Yu. A. Negulyaev

The permeability of aconitine-modified sodium channels to univalent cations in myelinated nerve.

1. Ionic currents through the sodium system of nodes of Ranvier treated with aconitine were measured under voltage clamp conditions in a Ringer solution containing Na+ or an equimolar amount of various test cations. 2. Average shifts in reversal potentials in nodes of Ranvier treated with aconitine with NH4+, Li+, K+, Rb+, Cs+ in place of Na+ in the Ringer solution are 7.6, --6.8, --25.0, --41.0 and --51.5 mV at 13--14degrees C. At 20--22degrees C the sequence of shifts is 7.5, --5.5, --13.5, --29.0 and --41.0 mV. For Tl+ the the average reversal potential shift is +3 mV at 20--22degrees C. 3. The slope of the instantaneous current-voltage relation at the reversal potential in nodes treated with aconitine changed with the various cations tested. The ratios are NH4+/Na+/K+/Rb+/Cs+/Li+ = 1.14 : 1.0 : 0.80 :0.67 :0.53 : 0.53. 4. Using a three energy barrier model some of the parameters for the aconitine-modified Na+ channels were estimated (Chizmadgev, Yu. A., Khodorov, B.I. and Aityan, S.Kh. (1974) Bioelectrochem. Bioenerg. 1, 301--312).

Evidence for existence of two acid groups controlling the conductance of sodium channel.

The inhibition of the sodium current in nodal membrane at low pH external solutions was studied under voltage clamp conditions. Analysis of the data for membrane potentials from +10 to +150 mV shows that the inhibition of the Na+ currents at high positive potentials cannot be described by a titration curve of a single acid group. The data can be explained on assumption that the conductance of each sodium channel is controlled by two acid groups: one is located within the pore, the other just near the outer mouth of the pore. The affinity of both groups for H+ is estimated.

TRPV5 and TRPV6 calcium channels in human T cells

The recent cloning of the special calcium channels TRPV5 and TRPV6 (transient receptor potential vanilloid channels) has provided a molecular basis for studying previously unidentified calcium influx channels in electrically nonexcitable cells. In the present work using RT-PCR, we obtained the endogenous expression of mRNAs of genes trpv5 and trpv6 in lymphoblast leukemia Jurkat cells and in normal human T lymphocytes. Additionally, by immunoblotting, the presence of the channel-forming TRPV5 proteins has been shown both in the total lysate and in crude membrane fractions from Jurkat cells and normal T lymphocytes. The use of immunoprecipitation revealed TRPV6 proteins in Jurkat cells, whereas in normal T lymphocytes, this protein was not detected. The expression pattern and the selective Ca2+ permeation properties of TRPV5 and TRPV6 channels indicate the important role of these channels in Ca2+ homeostasis, as well as most likely in malignant transformation of blood cells.

Several types of sodium-conducting channel in human carcinoma A-431 cells

Patch clamp method in outside-out configuration was used to search for cation channels which possibly mediate sodium influx through plasma membrane in A-431 carcinoma cells. We found four types of nonvoltage-gated Na-conducting channel. The first of 9-10 pS conductance (145 mM Na+, 30 degrees C) seems to be Na-selective; three others were characterized with conductance values of 24, 35 and 65 pS and lower selectivity among cations. Na-selective channels (9-10 pS) were not blocked by tetrodotoxin (1 microM). External application of amiloride (0.1-2 mM) resulted in a reversible inhibition of single currents through Na-selective channels.

[Functional properties of sodium channels in cholesterol-depleted K562 cells].

In the present paper, functional properties of nonvoltage-gated sodium channels in K562 cells were studied after cholesterol depletion, i.e., under conditions of the destruction of microdomains (rafts). For cholesterol depletion, cells were incubated with methyl-beta-cyclodextrin (MbCD), an oligosaccharide that selectively binds sterols. Single currents through sodium channels were recorded in cell-attached and inside-out experiments using the patch-clamp technique. After incubation with MbCD (2.5 or 5 mM), the activation of sodium channels in response to cytochalasin B or D was observed in both native cells and membrane fragments. Biophysical characteristics of sodium channels in cholesterol-depleted K562 cells were close to those in control; unitary conductance was 12 pS. Inside-out experiments with the use of globular actin have indicated that filament assembly on cytoplasmic membrane side causes an inactivation of sodium channels in the modified cells. These data imply that sodium channels in K562 cells are not associated with cholesterol-rich membrane microdomains. Possible mechanisms of the interaction of the plasma membrane and the cortical cytoskeleton are discussed.

Molecular and functional identification of sodium channels in K562 cells

Modulations of ion channel activity underlie rapid changes in membrane transport of cations in various nonexcitable cells. Previously, in smooth muscle cells, macrophages, lymphocytes, carcinoma and leukemia cell lines, non-voltage-gated sodium (NVGS) channels have been found. The activity of NVGS channels was shown to be critically dependent on the organization of actin cytoskeleton. The molecular identity of NVGS channels remains unclear. The present work is focused on molecular and functional identification of NVGS channels in human myeloid leukemia K562 cells. Degenerin/epithelial Na+ channels (DEG/ENaC) can be considered as possible molecular correlates. By using RT-PCR, expression of α-, β-, and γ-hENaC subunits in the K562 cells was detected. Various modes of the patch-clamp method were used to examine functional properties of sodium channels—specifically, to test the effect of amiloride on single channel and integral currents. The biophysical characteristics of the NVSG channels were close to those of ENaC; the channels have unitary conductance of 12 pS (145 mM Na+) and were impermeable to divalent cations (Ca2+ and Mg2+). We found that amiloride did not inhibit NVGS channels. Importantly, no amiloride-blockable sodium current was detected in the plasma membrane of K562 cells. Taken together, our observations suggest that amiloride-insensitive sodium channels in the K562 cells belong to the ENaC family.

Dynamics of L-929 cell spreading after mitosis

Changes in the cell shape of L-929 (NCTC, clone 929) during the cell cycle were analyzed with time-lapse microscopy. It was found that the cells pass through three spreading stages. The maximal cell spreading was observed during the first 1.5 h after mitosis. In this period, the cell area increases in correspondence with the sigmoid dependence and enlarges by approximately 3.0–3.5 times. After a short plateau, the cell area begins to increase, also correspondence with the sigmoid dependence. This period is longer (up to 6 h after the beginning of cell division), with an additional 1.5-fold increase in the cell size. Later, cell area enlargement continues linearly up to the beginning of the next mitosis.After the mother L-929 cell division, almost 100% of daughter cells remained to be bridged together in the site of the division furrow. Connected L-929 cells spread and migrate for 2.13 ± 0.06 h and then separate. The daughter-cell shape transition from round to the spread one and simultaneous maintenance of the intercellular bridge during a strictly determined time allows us to consider this phenomenon as independent and not related to mitosis. We suggest designating this junction between the daughter cells as the “posttelophase intercellular bridge.”

Average cell size is a factor reflecting the interaction of CHO cells during their proliferation

Changes in cell area during CHO cell cultivation have been studied with time-lapse microscopy. Capture was started the day after cell plating. It was found that the size of daughter cells after mitosis remained less than the size of the mother cell for a long time (up to 6 h). Nevertheless, the average cell area of the whole population was constant during the observation period (up to 18 h). We assume that this phenomenon is a result of interaction between dividing and nondividing cells. The experimental data we obtained confirm this conclusion.

The position of cleavage furrow in cultured L-929 and CHO cells

The position of the cleavage furrow (random or otherwise) was studied on cultured L-929 (NCTC, clone 929) and CHO cells. CHO cells were seeded uniformly on the surface of Petri dishes; L-929 cells were grown as colonies so that migrating cells could be watched. Cell behavior was registered by time-lapse imaging. Two parameters were analyzed on captured images: the angle between the cell polarization axis and cleavage furrow and the angle between the cell polarization axis or cleavage furrow and the horizontal axis of the image field. It was shown that the position of CHO cells in the dish plane and the value of the angle between the cell polarization axis and the cleavage furrow were random. The L-929 cells migrating from the colony were orientated such that their polarization axis was directed to the colony center and the cleavage furrow was perpendicular to this axis. The nonrandom position of cultivated cells during mitosis and their cleavage furrow during the telophase are discussed.

Coupled Activation of Mechanosensitive and Calcium-Dependent Potassium Channels in 3T3 and 3T3-SV40 Cells

Using the patch–clamp method, mechanosensitive regulation of ion channels was studied in cultivated 3T3 and 3T3-SV40 fibroblasts. The activity of mechanosensitive cation channels with a conductivity 25 pS in response to plasma-membrane stretching was observed in both cell lines. Despite obvious differences in the actin network in normal and transformed cells, the threshold values of the stimulus required for the channel activation were close and were approximately 55 mm Hg. The frequency of channels was significantly higher in transformed 3T3-SV40 fibroblasts than in their untransformed 3T3 analogs. Coupled activation of mechanosensitive calcium-permeable channels and potassium calcium-controlled channels was found in both cell lines. The analysis of flows through single channels allows to detect functional interaction of different channels: stretch-induced local calcium entry activates potassium channels that do not have their own mechanosensitivity. The results of a comparative study show that there is a fundamental similarity between the ion mechanisms of cellular mechanotransduction in normal and transformed fibroblasts. The quantitative differences, first of all, concern the level of functional activity of mechanosensitive channels that provide the development of the local calcium signal in the near-membrane cell region.