic daughter cells than those for any other pair of the cells. The results indicate a nonrandom pattern of change in the morphology of the cells during their movement along the substrate.     

Analysis of the cell cycle duration of cells of permanent line L-929

The direct measurement of the cell cycle duration in L-929 cells was performed using time-lapse photography. The cell cycle duration was 15.77 +/- 0.08 h with a standard deviation of 1.54 +/- 0.06 h. The experimental value fit to a normal distribution with a correlation coefficient 0.999. High homogeneity of this parameter and a wide range of variability of the karyotype (58-66 chromosomes) indicate that there is no correlation between these characteristics of L-929 cells. It is also shown that the difference between cell cycle durations of daughter cells tent to zero and fits by an exponent.