In blood cells, changes in intracellular Ca(2+) concentration ([Ca(2+)]i) are associated with multiple cellular events, including activation of cellular kinases and phosphatases, degranulation, regulation of cytoskeleton binding proteins, transcriptional control, and modulation of surface receptors. Although there is no doubt as to the significance of Ca(2+) signaling in blood cells, there is sparse knowledge about the molecular identities of the plasmalemmal Ca(2+) permeable channels that control Ca(2+) fluxes across the plasma membrane and mediate changes in [Ca(2+)]i in blood cells. Using RNA expression analysis, we have shown that human leukemia K562 cells endogenously co-express transient receptor potential vanilloid channels type 5 (TRPV5) and type 6 (TRPV6) mRNAs. Moreover, we demonstrated that TRPV5 and TRPV6 channel proteins are present in both the total lysates and the crude membrane preparations from leukemia cells. Immunoprecipitation revealed that a physical interaction between TRPV5 and TRPV6 may take place. Single-channel patch-clamp experiments demonstrated the presence of inwardly rectifying monovalent currents that displayed kinetic characteristics of unitary TRPV5 and/or TRPV6 currents and were blocked by extracellular Ca(2+) and ruthenium red. Taken together, our data strongly indicate that human myeloid leukemia cells co-express functional TRPV5 and TRPV6 calcium channels that may interact with each other and contribute into intracellular Ca(2+) signaling. Key words: Ca2+ channels, single-channel recording, blood cells, leukemia cells.

In blood cells, changes in intracellular Ca(2+) concentration ([Ca(2+)]i) are associated with multiple cellular events, including activation of cellular kinases and phosphatases, degranulation, regulation of cytoskeleton binding proteins, transcriptional control, and modulation of surface receptors. Although there is no doubt as to the significance of Ca(2+) signaling in blood cells, there is sparse knowledge about the molecular identities of the plasmalemmal Ca(2+) permeable channels that control Ca(2+) fluxes across the plasma membrane and mediate changes in [Ca(2+)]i in blood cells. Using RNA expression analysis, we have shown that human leukemia K562 cells endogenously co-express transient receptor potential vanilloid channels type 5 (TRPV5) and type 6 (TRPV6) mRNAs. Moreover, we demonstrated that TRPV5 and TRPV6 channel proteins are present in both the total lysates and the crude membrane preparations from leukemia cells. Immunoprecipitation revealed that a physical interaction between TRPV5 and TRPV6 may take place. Single-channel patch-clamp experiments demonstrated the presence of inwardly rectifying monovalent currents that displayed kinetic characteristics of unitary TRPV5 and/or TRPV6 currents and were blocked by extracellular Ca(2+) and ruthenium red. Taken together, our data strongly indicate that human myeloid leukemia cells co-express functional TRPV5 and TRPV6 calcium channels that may interact with each other and contribute into intracellular Ca(2+) signaling. Key words: Ca2+ channels, single-channel recording, blood cells, leukemia cells.

BACKGROUND: The Epithelial Na(+) Channel (ENaC) plays a central role in control of epithelial surface hydration and vascular volume. Similar to other ion channels, ENaC activity is regulated, in part, by cortical cytoskeleton. Besides, the cytoskeleton is an established target for small G proteins signaling. Here we studied whether ENaC activity is modulated by changes in the state of the cytoskeleton and whether cytoskeletal elements are involved in small G protein mediated increase of ENaC activity. METHODS AND FINDINGS: First, the functional importance of the cytoskeleton was established with whole-cell patch clamp experiments recording ENaC reconstituted in CHO cells. Pretreatment with Cytochalasin D (CytD; 10 microg/ml; 1-2 h) or colchicine (500 microM; 1-3 h) to disassembly F-actin and destroy microtubules, respectively, significantly decreased amiloride sensitive current. However, acute application of CytD induced rapid increase in macroscopic current. Single channel measurements under cell-attached conditions revealed similar observations. CytD rapidly increased ENaC activity in freshly isolated rat collecting duct, polarized epithelial mouse mpkCCD(c14) cells and HEK293 cells transiently transfected with ENaC subunits. In contrast, colchicine did not have an acute effect on ENaC activity. Small G proteins RhoA, Rac1 and Rab11a markedly increase ENaC activity. 1-2 h treatment with colchicine or CytD abolished effects of these GTPases. Interestingly, when cells were coexpressed with ENaC and RhoA, short-term treatment with CytD decreased ENaC activity. CONCLUSIONS: We conclude that cytoskeleton is involved in regulation of ENaC and is necessary for small G protein mediated increase of ENaC activity.

A jumonji domain containing gene 6 (Jmjd6), previously referred to as phosphatidylserine receptor (PSR) gene, plays an important role in cell differentiation and development of multiple organs, although mechanisms of its action are not known. The Jmjd6 gene product was initially identified as a membrane protein that participates in phagocytosis. However, the later findings that recombinant Jmjd6 in expression systems was targeted to the nucleus challenged the role of Jmjd6 as a membrane receptor. Using immunocytochemistry approach we studied the subcellular distribution of endogenous Jmjd6 protein in THP-1 cells activated with phorbol 12-myristate 13 acetate (PMA). We found that treatment with PMA stimulated Jmjd6 expression in the cytosol of activated cells. Furthermore, Jmjd6 initially appeared at the cell surface of immature phagocytes (1-2 days after activation) but then translocated into the nucleus of differentiated macrophage-like cells (5-9 days after activation). Anti-Jmjd6 antibodies suppressed the engulfment of dead cell corpses by THP-1 cells expressing the Jmjd6 at the cell surface. These data indicate that Jmjd6 serves as a membrane-associated receptor that regulates phagocytosis in immature macrophages but is dispensable for phagocytosis and has other functions when it is expressed in the cytosol and nucleus of mature macrophage-like cells. J. Cell. Physiol.(c) 2009 Wiley-Liss, Inc.

Stem cells, that is, cells that can both reproduce themselves and differentiate into functional cell types, attract much interest as potential aids to healing and disease therapy. Embryonic neural crest is pluripotent and generates the peripheral nervous system, melanocytes, and some connective tissues. Neural-crest-related stem cells have been reported previously in postnatal skin: committed melanocytic stem cells in the hair follicle, and pluripotent cell types from the hair follicle and papilla that can produce various sets of lineages. Here we describe novel pluripotent neural crest-like stem cells from neonatal mouse epidermis, with different potencies, isolated as 3 independent immortal lines. Using alternative regulatory factors, they could be converted to large numbers of either Schwann precursor cells, pigmented melanocytes, chondrocytes, or functional sensory neurons showing voltage-gated sodium channels. Some of the neurons displayed abundant active TRPV1 and TRPA1 receptors. Such functional neurons have previously been obtained in culture only with difficulty, by explantation. The system was also used to generate comparative gene expression data for the stem cells, melanocytes, and melanoblasts that sufficiently explain the lack of pigment in melanoblasts and provide a rationale for some genes expressed apparently ectopically in melanomas, such as ephrin receptors.-Sviderskaya, E. V., Easty, D. J., Lawrence, M. A., Sánchez, D. P., Negulyaev, Y. A., Patel, R. H., Anand, P., Korchev, Y. E., Bennett, D. C. Functional neurons and melanocytes induced from immortal lines of postnatal neural crest-like stem cells.

The most valuable property of stem cells (SCs) is their potential to differentiate into many or all cell types of the body. So far, monitoring SC differentiation has only been possible after cells were fixed or destroyed during sample preparation. It is, however, important to develop nondestructive methods of monitoring SCs. Scanning ion conductance microscopy (SICM) is a unique imaging technique that uses similar principles to the atomic force microscope, but with a pipette for the probe. This allows scanning of the surface of living cells noninvasively and enables measurement of cellular activities under more physiological conditions than is possible with other high-resolution microscopy techniques. We report here the novel use of the SICM for studying SCs to assess and monitor the status of SCs and various cell types differentiated from SCs.

The precise control of many T cell functions relies on cytosolic Ca2+ dynamics that is shaped by the Ca2+ release from the intracellular store and extracellular Ca2+ influx. The Ca2+ influx activated following T cell receptor (TCR)-mediated store depletion is considered to be a major mechanism for sustained elevation in cytosolic Ca2+ concentration ([Ca2+]i) necessary for T cell activation, whereas the role of intracellular Ca2+ release channels is believed to be minor. We observed, however, that in Jurkat T cells [Ca2+]i elevation observed upon activation of the store-operated Ca2+ entry (SOCE) by passive store depletion with cyclopiazonic acid (CPA), a reversible blocker of sarco-endoplasmic reticulum Ca2+ ATPase, inversely correlated with store-refilling. This indicated that intracellular Ca2+ release channels were activated in parallel with SOCE and contributed into global [Ca2+]i elevation. Pretreating cells with (-)-xestospongin C (10 M) or ryanodine (400 M), the antagonists of inositol 1,4,5-trisphosphate receptor (IP3R) or ryanodine receptor (RyR), respectively, facilitated store refilling and significantly reduced [Ca2+]i elevation evoked by the passive store depletion or TCR ligation. Although the Ca2+ release from the IP3R can be activated by TCR stimulation, the Ca2+ release from the RyR was not inducible via TCR engagement and was exclusively activated by the SOCE. We also established that inhibition of IP3R or RyR downregulated T cell proliferation and T-cell growth factor interleukin 2 (IL-2) production. These studies revealed a new aspect of [Ca2+]i signaling in T cells, that is SOCE-dependent Ca2+ release via IP3R and/or RyR and identified the IP3R and RyR as potential targets for manipulation of Ca2+-dependent functions of T lymphocytes.

Activated T lymphocytes release vesicles, termed exosomes, enriched in cholesterol and exposing phosphatidylserine (PS) at their outer membrane leaflet. Although CD4+ activated T lymphocytes infiltrate an atherosclerotic plaque, the effects of T cell exosomes on the atheroma-associated cells are not known. We report here that exosomes isolated from the supernatants of activated human CD4+ T cells enhance cholesterol accumulation in cultured human monocytes and THP-1 cells. Lipid droplets found in the cytosol of exosome-treated monocytes contained both cholesterol ester and free cholesterol. Anti-phosphatidylserine receptor antibodies recognized surface protein on the monocyte plasma membrane and prevented exosome-induced cholesterol accumulation, indicating that exosome internalization is mediated via endogenous phosphatidylserine receptor. The production of proinflammatory cytokine TNF-alpha enhanced in parallel with monocyte cholesterol accumulation. Our data strongly indicate that exosomes released by activated T cells may represent a powerful, previously unknown, atherogenic factor. J. Cell. Physiol.(c) 2007 Wiley-Liss, Inc.

Compelling evidence shows that intracellular free magnesium [Mg(2+)](i) may be a critical regulator of cell activity in eukaryotes. However, membrane transport mechanisms mediating Mg(2+) influx in mammalian cells are poorly understood. Here, we show that mechanosensitive (MS) cationic channels activated by stretch are permeable for Mg(2+) ions at different extracellular concentrations including physiological ones. Single-channel currents were recorded from cell-attached and inside-out patches on K562 leukaemia cells at various concentrations of MgCl(2) when Mg(2+) was the only available carrier of inward currents. At 2 mM Mg(2+), inward mechanogated currents representing Mg(2+) influx through MS channels corresponded to the unitary conductance of about 5 pS. At higher Mg(2+) levels, only slight increase of single-channel currents and conductance occurred, implying that Mg(2+) permeation through MS channels is characterized by strong saturation. At 20 and 90 mM Mg(2+), mean conductance values for inward currents carried by Mg(2+) were rather similar, being equal to 6.8 +/- 0.5 and 6.4 +/- 0.5 pS, respectively. The estimation of the channel-selective permeability according to constant field equation is obviously limited due to saturation effects. We conclude that the detection of single currents is the main evidence for Mg(2+) permeation through membrane channels activated by stretch. Our single-current measurements document Mg(2+) influx through MS channels in the plasma membrane of leukaemia cells.Cell Research advance online publication Jul 25 2006; doi:10.1038/sj.cr.7310084.

The mechanism of apoptotic cell volume decrease was studied in rat thymocytes treated with dexamethasone (Dex) or etoposide (Eto). Cell shrinkage, i.e. dehydration, was quantified by using buoyant density of the thymocytes in a continuous Percoll gradient. The K+ and Na+ content of cells from different density fractions were assayed by flame emission analysis. Apoptosis was tested by microscopy and flow cytometry of acridine orange stained cells as well as by flow DNA cytometry. Treatment of the thymocytes with 1 microM Dex for 4-5.5 h or 50 microM Eto for 5 h resulted in the appearance of a new distinct high-density cell subpopulation. The cells from this heavy subpopulation but not those with normal buoyant density had typical features of apoptosis. Apoptotic increase of cell density was accompanied by a decrease in cellular K+ content, which exceeded the simultaneous increase in cellular Na+ content. Cellular loss of K+ contributed to most of the estimated loss of cellular osmolytes, but owing to the parallel loss of cell water, the decrease in cytosolic K+ concentration was less than one third. Due to gain of Na+ and loss of cell water the cytosolic Na+ concentration in thymocytes rose following treatment with Dex (5.5 h) or Eto (5 h) by a factor of about 3.6 and 3.1, respectively.

Mechanosensitive channels in various eucaryotic cells are thought to be functionally and structurally coupled to the cortical cytoskeleton. However, the results of electrophysiological studies are rather controversial and the functional impact of cytoskeleton assembly-disassembly on stretch-activated channel properties remains unclear. Here, the possible involvement of cytoskeletal elements in the regulation of stretch-activated Ca2+-permeable channels was studied in human leukaemia K562 cells with the use of agents that selectively modify the actin or tubulin system. F-actin disassembly resulted in a considerable reduction of the amplitude of stretch-activated currents without significant change in channel open probability. The effects of treatments with cytochalasins or latrunculin were principally similar, developed gradually and consisted a strong decrease of single channel conductance. Microtubule disruption did not affect stretch-activated channels. The data presented here are in principal agreement with the general conclusion that mechanosensitive channel functions are largely dependent on the integrity of the cortical actin cytoskeleton. Specifically, changes in conductive properties of the pore may provide an essential mechanism of channel regulation underlying functional modulation of membrane currents. Our results allow one to speculate that microfilament organization may be an important determinant in modulating biophysical characteristics of stretch-activated cation channels in cells of blood origin.

Voltage-induced impedance variation of the minicolumn (i.d. 0.53 mm, length 2 mm) packed with cation exchanger was investigated to develop a sensing method. An aqueous sample solution containing the metal cations was continuously supplied to the minicolumn during the impedance measurement with the simultaneous application of both alternating current voltage (amplitude, 1.0 V; frequency, 200 kHz to 6 Hz) and direct current (DC) offset voltage (0.1 to 1.0 V). On a complex plane plot, the profile of the column impedance consisted of a semicircle (200 kHz to 100 Hz) and a straight line (<100 Hz), of which slope varied with the magnitude of the applied DC offset voltage (V(DC)). The slope-V(DC) relation depended on the kind of the metal cation and its concentration; in particular, the slope-V(DC) relations of monovalent cations (Na(+) and K(+)) and divalent ones (Mg(2+) and Ca(2+)) were significantly different. With the change in the concentration of minor divalent salt of MgCl(2) or CaCl(2)(60 to 140 microM) in the sample solution containing 10 mM NaCl, the slopes showed almost linear relationships between those with application of V(DC)= 0.1 V and 1.0 V both for magnesium and calcium additions. In the case of plural addition of both MgCl(2) and CaCl(2) to the solution, the data points in the slope(0.1 V)-slope(1.0 V) plot were located between the two proportional lines for single additions of magnesium and calcium, reflecting both the mixing ratio and net concentrations of the divalent cations. Thus, simulations determination of Mg(2+) and Ca(2+) can be attained on the basis of the slope(0.1 V)-slope(1.0 V) relation obtained by the impedance measurements of the minicolumn. Actually, the contents of both magnesium and calcium cations in the bottled mineral waters determined simultaneously using the proposed method were almost equivalent to those obtained by the atomic absorption spectrometric measurement.

In blood cells, changes in intracellular Ca(2+) concentration ([Ca(2+)]i) are associated with multiple cellular events, including activation of cellular kinases and phosphatases, degranulation, regulation of cytoskeleton binding proteins, transcriptional control, and modulation of surface receptors. Although there is no doubt as to the significance of Ca(2+) signaling in blood cells, there is sparse knowledge about the molecular identities of the plasmalemmal Ca(2+) permeable channels that control Ca(2+) fluxes across the plasma membrane and mediate changes in [Ca(2+)]i in blood cells. Using RNA expression analysis, we have shown that human leukemia K562 cells endogenously co-express transient receptor potential vanilloid channels type 5 (TRPV5) and type 6 (TRPV6) mRNAs. Moreover, we demonstrated that TRPV5 and TRPV6 channel proteins are present in both the total lysates and the crude membrane preparations from leukemia cells. Immunoprecipitation revealed that a physical interaction between TRPV5 and TRPV6 may take place. Single-channel patch-clamp experiments demonstrated the presence of inwardly rectifying monovalent currents that displayed kinetic characteristics of unitary TRPV5 and/or TRPV6 currents and were blocked by extracellular Ca(2+) and ruthenium red. Taken together, our data strongly indicate that human myeloid leukemia cells co-express functional TRPV5 and TRPV6 calcium channels that may interact with each other and contribute into intracellular Ca(2+) signaling. Key words: Ca2+ channels, single-channel recording, blood cells, leukemia cells.

The recent cloning of the special calcium channels TRPV5 and TRPV6 (transient receptor potential vanilloid channels) provided the molecular base for the studying of new candidate of calcium influx in non-excitable cells. Using RT-PCR technique we obtained endogenous expression of the mRNAs trpv5 and trpv6 in lymphoblast leukemia Jurkat cells and in human blood primary T lymphocytes. Additionally, Western blot analysis showed TRPV5 proteins in both the whole lysate and in the crude membrane preparations from Jurkat cells and normal T lymphocytes. The using of the immunoprecipitation revealed TRPV6 proteins in Jurkat cells, whereas in normal T lymphocytes TRPV6 was not detected. The expression pattern and the selective Ca2+ permeation properties of TRPV5 and TRPV6 channels indicate an important role of these channels in the Ca2+ homeostasis and probably in malignant transformation of blood cells.

TRPA1, a poorly selective Ca(2+)-permeable cation channel, is expressed in peripheral sensory neurones where it is considered to contribute to a variety of sensory processes such as the detection of painful stimuli. Furthermore, TRPA1 was also identified in hair cells of the inner ear but its involvement in sensing mechanical forces is still being controversially discussed. Amphipathic molecules such as trinitrophenol and chlorpromazine have been shown to provide useful tools to study mechanosensitive channels. Depending on their charge, they partition in the inner or outer sheets of the lipid bilayer causing a curvature of the membrane which has been demonstrated to activate or inhibit mechanosensitive ion channels. In the present study we investigated the effect of these molecules on TRPA1 gating. TRPA1 is robustly activated by the anionic amphipathic molecule trinitrophenol. The whole-cell and single channel properties resemble those previously described for TRPA1. Moreover, we could show that the toxin GsMTx-4 acts on TRPA1. In addition to its recently described role as an inhibitor of stretch-activated ion channels, it serves as a potent activator of TRPA1 channels. On the other hand, the positively charged drug chlorpromazine modulates activated TRPA1 currents in a voltage-dependent way. The exposure of activated TRPA1 channels to chlorpromazine led to a block at positive potentials and an increased open probability at negative potentials. The variability in the shape of the I-V curve gives a first indication that native mechanically activated TRPA1 currents must not necessarily exhibit the same biophysical properties as ligand-activated TRPA1 currents.

Ion channels play key roles in cell physiology and underlie a broad spectrum of disorders. To this day, the gold standard for studying ion channels is the patch clamp technique. Patch clamping involves careful positioning of a fine-tipped glass micropipette onto the surface of the cell to form a high-resistance (>1 GOmega) seal ("gigaseal"), a procedure that is laborious, vibration-sensitive, and not easily amenable to automation. In addition, the solution inside the pipette cannot be easily exchanged. Recently reported patch clamp chips offer the potential of increased throughput, but to date the overall per-cell performance of most designs has been very low when compared to pipettes, and/or the fabrication process is prohibitively expensive. Here we demonstrate a replica-molded elastomeric patch clamp chip incorporating nanofabricated constrictions, which delivers high-stability gigaseals, with success rates comparable to those of pipettes, using rat basophilic leukemia (RBL) cells. The high stability enables exchanges of both the extracellular and intracellular solution during whole-cell recordings. In a sample of 103 experiments, 66 cells (64%) were successfully immobilized at the patch aperture; 38 cells (58% of immobilized cells, 37% of all cells) were successfully gigasealed; and 25 cells (65% of gigasealed cells, 34% of immobilized cells, 24% of all cells) were successfully perforated for whole-cell access. In the last group of 27 experiments, 79% of the cells could be immobilized, of which 68% could be gigasealed and 46% perforated for whole-cell access, indicating that dexterity is important.

Compelling evidence shows that intracellular free magnesium [Mg(2+)](i) may be a critical regulator of cell activity in eukaryotes. However, membrane transport mechanisms mediating Mg(2+) influx in mammalian cells are poorly understood. Here, we show that mechanosensitive (MS) cationic channels activated by stretch are permeable for Mg(2+) ions at different extracellular concentrations including physiological ones. Single-channel currents were recorded from cell-attached and inside-out patches on K562 leukaemia cells at various concentrations of MgCl(2) when Mg(2+) was the only available carrier of inward currents. At 2 mM Mg(2+), inward mechanogated currents representing Mg(2+) influx through MS channels corresponded to the unitary conductance of about 5 pS. At higher Mg(2+) levels, only slight increase of single-channel currents and conductance occurred, implying that Mg(2+) permeation through MS channels is characterized by strong saturation. At 20 and 90 mM Mg(2+), mean conductance values for inward currents carried by Mg(2+) were rather similar, being equal to 6.8 +/- 0.5 and 6.4 +/- 0.5 pS, respectively. The estimation of the channel-selective permeability according to constant field equation is obviously limited due to saturation effects. We conclude that the detection of single currents is the main evidence for Mg(2+) permeation through membrane channels activated by stretch. Our single-current measurements document Mg(2+) influx through MS channels in the plasma membrane of leukaemia cells.Cell Research advance online publication Jul 25 2006; doi:10.1038/sj.cr.7310084.

Intracellular Mg2+ and natural polyamines block outward currents in BK channels in a highly voltage-dependent manner. Here we investigate the contribution of the ring of eight negatively charged residues (4 x E321/E324) at the entrance to the inner vestibule of BK channels to this block. Channels with or without (E321N/E324N) the ring of negative charge were expressed in oocytes and unitary currents were recorded from inside-out patches over a range of intracellular Mg2+ and polyamine concentrations. Removing the ring of charge greatly decreased the block, increasing K(B)(ap)(0 mV) for Mg2+ block from 48.3 +/- 3.0 to 143 +/- 8 mM, and for spermine block from 8.0 +/- 1.0 to 721 +/- 9 mM (150 mM symmetrical KCl). Polyamines with fewer amine groups blocked less: putrescine < spermidine < spermine. An equation that combined an empirical Hill function for block together with a Boltzmann function for the voltage dependence of K(B)(ap) described the voltage and concentration dependence of the block for channels with and without the ring of charge. The Hill coefficients for these descriptions were <1 for both Mg2+ and spermine block, and were unchanged by removing the ring of charge. When KCl(i) was increased from 150 mM to 3 M, the ring of charge no longer facilitated block, Mg2+ block was reduced, spermine block became negligible, and the Hill coefficients became approximately 1.0. BK channels in cell-attached oocyte patches displayed inward rectification, which was reduced for channels without the ring of charge. Taken together, these observations suggest that the ring of negative charge facilitates block through a preferential electrostatic attraction of Mg2+ and polyamine over K+. This preferential attraction of multivalent blockers over monovalent K+ would decrease the K+ available at the inner vestibule to carry outward current in the presence of Mg2+ or polyamines, while increasing the concentration of blocker available to enter and block the conduction pathway.

Plantago media L. and Plantago maritima L. differ in their strategy toward salt stress, a major difference being the uptake and distribution of ions. Patch clamp techniques were applied to root cell vacuoles to study the tonoplast channel characteristics. In both species the major channel found was a 60 to 70 picosiemens channel with a low ion selectivity. The conductance of this channel for Na(+) was the same as for K(+), P(K)(+)/P(Na)(+)= 1, whereas the cation/anion selectivity (P(K)(+)/P(c1)(-)) was about 5. Gating characteristics were voltage and calcium dependent. An additional smaller channel of 25 picosiemens was present in P. maritima. In the whole vacuole configuration, the summation of the single channel currents resulted in slowly activated inward currents (t((1/2))= 1.2 second). Inwardly directed, ATP-dependent currents could be measured against a DeltapH gradient of 1.5 units over the tonoplast. This observation strongly indicated the physiological intactness of the used vacuoles. The open probability of the tonoplast channels dramatically decreased when plants were grown on NaCl, although single channel conductance and selectivity were not altered.

There are little data on the properties of ion channels in the inner nuclear membrane of lymphocytes, though the transport system between cytoplasm and karyoplasm is of a great importance for a complex genetic regulation in these cells. Using the patch-clamp technique we have investigated ion channels of the inner membrane of nuclei isolated from cultured T-lymphoblasts. Our research has shown that there are anionic (370 pS) and cationic (152 pS) channels on the inner nuclear membrane. The latter were characterized by fast kinetics and rapid fluctuations; they had high open probability and were inactivated at large negative potentials. These channels were permeable for K+ and Na+, but impermeable for Cl-. The physiological role of the channels is not clear, but they may play an important role in the ion balance between the cytoplasm and the lumen of the endoplasmatic reticulume of the cell.