Biomedical subjects

V Valiunas

Publications and source records attributed to V Valiunas.

15 recordsLinked to original sources

PubMed2005

Connexin-specific cell-to-cell transfer of short interfering RNA by gap junctions.

The purpose of this study was to determine whether oligonucleotides the size of siRNA are permeable to gap junctions and whether a specific siRNA for DNA polymerase beta (pol beta) can move from one cell to another via gap junctions, thus allowing one cell to inhibit gene expression in another cell directly. To test this hypothesis, fluorescently labelled oligonucleotides (morpholinos) 12, 16 and 24 nucleotides in length were synthesized and introduced into one cell of a pair using a patch pipette. These probes moved from cell to cell through gap junctions composed of connexin 43 (Cx43). Moreover, the rate of transfer declined with increasing length of the oligonucleotide. To test whether siRNA for pol beta was permeable to gap junctions we used three cell lines: (1) NRK cells that endogenously express Cx43; (2) Mbeta16tsA cells, which express Cx32 and Cx26 but not Cx43; and (3) connexin-deficient N2A cells. NRK and Mbeta16tsA cells were each divided into two groups, one of which was stably transfected to express a small hairpin RNA (shRNA), which gives rise to siRNA that targets pol beta. These two pol beta knockdown cell lines (NRK-kcdc and Mbeta16tsA-kcdc) were co-cultured with labelled wild type, NRK-wt or Mbeta16tsA-wt cells or N2A cells. The levels of pol beta mRNA and protein were determined by semiquantitative RT-PCR and immunoblotting. Co-culture of Mbeta16tsA-kcdc cells with Mbeta16tsA-wt, N2A or NRK-wt cells had no effect on pol beta levels in these cells. Similarly, co-culture of NRK-kcdc with N2A cells had no effect on pol beta levels in the N2A cells. In contrast, co-culture of NRK-kcdc with NRK-wt cells resulted in a significant reduction in pol beta in the wt cells. The inability of Mbeta16tsA-kcdc cells to transfer siRNA is consistent with the fact that oligonucleotides of the 12 nucleotide length were not permeable to Cx32/Cx26 channels. This suggested that Cx43 but not Cx32/Cx26 channels allowed the cell-to-cell movement of the siRNA. These results support the novel hypothesis that non-hybridized and possible hybridized forms of siRNA can move between mammalian cells through connexin-specific gap junctions.

Animals↗

PubMed2005

Non-stationary fluctuation analysis of macroscopic gap junction channel records.

Non-stationary fluctuation analysis was applied to macroscopic records of junctional currents arising from homotypic Cx37 and Cx43 gap junction channels expressed in RIN cells. The data were analyzed by a modification of existing analytical methods that takes endemic uncoupling into account. The results are consistent with both channels having open probabilities ranging from 0.7 to near unity for low transjunctional voltages. The analysis also yielded estimates of single-channel conductances for the two channel types similar to those seen in single-channel recordings. The results presented here show that fluctuation analysis can be used to extract single-channel gap junctional conductances from macroscopic double whole-cell recordings. These results also constitute empirically determined estimates of the open probability that are not model-dependent.

Animals↗

PubMed2001

Site-directed mutations in the transmembrane domain M3 of human connexin37 alter channel conductance and gating.

Connexin37 (Cx37) is expressed principally in endothelial cells. We have introduced individual point mutations (Cx37-V156D or Cx37-K162E) in the putative pore lining segment M3 of a polymorphic human Cx37 (Cx37-S319) and expressed them in N2A and RIN cells. RT-PCR and immunofluorescence microscopy were used to confirm the expression of the proteins. Stably transfected cells were subjected to electrophysiological studies. Experiments were performed on cell pairs using the dual whole cell patch-clamp method. Single channel records showed that both mutants display a variety of conductive states (Cx37-V156D, 47-250 pS; Cx37-K162E, 58-342 pS) in contrast to the typical high conductance of 340-375 pS and subconductive state of 60-80 pS reported for Cx37-S319. Analysis of the macroscopic data for Cx37-K162E revealed a broadened Vo indicating the influence of the mutation on voltage gating. Our data indicate that substitution of a conserved residue with a charged residue could cause changes in the main state and/or in the size of the pore. It is possible that these particular residues in the M3 domain interact electrostatistically with several of the other domains in the Cx37 protein.

Animals↗

PubMed2001

Co-operativity between mouse connexin30 gap junction channels.

HeLa cells stably transfected with mouse cDNA coding for connexin30 (Cx30) were used to study the electrical properties of gap junction channels. The experiments involved the measurement of intercellular currents (Ij) from cell pairs using dual whole-cell recording with the patch-clamp method. The aim was to compare Ij from cell pairs whose gap junctions consisted of a single channel and cell pairs whose gap junctions consisted of many channels. We found that both the ensemble average currents gained from single-channel records and the currents obtained from multichannel records inactivated exponentially with time. However, the former inactivated significantly slower than the latter. At ajunctional voltage (Vj) of 50 mV, the time constants of inactivation (tau(i)) were 8.1 s and 1.6 s, respectively. Moreover, the ratio tau(i)(single-channel)/tau(i)(multichannel) turned out to be voltage sensitive, i.e. it decreased with increasing V(j) These observations suggest that the operation of Cx30 gap junction channels in the multichannel configuration involves co-operative interactions.

Animals↗

PubMed2001

Gap junction channels formed by coexpressed connexin40 and connexin43.

Many cardiovascular cells coexpress multiple connexins (Cx), leading to the potential formation of mixed (heteromeric) gap junction hemichannels whose biophysical properties may differ from homomeric channels containing only one connexin type. We examined the potential interaction of connexin Cx43 and Cx40 in HeLa cells sequentially stably transfected with these two connexins. Immunoblots verified the production of comparable amounts of both connexins, cross-linking showed that both connexins formed oligomers, and immunofluorescence showed extensive colocalization. Moreover, Cx40 copurified with (His)(6)-tagged Cx43 by affinity chromatography of detergent-solubilized connexons, demonstrating the presence of both connexins in some hemichannels. The dual whole cell patch-clamp method was used to compare the gating properties of gap junctions in HeLa Cx43/Cx40 cells with homotypic (Cx40-Cx40 and Cx43-Cx43) and heterotypic (Cx40-Cx43) gap junctions. Many of the observed single channel conductances resembled those of homotypic or heterotypic channels. The steady-state junctional conductance (g(j,ss)) in coexpressing cell pairs showed a reduced sensitivity to the voltage between cells (V(j)) compared with homotypic gap junctions and/or an asymmetrical V(j) dependence reminiscent of heterotypic gap junctions. These gating properties could be fit using a combination of homotypic and heterotypic channel properties. Thus, whereas our biochemical evidence suggests that Cx40 and Cx43 form heteromeric connexons, we conclude that they are functionally insignificant with regard to voltage-dependent gating.

Animals↗

PubMed2001

Heteromeric mixing of connexins: compatibility of partners and functional consequences.

Cx43 is widely expressed in many different cell types, and many of these cells also express other connexins. If these connexins are capable of mixing, the functional properties of channels containing heteromeric connexons may substantially influence intercellular communication between such cells. We used biochemical strategies (sedimentation through sucrose gradients, co-immunoprecipitation, or co-purification by Ni-NTA chromatography) to examine heteromeric mixing of Cx43 with other connexins (including Cx26, Cx37, Cx40, Cx45, and Cx56) in transfected cells. These analyses showed that all of the tested connexins except Cx26 formed heteromeric connexons with Cx43. We used the double whole-cell patch-camp technique to analyze the electrophysiological properties of gap junction channels in pairs of co-expressing cells. Cx37 and Cx45 made a large variety of functional heteromeric combinations with Cx43 based on detection of many different single channel conductances. Most of the channel event sizes observed in cells co-expressing Cx40 and Cx43 were similar to those of homomeric Cx43 or Cx40 hemichannels in homo- or hetero-typic configurations. Our data suggest several different possible consequences of connexin co-expression: (1) some combinations of connexins may form heteromeric connexons with novel proeprties; (2) some connexins may form heteromeric channels that do not have unique properties, and (3) some connexins may be incompatible for heteromeric mixing.

Animals↗

PubMed2000

Functional expression and biophysical properties of polymorphic variants of the human gap junction protein connexin37.

Connexin37 (Cx37) forms gap junction channels between endothelial cells, and two polymorphic Cx37 variants (Cx37-S319 and Cx37-P319) have been identified with a possible link to atherosclerosis. We studied the gap junction channel properties of these hCx37 polymorphs by expression in stably transfected communication-deficient cells (N2A and RIN). We also expressed a third, truncated variant (Cx37-fs254Delta293) and Cx37 constructs containing epitope tags added to their amino or carboxyl termini. All Cx37 constructs were produced by the transfected cells as demonstrated by RT-PCR and immunoblotting and trafficked to appositional surfaces between cells as demonstrated by immunofluorescence microscopy. Dual whole cell patch-clamping studies demonstrated that Cx37-P319, Cx37-S319, and Cx37-fs254Delta293 had large unitary conductances ( approximately 300 pS). However, addition of an amino terminal T7 tag (T7-Cx37-fs254Delta293) produced a single channel conductance of 120-145 pS with a 24-30 pS residual state. Moreover, the kinetics of the voltage-dependent decline in junctional current for T7-Cx37-fs254Delta293 were significantly slower than for the wild type, implying a destabilization of the transition state. These data suggest that the amino terminus of Cx37 plays a significant role in gating as well as conductance. The carboxyl terminal tail has lesser influence on unitary conductance and inactivation kinetics.

Amino Acid Sequence↗

PubMed2000

Formation of heterotypic gap junction channels by connexins 40 and 43.

Gap junctions formed between transfected cells expressing connexin (Cx) 40 and Cx43 (Cx43-RIN, Cx40-HeLa, and Cx43-HeLa) revealed a relationship, g(j)=f(V(j)), at steady state, that is typified by a nonsymmetrical behavior similar to that previously reported for other heterotypic channels (gap junction conductance [g(j)]; transjunctional voltage [V(j)]). The unitary conductance of the channels was sensitive to the polarity of V(j). A main state conductance of 61 pS was found when the Cx43 cell was stepped positively or the Cx40 cell negatively (V(j)=70 mV); the reverse polarities yielded a conductance of 100 pS. These heterotypic channels were permeable to carboxyfluorescein. In addition, two other heterotypic forms are illustrated to demonstrate that endogenous Cx45 expression cannot explain the results. The demonstration of heterotypic Cx40-Cx43 channels may have implications for the propagation of the electrical impulse in heart. For example, they may contribute to the slowing of the impulse propagation through the junctions between Purkinje fibers and ventricular muscle.

Animals↗

PubMed2000

Electrical properties of gap junction hemichannels identified in transfected HeLa cells.

Human HeLa cells transfected with mouse connexin Cx30, Cx46 or Cx50 were used to study the electrical properties of gap junction hemichannels. With no extracellular Ca2+, whole-cell recording revealed currents arising from hemichannels. Multichannel currents showed a time-dependent inactivation sensitive to voltage, Vm. Plots of the instantaneous conductance, ghc,inst, versus Vm were constant; plots of the steady-state conductance, ghc,ss, versus Vm were bell-shaped. Single-channel currents showed two conductances, gammahc,main and gammahc,residual, the latter approximately or approximately equals=1/6 of the former. Single-channel currents exhibited fast transitions (1-2 ms) between the main state and residual state. Late during wash-in and early during wash-out of 2 mM heptanol, single-hemichannel currents showed slow transitions between an open state and closed state. The open channel probability, Po, was Vm-dependent. It declined from approximately =1 at Vm= 0 mV to 0 at large Vm of either polarity. Hemichannel currents showed a voltage-dependent gammahc,main, i.e., it increased/decreased with hyperpolarization/depolarization. Extrapolation to Vm=0 mV led to a gammahc,main of 283, 250 and 352 pS for Cx30, Cx46 and Cx50, respectively. The hemichannels possess two gating mechanisms. Gating with positive voltage reflects Vj-gating of gap junction channels, gating with negative voltage reflects a property inherent to hemichannels, i.e., Vm or "loop" gating. We conclude that Cx30, Cx46 and Cx50 form voltage-sensitive hemichannels in single cells which are closed under physiological conditions.

Animals↗

PubMed2000

The kinetics of gap junction currents are sensitive to the ionic composition of the pipette solution.

Myocytes were isolated from neonatal rat hearts using an enzymatic procedure. Cell pairs were used to control the junctional voltage, V(j), and to measure the transjunctional current, I(j), using the dual voltage-clamp method. V(j) gradients provoked I(j) signals with voltage-dependent inactivation. During voltage pulses, I(j) remained virtually constant at ¿V(j)¿ <40 mV. At ¿V(j)¿>40 mV, it inactivated with time to a residual level. The inactivation followed a single exponential. The time constant of I(j) inactivation, taui, and the size of I(j) at steady state, I(j,ss), were both sensitive to the ions in the pipette solution. I(j,ss) was smaller in the presence of tetraethylammonium aspartate (TEA+ aspartate-) than KC1, while taui was smaller in the presence of KC1 than TEA+ aspartate-. The modification of I(j,ss) is readily explained by a change in the residual conductance of the gap junction channels, gammaj,residual x The alterations in taui are correlated with a change in beta, the rate constant that describes the transition of the channel from the main state to the residual state. Pipette solutions may affect the kinetics of gap junction currents by altering the conductive and/or kinetic parameters. Computer simulations revealed a substantial influence of the latter, but only a marginal effect of the former. Conceivably, ions of the pipette solution may affect the kinetics of gap junction channels by screening surface charges of the channel wall.

Animals↗

PubMed1999

Biophysical properties of mouse connexin30 gap junction channels studied in transfected human HeLa cells.

1. Human HeLa cells expressing mouse connexin30 (Cx30) were used to study the electrical properties of Cx30 gap junction channels. Experiments were performed on cell pairs with the dual voltage-clamp method. 2. The gap junction conductance (gj) at steady state showed a bell-shaped dependence on junctional voltage (Vj; Boltzmann fit: Vj,0 = 27 mV, gj,min = 0.15, z = 4). The instantaneous gj decreased slightly with increasing Vj. 3. The gap junction currents (Ij) declined with time following a single exponential. The time constants of Ij inactivation (taui) decreased with increasing Vj. 4. Single channels exhibited a main state, a residual state and a closed state. The conductances gammaj,main and gammaj,residual were 179 and 48 pS, respectively (pipette solution, potassium aspartate; temperature, 36-37 degrees C; extrapolated to Vj = 0 mV). 5. The conductances gammaj,residual and gammaj,main showed a slight Vj dependence and were sensitive to temperature (Q10 values of 1.28 and 1.16, respectively). 6. Current transitions between open states (i.e. main state, substates, residual state) were fast (< 2 ms), while those between an open state and the closed state were slow (12 ms). 7. The open channel probability (Po) at steady state decreased from 1 to 0 with increasing Vj (Boltzmann fit: Vj,0 = 37 mV; z = 3). 8. Histograms of channel open times implied the presence of a single main state; histograms of channel closed times suggested the existence of two closed states (i.e. residual states). 9. We conclude that Cx30 channels are controlled by two types of gates, a fast one responsible for Vj gating involving transitions between open states (i.e. residual state, main state), and a slow one correlated with chemical gating involving transitions between the closed state and an open state.

Animals↗

PubMed1999

Electrophysiological properties of gap junction channels in hepatocytes isolated from connexin32-deficient and wild-type mice.

Hepatocytes were isolated from wild-type and connexin32-deficient (Cx32-deficient) mice. Pairs of cells were chosen to study the electrical properties of gap junction channels using the dual voltage-clamp method. The total gap junction currents revealed that Cx32-deficient hepatocytes express one type of connexin (Cx26) and wild-type hepatocytes express two types of connexins (Cx26 and Cx32). The unitary gap junction currents suggest that Cx32-deficient cells have homotypic channels (Cx26-Cx26) while wild-type cells form homotypic (Cx26-Cx26, Cx32-Cx32) and heterotypic channels (Cx26-Cx32). Homotypic channels exhibited a main conductance and a residual conductance, both virtually insensitive to gap junction voltage (Vj) (Cx32-Cx32: gammaj,main=31 pS, gammaj,residual=9 pS; Cx26-Cx26: gammaj,main=102 pS, gammaj,residual=17 pS). Residual states were regularly seen in Cx32-Cx32 channels, but rarely in Cx26-Cx26 channels. Heterotypic channels showed a main conductance and a residual conductance. The former was sensitive to Vj (average gammaj,main=52 pS). The electrophysiological data suggest that Cx32 hemichannels are more abundant than Cx26 hemichannels in prenatal (ratio 4:1) and adult wild-type hepatocytes (ratio 23:1) and that the total number of gap junction channels is larger in prenatal cells than in adult cells. The diversity of the relationship gj, ss/gj,inst=f(Vj) (gj,ss: gap junction conductance at steady state; gj,inst: instantaneous gap junction conductance; Vj: transjunctional voltage) seen in wild-type cells suggests that the ratio Cx26/Cx32 hemichannels is variable among hepatocytes. A comparison of total and unitary conductances implies that Cx26 hemichannels are down-regulated in Cx32-deficient cells and that docking between Cx26 and Cx32 hemichannels occurs randomly. While the gap junction currents are compatible with homotypic and heterotypic channels, the presence of heteromeric channels cannot be excluded.

Animals↗

PubMed1997

Modulation of cardiac gap junctions: the mode of action of arachidonic acid.

Myocytes isolated from neonatal rat hearts were grown in culture dishes. Cell pairs were selected to examine the mode of action of arachidonic acid (AA) on gap junctions. The dual voltage-clamp method was used to measure intercellular currents and determine the gap junction conductance, gj. Exposure of cell pairs to 10 microM AA produced reversible uncoupling. Pretreatment with 10 microM POCA (sodium-2-[5-(4-chlorophenyl)-pentyl]-oxirane-2-carboxylate; which inhibits mitochondrial beta-oxidation) did not prevent AA-dependent uncoupling. Thus, it seems that metabolites of beta-oxidation are not involved in AA-induced impairment of gj. Pre-exposure to 10 microM indomethacin (which blocks the cyclooxygenase pathway of the AA-cascade) had no effect on AA-dependent uncoupling. This suggests that cyclooxygenase products such as prostaglandins or thromboxanes play no role in gj modulation. Exposure to 5 microM NDGA (nordihydroguaiaretic acid; which inhibits the 5-lipoxygenase pathway) or 10 microM ETYA (5,8,11,14-eicosatetrynoic acid: which inhibits the 12- and 15-lipoxygenase pathway) led to a reversible decrease in gj. Pre-treatment with 4-BPB (4-bromophenacyl bromide: which inhibits phospholipase A2) did not prevent the effects on gj by NDGA or ETYA. This renders it unlikely that gj is regulated by eicosanoids. Also, accumulation of endogenous AA cannot be responsible for NDGA- and ETYA-dependent uncoupling. Exposure to 75 microM SKF-525A (inhibits the epoxygenase pathway) reversibly impaired gj. This is consistent with a direct action of SKF-525A on gj, but leaves open the possibility of an involvement of epoxides. The data gathered will be discussed in terms of molecular mechanisms. Due to their amphipathic character. AA, NDGA, ETYA and SKF-525A may interfere with gj by disturbing the lipid-protein interface of the cell membranes and thereby impair gap junction channels.

Animals↗

PubMed1997

Conductances and selective permeability of connexin43 gap junction channels examined in neonatal rat heart cells.

Myocytes from neonatal rat hearts were used to assess the conductive properties of gap junction channels by means of the dual voltage-clamp method. The experiments were carried out on three types (groups) of preparations: (1) induced cell pairs, (2) preformed cell pairs with few gap junction channels (1 to 3 channels), and (3) preformed cell pairs with many channels (100 to 200 channels) after treatment with uncoupling agents such as SKF-525A (75 micromol/L), heptanol (3 mmol/L), and arachidonic acid (100 micromol/L). In group 1, the first opening of a newly formed channel was slow (20 to 65 ms) and occurred 7 to 25 minutes after physical cell contact. The rate of channel insertion was 1.3 channels/min. Associated with a junctional voltage gradient (Vj), the channels revealed multiple conductances, a main open state [gamma(j)(main state)], several substates [gamma(j)(substates)], and a residual state [gamma(j)(residual state)]. On rare occasions, the channels closed completely. The same phenomena were observed in groups 2 and 3. The existence of gamma(j)(residual state) provides an explanation for the incomplete inactivation of the junctional current (Ij) at large values of Vj in cell pairs with many gap junction channels. The values of gamma(j)(main state) and gamma(j)(residual state) gained from groups 1, 2, and 3 turned out to be comparable and hence were pooled. The fit of the data to a Gaussian distribution revealed a narrow single peak for both conductances. The values of gamma(j) were dependent on the composition of the pipette solution. Solutions were as follows: (1) KCl solution, gamma(j)(main state)=96 pS and gamma(j)(residual state)=23 pS; (2) Cs+ aspartate solution, gamma(j)(main state)=61 pS and gamma(j)(residual state)=12 pS; and (3) tetraethylammonium+ aspartate solution, gamma(j)(main state)=19 pS and gamma(j)(residual state)=3 pS. The respective gamma(j)(main state)-to-gamma(j)(residual state) ratios were 4.2, 5.1, and 6.3. This indicates that the residual state restricts ion permeation more efficiently than does the main state. Transitions of Ij between open states (main open state, substates, and residual state) were fast (<2 ms), and transitions involving the closed state and an open state were slow (15 to 65 ms). This implies the existence of two gating mechanisms. The residual state may be regarded as the ground state of electrical gating controlled by Vj; the closed state, as the ground state of chemical gating.

Animals↗

PubMed1994

Use of cluster analysis technique for computerized recognition and prognosis of myocardial vulnerability.

Variability of factors exerting influence on arrhythmia origination leads to the appearance of polymodal distribution in sample space. Therefore, for the approximation of feature distribution, distribution mixture is used. To estimate the number of mixture components and to determine its parameters, cluster algorithm is used. The basic task of the algorithm is to identify the accumulation of vectors in the parallelepiped of their distribution. The accumulations of points are determined by testing statistical hypothesis of uniformity. On the basis of accumulations, clusters are formed and the parameters of normal mixtures of classes are estimated. Analysis of error matrix for recognition of mixture, enable to establish the parameters of the decision rule. The above algorithm was applied for recognition and prognosis of vulnerability of reentry and focal source in experiments on the right rabbit's atrium by using the electrophysiological parameters. We studied 30 cases of reentry, 36 cases of focal sources and 165-arrhythmia-free cases. As a result, we established the 7-class normal mixture which enabled a more effective (96.4%) recognition of the vulnerability types and 89.9% prognosis by features: increase in latency (theta), width of the interval of latency distribution, ratio theta/R, where R-refractory period.

Algorithms↗