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1.
E A Negulyaev YuAVedernikova 《General physiology and biophysics》1985,4(4):359-365
Ionic currents through fast sodium channels in the neuronal somatic membrane were measured under voltage clamp conditions using external solutions of normal and low pH. Voltage-dependent inhibition of ionic currents through open channels was observed in acidic solutions. The voltage-dependent block of sodium channels may be explained by the presence of two acid groups at the channel. The parameters of the inner and outer acid groups calculated according to this model are similar to those reported for the nodal membrane. 相似文献
2.
Aconitine-induced modification of single sodium channels in neuroblastoma cell membrane 总被引:1,自引:0,他引:1
Aconitine-modified sodium channels in the neuroblastoma cell membrane were investigated with patch-clamp technique in outside-out configuration. When aconitine (0.1 mmol/l) was present in the pipette solution two types of modified single sodium channels were observed. The first type showed openings with normal amplitude (slope conductance 15.5 pS) and bursting behaviour. The second type of modified channel openings was characterized with low amplitude (slope conductance 2.8 pS) and longer open time as comparing to unmodified channels. The low-amplitude channels were shown to have altered ion selectivity: they were permeable to NH4+. Both populations of aconitine-modified channels could be blocked by tetrodotoxin. In contrast to macroscopic current experiments (Mozhayeva et al. 1977) the development of aconitine modification was not affected by repetitive stimulation and external application of the agent had no effect on single sodium channels in outside-out membrane patch. 相似文献
3.
Disruption of actin filaments increases the activity of sodium-conducting channels in human myeloid leukemia cells. 总被引:3,自引:0,他引:3
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With the use of the patch clamp technique, the role of cytoskeleton in the regulation of ion channels in plasma membrane of leukemic K562 cells was examined. Single-channel measurements have indicated that disruption of actin filaments with cytochalasin D (CD) resulted in a considerable increase of the activity of non-voltage-gated sodium-permeable channels of 12 pS unitary conductance. Background activity of these channels was low; open probability (po) did not exceed 0.01-0.02. After CD, po grew at least 10-20 times. Cell-attached and whole-cell recordings showed that activation of sodium channels was elicited within 1-3 min after the addition of 10-20 micrograms/ml CD to the bath extracellular solution or in the presence of 5 micrograms/ml CD in the intracellular pipette solution. Preincubation of K562 cells with CD during 1 h also increased drastically the activity of 12 pS sodium channels. Whole-cell measurements confirmed that CD-activated channels were permeable to monovalent cations (preferentially to Na+ and Li+), but not to bivalent cations (Ca2+, Ba2+). Colchicine (1 microM), which affect microtubules, did not alter background channel activity. Our data indicate that actin filaments organization plays an important role in the regulation of sodium-permeable channels which may participate in providing passive Na+ influx in red blood cells. 相似文献
4.
Evidence for existence of two acid groups controlling the conductance of sodium channel 总被引:2,自引:0,他引:2
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. 相似文献
5.
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. 相似文献
6.
D. V. Ilatovskaya T. S. Pavlov Yu. A. Negulyaev A. Staruschenko 《Cell and Tissue Biology》2012,6(1):52-59
We have recently shown that epithelial sodium channels (ENaCs) are regulated by the actin-binding protein cortactin via the
Arp2/3 protein complex. It has been also demonstrated that a 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. Thus, treatment of mpkCCDc14 cells monolayers with dynasore (in concentrations of 10 and 100 μM) resulted in a decrease in ENaC-mediated transepithelial
currents. Besides, dynasore caused reduced amiloride-sensitive currents in CHO cells transfected with ENaC subunits. Therefore,
the data demonstrated that dynasore down regulates both native and overexpressed channel’s activity and use of this drug is
not appropriate for studies of ENaC endocytosis. We hypothesize that this effect is most likely caused either by dynasore’s
toxic actions upon the cells or by enhanced endocytosis of ENaC-activating proteins. In the following experiments 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 only
(control); additional transfection of cortactin together with the K44A dynamin 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 of ENaC current. Thus, we have shown in this study that cortactin can mediate ENaC activity not only via
the Arp2/3 complex, but also through the dynamin-mediated processes. 相似文献
7.
D. V. Ilatovskaya T. S. Pavlov Y. A. Negulyaev A. Staruschenko 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2012,6(3):265-272
Family focal segmental glomerulosclerosis (FSGS) is characterized by sclerosis and hyalinosis of particular loops of glomeruli and is one of the causes of the nephrotic syndrome. Certain mutations in the structure of TRPC6 channels are the genetic impetus for FSGS development resulting in podocytes functional abnormalities and various nephropathies. We have recently demonstrated that non-steroid antiinflammatory drugs (NSAID) ibuprofen and diclofenac decrease the activity of endogenous TRPC-like calcium channels in the podocytes of the freshly isolated rat glomeruli. It has also been shown that TRPC6 channels are expressed in the podocytes. In the current study we have functionally reconstituted TRPC6 channels in mammalian cells to investigate the effects of diclofenac on the activity of wild type TRPC6 channel and TRPC6P112Q channel containing a mutation in the N-terminus that was described in FSGS patients. Intracellular calcium level measurements in transfected cells revealed a more intensive carbachol-induced increase of calcium concentration in HEK-293 cells expressing TRPC6P112Q versus the cells expressing wild-type TRPC6. We also performed patch-clamp experiments to study TRPC6 channels reconstituted in Chinese hamster ovary (CHO) cell line and found that application of diclofenac (500 μM) acutely reduced single channel activity. Preincubation with diclofenac (100 μM) also decreased the whole-cell current in CHO cells overexpressing TRPC6P112Q. Therefore, our previously published data on the effects of NSAID on TRPC-like channels in the isolated rat glomeruli, along with this current investigation on the cultured overexpressed mammalian cells, allows hypothesizing that TRPC6 channels may be a target for NSAID that can be important in the treatment of FSGS. 相似文献
8.
I. O. Vassilieva Yu. A. Negulyaev I. I. Marakhova S. B. Semenova 《Cell and Tissue Biology》2008,2(6):584-589
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. 相似文献
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