共查询到20条相似文献,搜索用时 0 毫秒
1.
Molecular analysis of voltage dependence of heterotypic gap junctions formed by connexins 26 and 32. 总被引:6,自引:0,他引:6
Heterotypic gap junctions formed by pairing Xenopus oocytes expressing hemichannels formed of Cx32 with those expressing hemichannels formed of Cx26 displayed novel transjunctional voltage (Vj) dependence not predicted by the behavior of these connexins in homotypic configurations. Rectification of initial and steady-state currents was observed. Relative positivity and negativity on the Cx26 side of the junction resulted in increased and decreased initial conductance (gj0), respectively. Only relative positivity on the Cx26 decreased steady-state conductance (gj infinity). This behavior suggested that interactions between hemichannels influences gap junction gating. The role of the first extracellular loop (E1) in these interactions was examined by pairing Cx32 and Cx26 with a chimeric connexin in which Cx32 E1 was replaced with Cx26 E1 (Cx32*26E1). Both junctions rectified with gj0/Vj relations that were less steep than that observed for Cx32/Cx26. Decreases in gj infinity occurred for either polarity Vj in the Cx32/Cx32*26E1 junction. Mutation of two amino acids in Cx26 E1 increased the steepness of both the gj0/Vj and gj infinity/Vj relations. These data demonstrate that fast rectification can arise from mismatched E1 domains and that E1 may contribute to the voltage sensing mechanisms underlying both fast and slow Vj-dependent processes. 相似文献
2.
Summary We used the double whole-cell voltage-clamp technique on ventricle cell pairs isolated from 7-day chick heart to measure the conductance of their gap junctions (G
j) and junctional channels (
j) with a steady-state voltage difference (V
j) applied across the junction. Currents were recorded from single gap junction channels (i
j) as symmetrical rectangular signals of equal size and opposite sign in the two cells, and
j was measured from i
j/V
j. We observed channel openings at six reproducible conductance levels with means of 42.6, 80.7, 119.6, 157.7, 200.4 and 240.3 pS. More than half of all openings were to the 80-and 160-pS conductance levels. The probability that a high conductance event (e.g., 160 or 240 pS) results from the random simultaneous opening of several 40-pS channels is small, based on their frequency of occurrence and on the prevalence of shifts between small and large conductance states with no intervening 40-pS steps. Our results are consistent with three models of embryonic cardiac gap junction channel configuration: a homogeneous population of 40-pS channels that can open cooperatively in groups of up to six; a single population of large channels with a maximal conductance near 240 pS and five smaller substates; or several different channel types, each with its own conductance.
G
j was determined from the junctional current (I
j) elicited by rectangular pulses of applied transjunctional voltage as I
j/V
j. It was highest near 0 V
j and was progressively reduced by application of V
j between 20 and 80 mV or –20 and –80 mV. In response to increases in V
j, G
j decayed in a voltage-and timedependent fashion. After a 6-sec holding period at 0 V
j, the initial conductance (G
init) measured immediately after the onset of an 80-mV step in V
j was nearly the same as that measured by a 10-mV prepulse. However, during 6-sec pulses of V
j>±20 mV, G
j declined over several seconds from G
init to a steady-state value (G
ss). At potentials greater than ±20 mV the current decay could be fit with biexponential curves with the slow decay time constant (
2) 5–20 times longer than
1. For the response to a step to 80 mV V
j, for example,
1=127 msec and
2=2.6 sec. The rate of current decay in response to smaller positive or negative steps in V
j was slower, the magnitude of the decline was smaller, and the ratio
2/
1 decreased. The relationship between G
init and V
j was approximately linear between 0 and 80 mV or –80 mV. whereas the relationship between G
ss and V
j was nonlinear beyond ±20 mV. Upon returning to 0 V
j, G
j recovered with a biexponential time course, reaching its maximal value after several seconds; recovery time constants after a step in V
j from 80 to 0 mV were 225 msec and 1.9 sec. In the resting state, at low junctional voltage, high conductance channel activity (160–240 pS) is favored. Voltage-dependent decline of G
j results in part from a shift from high to lower conductance states.We thank Ms. B.J. Duke for technical assistance and for preparation of the cell cultures and Drs. L.J. DeFelice and D. Eaton for stimulating and helpful discussions of the results. 相似文献
3.
4.
《Channels (Austin, Tex.)》2013,7(5):390-392
In the mammalian CNS, excessive release of glutamate and overactivation of glutamate receptors are responsible for the secondary (delayed) neuronal death following neuronal injury, including ischemia, traumatic brain injury (TBI) and epilepsy. The coupling of neurons by gap junctions (electrical synapses) increases during neuronal injury. In a recent study with the use of in vivo and in vitro models of cortical ischemia in mice, we have demonstrated that the ischemic increase in neuronal gap junction coupling is regulated by glutamate via group II metabotropic glutamate receptors (mGluR). Specifically, we found that activation of group II mGluRs increases background levels of neuronal gap junction coupling and expression of connexin 36 (Cx36; neuronal gap junction protein), whereas inactivation of group II mGluRs prevents the ischemia-mediated increases in the coupling and Cx36 expression. Using the analysis of neuronal death, we also established that inactivation of group II mGluRs or genetic elimination of Cx36 both dramatically reduce ischemic neuronal death in vitro and in vivo. Similar results were obtained using in vitro models of TBI and epilepsy. Our study demonstrated that mechanisms for the injury-mediated increase in neuronal gap junction coupling are part of the mechanisms for glutamate-dependent neuronal death. 相似文献
5.
Andrei B. Belousov 《Channels (Austin, Tex.)》2012,6(5):390-392
In the mammalian CNS, excessive release of glutamate and overactivation of glutamate receptors are responsible for the secondary (delayed) neuronal death following neuronal injury, including ischemia, traumatic brain injury (TBI) and epilepsy. The coupling of neurons by gap junctions (electrical synapses) increases during neuronal injury. In a recent study with the use of in vivo and in vitro models of cortical ischemia in mice, we have demonstrated that the ischemic increase in neuronal gap junction coupling is regulated by glutamate via group II metabotropic glutamate receptors (mGluR). Specifically, we found that activation of group II mGluRs increases background levels of neuronal gap junction coupling and expression of connexin 36 (Cx36; neuronal gap junction protein), whereas inactivation of group II mGluRs prevents the ischemia-mediated increases in the coupling and Cx36 expression. Using the analysis of neuronal death, we also established that inactivation of group II mGluRs or genetic elimination of Cx36 both dramatically reduce ischemic neuronal death in vitro and in vivo. Similar results were obtained using in vitro models of TBI and epilepsy. Our study demonstrated that mechanisms for the injury-mediated increase in neuronal gap junction coupling are part of the mechanisms for glutamate-dependent neuronal death. 相似文献
6.
Crustecdysone, the hormone responsible for onset and regulation of the molt cycle in Crustacea, causes an increase in ionic coupling of cells of the hepatopancreas concomitant with the events of the molt. Hepatopancreatic tissue incubated for up to 4 hr in modified Eagle Basal Medium containing crustecdysone, exhibited an approximate 29% decrease in intercellular resistance as compared with tissue incubated in control medium. This represents a 29% increase in ionic coupling between hepatopancreatocytes following treatment with crustecdysone. Examination of platinum replicas of freeze-fractured, crustecdysone-treated hepatocyte plasma membrane revealed that most of the gap junction plaques were round with tightly packed intramembrane particles; a condition indicative of highly coupled cells. Similar preparations of control plasmalemmae demonstrated many gap junction plaques which were round or irregular in shape with very loosely packed particles and were indicative of uncoupled junctions. Results of this study are identical to those from a previous investigation of the electrophysiology and freeze-fracture morphology of hepatopancreatocytes during the molt cycle (McVicar and Shivers, 1984), and are thus presumed to reflect a crustecdysone-controlled increase in cell communications in vivo. 相似文献
7.
We study the dynamics of a pair of intrinsically oscillating leaky integrate-and-fire neurons (identical and noise-free) connected by combinations of electrical and inhibitory coupling. We use the theory of weakly coupled oscillators to examine how synchronization patterns are influenced by cellular properties (intrinsic frequency and the strength of spikes) and coupling parameters (speed of synapses and coupling strengths). We find that, when inhibitory synapses are fast and the electrotonic effect of the suprathreshold portion of the spike is large, increasing the strength of weak electrical coupling promotes synchrony. Conversely, when inhibitory synapses are slow and the electrotonic effect of the suprathreshold portion of the spike is small, increasing the strength of weak electrical coupling promotes antisynchrony (see Fig. 10). Furthermore, our results indicate that, given a fixed total coupling strength, either electrical coupling alone or inhibition alone is better at enhancing neural synchrony than a combination of electrical and inhibitory coupling. We also show that these results extend to moderate coupling strengths. 相似文献
8.
Camillo Peracchia 《The Journal of membrane biology》1984,81(1):49-58
Summary This paper reports the inhibitory effects of calmidazolium (CDZ), a calmodulin inhibitor, on electrical uncoupling by CO2. Membrane potential and coupling ratio (V
2/V1) are measured in two neighboring cells ofXenopus embryos (16 to 64 cell stage) for periods as long as 5.5 hr. Upon exposure to 100% CO2, control cells consistently uncouple even if the CO2 treatments are repeated every 15 min for 2.5 hr. CDZ (5×10–8–1×10–7
m) strongly inhibits uncoupling. The inhibition starts after 30, 50 and 60 min of treatment with 1×10–7, 7×10–8 and 5×10–8
m CDZ, respectively, is concentration-dependent and partially reversible. In the absence of CO2, CDZ also improves electrical coupling. CDZ has no significant effect on membrane potential and nonjunctional membrane resistance. These data suggest that calmodulin or a calmodulin-like protein participates in the uncoupling mechanism. 相似文献
9.
目的:探讨缝隙连接(GJ)是否参与同型半胱氨酸(Hcy)介导的自发性高血压(SHR)大鼠血管平滑肌细胞(VSMCs)增殖及可能的分子机制。方法:原代培养SHR VSMCs,细胞分四组:①对照组,②Hcy组,③缝隙连接阻断剂(18α-GA)组和④Hcy+18α-GA组。MTT法及流式细胞仪检测细胞的增殖活性,免疫荧光技术观察细胞中Cx43、Cx40蛋白表达及定位,Western blot法检测细胞中Cx43、Cx40蛋白表达,染料示踪分子传递法(划痕标记染料传输法)检测细胞的缝隙连接功能。结果:①与对照组相比,Hcy组MTT法测得A值及细胞周期测得S值增高(P0.05),18α-GA组降低(P0.05);与Hcy组比,TGFβ-1+18α-GA组A值及S值均降低(P0.05)。②免疫荧光技术检测细胞中Cx43、Cx40蛋白表达呈阳性,两者共定位于胞浆。③与对照组相比,Hcy组Cx43、Cx40蛋白表达增强(P0.05),18α-GA组Cx43、Cx40表达均减弱(P0.05);与Hcy组比,Hcy+18α-GA组Cx43、Cx40表达均减弱(P0.05)。④与对照组相比,Hcy组缝隙连接功能明显增强(P0.05),18α-GA组缝隙连接功能明显减弱(P0.05),与Hcy组比,Hcy+18α-GA组缝隙连接功能显著降低(P0.05)。结论:Hcy通过上调SHR VSMCs Cx43和Cx40的蛋白表达,引起缝隙连接通讯功能的增强,促进了SHR VSMCs增殖。 相似文献
10.
Summary Lens fibers are coupled by communicating junctions which contain a 28-kDalton protein (MIP26) believed to be the main component of the cell-to-cell channel. To study the permeability properties and regulation of these channels, anin vitro system has been developed in which MIP26 isolated from calf lens is incorporated into liposomes and the resulting channels are studied spectrophotometrically by a swelling assay. Liposome vesicles were prepared using a sonication/resuspension method. Incorporation efficiency was monitored by freeze-fracture. Vesicles were resuspended in 6% Dextran T-10. Assay buffer was identical, except for isotonic substitution of sucrose for T-10. MIP26-incorporated (but not control) vesicles swell under isotonic conditions indicating sucrose entry (via channels) followed by water to maintain osmotic balance. In the absence of calmodulin, calcium ion has no effect on channel permeability. On the contrary, vesicles prepared with equimolar amounts of MIP26 and CaM do not swell in the presence of calcium ion, indicating that the channels can be closed. Addition of EGTA to these vesicles reinitiates swelling—evidence that the channel gating mechanism is reversible. Magnesium ion has no effect on either type of vesicle. 相似文献
11.
Michael M. Atkinson Susan K. Anderson Judson D. Sheridan 《The Journal of membrane biology》1986,91(1):53-64
Summary Prompted by our observation that a reduction in junctional permeance is one of the earlier events in the process of neoplastic transformation of a cell line by Rous sarcoma virus, we analyzed the gap junctions, from these cells to determine if the basis of the reduction is a loss of junctional channels. The cells (normal rat kidney, or NRK) are infected with a temperature-sensitive mutant of Rous sarcoma virus, allowing one easily to manipulate the cells into and out of the transformed state, and hence also to manipulate the junctional permeance. Using freeze-fracture electron microscopy, we found that the number and size of the junctions did not change in parallel with the permeance changes we had previously characterized. There is, however, a significant rearrangement of the junctional particles to a more random configuration when the cells are transformed and a reversal to the more ordered pattern when the cells are shifted back to the normal phenotype. These changes do parallel the changes in junctional permeance. We conclude that the permeance of existing junctional channels is modified and that the change in permeance may involve a change in the interaction of the junctional channels with each other and/or the surrounding lipid domain. 相似文献
12.
A. J. Oakley B. Martinac M. C. Wilce 《Protein science : a publication of the Protein Society》1999,8(10):1915-1921
Mechanosensation in bacteria involves transducing membrane stress into an electrochemical response. In Escherichia coli and other bacteria, this function is carried out by a number of proteins including MscL, the mechanosensitive channel of large conductance. MscL is the best characterized of all mechanosensitive channels. It has been the subject of numerous structural and functional investigations. The explosion in experimental data on MscL recently culminated in the solution of the three-dimensional structure of the MscL homologue from Mycobacterium tuberculosis. In this review, much of these data are united and interpreted in terms of the newly published M. tuberculosis MscL crystal structure. 相似文献
13.
The importance of connexins (Cxs) in the cochlear functions has been indicated by the finding that mutations in connexin genes cause a large proportion of sensorineural deafness cases. However, functional roles of connexins in the cochlea are still unclear. In this study, we compared the relative expression levels of 16 different subtypes of mouse connexins in the cochlea. cDNA macroarray hybridizations identified four most prominently expressed connexins (listed in descending order): Cxs 26, 29, 30, and 43. Two of these connexins (Cx26 and Cx30), both belonging to the beta-group, were investigated for their molecular assemblies in the cochlea. Co-immunostaining showed expressions of Cxs 26 and 30 in the same gap junction plaques and their co-assembly was confirmed by co-immunoprecipitation of proteins extracted from the cochlear tissues. The heterologous molecular assembly of connexins is expected to produce gap junctions with biophysical characteristics appropriate for maintaining ionic homeostasis in the cochlea. 相似文献
14.
Ann M. G. L. De Mazière Dietrich W. Scheuermann Philip A. P. M. Aertgeerts 《The Journal of membrane biology》1987,97(2):107-115
Summary Particles and pits of freeze-fractured gap junctions are considered as complementary structures despite the frequent observations of more regular and closer spacings of pits, ascribed to plastic deformation of particle arrays. Recently, however, the noncomplementarity of pits and particles in Purkinje fibers has been reported. To ascertain the relationship between both structures, gap junctions from fixed, cryoprotected liver and myocardium were investigated using spacing and density measurements and complementary replicas.In hepatocyte gap junctions, the center-to-center distances (mean±sd) among pits, 9.57±1.49 nm, and particles, 9.70±1.77 nm, are not significantly different. Density determinations yielded a slightly higher value for the pits, (11,510±830)/m2, than for the particles, (11,230±950)/m2. In the myocardium, the spacing of the regularly arrayed pits, 9.55±1.33 nm barely exceeds the value of 9.44±1.62 nm for the particles, which show some clustering. However, the packing density for the pits, (10,090±740)/m2, appears a little higher than that of the particles (9,890±920)/m2. As density and spacing measurements provided no decisive answers, the positions of individual pits and particles of complementary junctional faces were recorded on transparent sheets and compared. In this fashion, a one-to-one correspondence between particles and pits could be established, while small discrepancies may be attributed to plastic deformation. Moreover, the collinearity of pits and particles may be suggested by the observation of a platinum grain in the center of many pits. 相似文献
15.
Molecular basis of inward rectification: polyamine interaction sites located by combined channel and ligand mutagenesis
下载免费PDF全文
![点击此处可从《The Journal of general physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Kurata HT Phillips LR Rose T Loussouarn G Herlitze S Fritzenschaft H Enkvetchakul D Nichols CG Baukrowitz T 《The Journal of general physiology》2004,124(5):541-554
Polyamines cause inward rectification of (Kir) K+ channels, but the mechanism is controversial. We employed scanning mutagenesis of Kir6.2, and a structural series of blocking diamines, to combinatorially examine the role of both channel and blocker charges. We find that introduced glutamates at any pore-facing residue in the inner cavity, up to and including the entrance to the selectivity filter, can confer strong rectification. As these negative charges are moved higher (toward the selectivity filter), or lower (toward the cytoplasm), they preferentially enhance the potency of block by shorter, or longer, diamines, respectively. MTSEA+ modification of engineered cysteines in the inner cavity reduces rectification, but modification below the inner cavity slows spermine entry and exit, without changing steady-state rectification. The data provide a coherent explanation of classical strong rectification as the result of polyamine block in the inner cavity and selectivity filter. 相似文献
16.
17.
《Cell communication & adhesion》2013,20(4):57-65
AbstractGαq-stimulation reduces intercellular coupling within 10 min via a decrease in the membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2), but the mechanism is unknown. Here we show that uncoupling in rat cardiomyocytes after stimulation of α-adrenergic Gαq-coupled receptors with norepinephrine is prevented by proteasomal and lysosomal inhibitors, suggesting that internalization and possibly degradation of connexin43 (Cx43) is involved. Uncoupling was accompanied by increased Triton X-100 solubility of Cx43, which is considered a measure of the non-junctional pool of Cx43. However, inhibition of the proteasome and lysosome further increased solubility while preserving coupling, suggesting that communicating gap junctions can be part of the soluble fraction. Ubiquitination of Cx43 was also increased, and Cx43 co-immunoprecipitated with the ubiquitin ligase Nedd4. Conclusions: Norepinephrine increases ubiquitination of Cx43 in cardiomyocytes, possibly via Nedd4. We suggest that Cx43 is subsequently internalized, which is preceded by acquired solubility in Triton X-100, which does not lead to uncoupling per se. 相似文献
18.
Regulation of cardiac gap junction channel permeability and conductance by several phosphorylating conditions 总被引:11,自引:0,他引:11
Short term (15 min) effects of activators of protein kinase A (PKA), PKC and PKG on cardiac macroscopic (gj) and single channel (j) gap junctional conductances were studied in pairs of neonatal rat cardiomyocytes. Under dual whole-cell voltage-clamp, PKC activation by 100 nM TPA increased gj by 16 ± 2% (mean ± S.E.M, n=9), 1.5 mM of the PKG activator 8-bromo-cGMP (8Br-cGMP) decreased gj by 26 ± 2% (n=4), whereas 1.5 mM of the PKA activator 8Br-cAMP did not affect gj (1 ± 5%, n=11). Single cardiac gap junction channel events, resolved in the presence of heptanol, indicated two j sizes of 20 pS and 40–45 pS. Under control conditions, the larger events were most frequently observed. Whereas 8Br-cAMP did not change this distribution, TPA or 8Br-cGMP shifted the j distribution to the lower sizes. Diffusion of 6-carboxyfluorescein (6-CF), a gap junction permeant tracer, from the injected cell to neighboring cells was studied on small clusters of neonatal rat cardiomyocytes. Under control conditions, 6-CF labeled 8.4 ± 0.4 cells (mean ± S.E.M, n=31). Whereas 8Br-cAMP did not change the extent of dye transfer (8.1 ± 0.5 cells, n=10), TPA restricted the diffusion of 6-CF to 2.2 ± 0.2 cells (n=30) and 8Br-cGMP to 3.5 ± 0.3 cells (n=10). This suggests that permeability and single channel conductance of Cx43 gap junction channels are parallel related. Altogether, these results point to the differential modulation of electrical and metabolic coupling of cardiac cells by various phosphorylating conditions. 相似文献
19.
Jennifer Petsche Connell Emily Augustini Kenneth J. Moise Jr. Anthony Johnson Jeffrey G. Jacot 《Journal of cellular and molecular medicine》2013,17(6):774-781
Amniotic fluid‐derived stem cells (AFSC) have been reported to differentiate into cardiomyocyte‐like cells and form gap junctions when directly mixed and cultured with neonatal rat ventricular myocytes (NRVM). This study investigated whether or not culture of AFSC on the opposite side of a Transwell membrane from NRVM, allowing for contact and communication without confounding factors such as cell fusion, could direct cardiac differentiation and enhance gap junction formation. Results were compared to shared media (Transwell), conditioned media and monoculture media controls. After a 2‐week culture period, AFSC did not express cardiac myosin heavy chain or troponin T in any co‐culture group. Protein expression of cardiac calsequestrin 2 was up‐regulated in direct transmembrane co‐cultures and media control cultures compared to the other experimental groups, but all groups were up‐regulated compared with undifferentiated AFSC cultures. Gap junction communication, assessed with a scrape‐loading dye transfer assay, was significantly increased in direct transmembrane co‐cultures compared to all other conditions. Gap junction communication corresponded with increased connexin 43 gene expression and decreased phosphorylation of connexin 43. Our results suggest that direct transmembrane co‐culture does not induce cardiomyocyte differentiation of AFSC, though calsequestrin expression is increased. However, direct transmembrane co‐culture does enhance connexin‐43‐mediated gap junction communication between AFSC. 相似文献
20.