scholarly journals Single-channel events and gating behavior of the cardiac gap junction channel.

1988 ◽  
Vol 85 (10) ◽  
pp. 3431-3434 ◽  
Author(s):  
J. M. Burt ◽  
D. C. Spray
Keyword(s):  
Gap Junction ◽  
Single Channel ◽  
Gap Junction Channel

Distinctive gap junction channel types connect WB cells, a clonal cell line derived from rat liver

1991 ◽  
Vol 260 (3) ◽  
pp. C513-C527 ◽  
Author(s):  
D. C. Spray ◽  
M. Chanson ◽  
A. P. Moreno ◽  
R. Dermietzel ◽  
P. Meda
Keyword(s):  
Gap Junction ◽  
Cell Line ◽  
Rat Liver ◽  
Connexin 43 ◽  
Single Channel ◽  
Freeze Fracture ◽  
Particle Sizes ◽  
Frequency Distributions ◽  
Gap Junction Channel ◽  
Junctional Conductance

Gap junctions, dye coupling, and junctional conductance were studied in a cell line (WB) that is derived from rat liver and displays a phenotype similar to “oval” cells. In freeze-fracture replicas, two distinctive particle sizes were detected in gap junctional plaques. Immunocytochemical studies indicated punctate staining at membrane appositions using antibodies to connexin 43 and to a brain gap junction-associated antigen (34 kDa). No staining was observed using antibodies prepared against rat liver gap junction proteins (connexins 32 and 26). Pairs of WB cells were electrically and dye coupled. Junctional conductance (gj) between cell pairs averaged approximately 10 nS; occasionally, gj was low enough that unitary junctional conductances (gamma j) could be detected. Using a CsCl-containing electrode solution, distinctive gamma j values were recorded: approximately 20-30 pS, approximately 80-90 pS, and the sum of the other sizes. The largest gamma j events were apparently due to random coincident openings or closures of the smaller channels. Several treatments reduced gj. Frequency distributions of gamma j were unaltered by 2 mM halothane or 3.5 heptanol, but the sizes of intermediate and largest events were reduced slightly by 100 nM phorbol ester, and the relative frequency of the largest events was increased by 10 microM glutaraldehyde. We conclude that the distinctive gamma j values represent openings and closures of two distinct types of gap junction channels rather than substates of a single channel type; these unitary conductances may correspond to the dual immunoreactivity and to the two particle sizes seen in freeze fracture.

scholarly journals Calmodulin-Connexin Partnership in Gap Junction Channel Regulation-Calmodulin-Cork Gating Model

2021 ◽  
Vol 22 (23) ◽  
pp. 13055
Author(s):  
Camillo Peracchia ◽  
Lillian Mae Leverone Peracchia
Keyword(s):  
Gap Junction ◽  
Single Channel ◽  
Wild Type ◽  
X Ray Diffraction ◽  
The Past ◽  
Channel Regulation ◽  
Gating Model ◽  
Gap Junction Channel ◽  
Chemical Gating ◽  
Nanomolar Range

In the past four decades numerous findings have indicated that gap junction channel gating is mediated by intracellular calcium concentrations ([Ca2+i]) in the high nanomolar range via calmodulin (CaM). We have proposed a CaM-based gating model based on evidence for a direct CaM role in gating. This model is based on the following: CaM inhibitors and the inhibition of CaM expression to prevent chemical gating. A CaM mutant with higher Ca2+ sensitivity greatly increases gating sensitivity. CaM co-localizes with connexins. Connexins have high-affinity CaM-binding sites. Connexin mutants paired to wild type connexins have a higher gating sensitivity, which is eliminated by the inhibition of CaM expression. Repeated trans-junctional voltage (Vj) pulses progressively close channels by the chemical/slow gate (CaM’s N-lobe). At the single channel level, the gate closes and opens slowly with on-off fluctuations. Internally perfused crayfish axons lose gating competency but recover it by the addition of Ca-CaM to the internal perfusion solution. X-ray diffraction data demonstrate that isolated gap junctions are gated at the cytoplasmic end by a particle of the size of a CaM lobe. We have proposed two types of CaM-driven gating: “Ca-CaM-Cork” and “CaM-Cork”. In the first, the gating involves Ca2+-induced CaM activation. In the second, the gating occurs without a [Ca2+]i rise.

scholarly journals Differential regulation of distinct types of gap junction channels by similar phosphorylating conditions.

1995 ◽  
Vol 6 (12) ◽  
pp. 1707-1719 ◽  
Author(s):  
B R Kwak ◽  
M M Hermans ◽  
H R De Jonge ◽  
S M Lohmann ◽  
H J Jongsma ◽  
...  
Keyword(s):  
Gap Junction ◽  
Single Channel ◽  
Cell Communication ◽  
Differential Regulation ◽  
Cell Coupling ◽  
Gap Junction Channels ◽  
Physiological Importance ◽  
Gap Junction Channel ◽  
Conductance States ◽  
Channel Properties

Studies on physiological modulation of intercellular communication mediated by protein kinases are often complicated by the fact that cells express multiple gap junction proteins (connexins; Cx). Changes in cell coupling can be masked by simultaneous opposite regulation of the gap junction channel types expressed. We have examined the effects of activators and inhibitors of protein kinase A (PKA), PKC, and PKG on permeability and single channel conductance of gap junction channels composed of Cx45, Cx43, or Cx26 subunits. To allow direct comparison between these Cx, SKHep1 cells, which endogenously express Cx45, were stably transfected with cDNAs coding for Cx43 or Cx26. Under control conditions, the distinct types of gap junction channels could be distinguished on the basis of their permeability and single channel properties. Under various phosphorylating conditions, these channels behaved differently. Whereas agonists/antagonist of PKA did not affect permeability and conductance of all gap junction channels, variable changes were observed under PKC stimulation. Cx45 channels exhibited an additional conductance state, the detection of the smaller conductance states of Cx43 channels was favored, and Cx26 channels were less often observed. In contrast to the other kinases, agonists/antagonist of PKG affected permeability and conductance of Cx43 gap junction channels only. Taken together, these results show that distinct types of gap junction channels are differentially regulated by similar phosphorylating conditions. This differential regulation may be of physiological importance during modulation of cell-to-cell communication of more complex cell systems.

Gap junction channel activity in short-term cultured human detrusor myocyte cell pairs: gating and unitary conductances

2006 ◽  
Vol 291 (6) ◽  
pp. C1366-C1376 ◽  
Author(s):  
H.-Z. Wang ◽  
Peter R. Brink ◽  
George J. Christ
Keyword(s):  
Gap Junction ◽  
Intercellular Communication ◽  
Single Channel ◽  
Short Term ◽  
Bladder Tissue ◽  
Western Blots ◽  
Gap Junction Channel ◽  
Junctional Communication ◽  
Subconductance States ◽  
Human Detrusor

Several independent lines of investigation indicate that intercellular communication through gap junctions modulates bladder physiology and, moreover, that altered junctional communication may contribute to detrusor overactivity. However, as far as we are aware, there are still no direct recordings of gap junction-mediated intercellular currents between human or rat detrusor myocytes. Northern and Western blots were used to identify connexin expression in frozen human bladder tissue and short-term cultured human detrusor myocytes. Double whole cell patch (DWCP) recording revealed that human detrusor myocyte cell pairs were well coupled with an average junctional conductance of 6.5 ± 4.6 nS (ranging from 0.1 to 15 nS, n = 22 cell pairs). Macroscopic gap junction channel currents in human detrusor myocytes exhibited voltage dependence similar to homotypic connexin43. The normalized transjunctional conductance-voltage ( Gj- Vj) relationship was symmetrical and well described by a two-state Boltzmann relation ( Gmin≈ 0.33, V0= 63.6 mV, Z = 0.117 or equal to 2.95 gating charges), suggestive of a bilateral voltage-gated mechanism. In symmetric 165 mM CsCl, the measured single-channel slope conductance was ∼120 pS for the fully open channel and ∼26 pS for the major substate. Occasionally, other subconductance states were also observed. The single-channel mean open time declined with increasing Vj, accounting for the Vj-dependent decline of macroscopic junctional current. Qualitatively similar electrophysiological characteristics were observed in DWCP of freshly isolated rat detrusor myocytes. These data confirm and extend previous observations and are consistent with reports in other smooth muscle cells types in which Cx43-mediated intercellular communication has been identified.

Intercellular communication in cultured human vascular smooth muscle cells

2001 ◽  
Vol 281 (1) ◽  
pp. C75-C88 ◽  
Author(s):  
Hong-Zhan Wang ◽  
Nancy Day ◽  
Mira Valcic ◽  
Ken Hsieh ◽  
Scott Serels ◽  
...  
Keyword(s):  
Gap Junction ◽  
Intercellular Communication ◽  
Cultured Cells ◽  
Single Channel ◽  
Western Blots ◽  
Gap Junction Channel ◽  
Whole Cell Patch Clamp ◽  
Channel Expression ◽  
Single Channel Activity ◽  
Internal Mammary

Intercellular communication through gap junction channels plays a fundamental role in regulating vascular myocyte tone. We investigated gap junction channel expression and activity in myocytes from the physiologically distinct vasculature of the human internal mammary artery (IMA, conduit vessel) and saphenous vein (SV, capacitance vessel). Northern and Western blots documented the presence of connexin43 (Cx43) in frozen tissues and cultured cells from both vessels. Northern blots also confirmed the presence of Cx40 mRNA in cultured IMA and SV myocytes. Dual whole cell patch-clamp experiments revealed that macroscopic junctional conductance was voltage dependent and characteristic of that observed for Cx43. In the majority of records, in both vessels, single-channel activity was dominated by a main-state conductance of 120 pS, with subconducting events comprising less than 10% of the amplitude histograms. However, some records showed “atypical” unitary events that had a conductance similar to Cx40 (∼140–160 pS), but gating behavior like that of Cx43. As such, it is conceivable that the presence and coexpression of Cx40 and Cx43 in IMA and SV myocytes may result in heteromeric channel formation. Nonetheless, in terms of gating, Cx43-like behavior clearly dominates.

Cardiac gap junction channel activity in embryonic chick ventricle cells

1988 ◽  
Vol 254 (1) ◽  
pp. H170-H180 ◽  
Author(s):  
R. D. Veenstra ◽  
R. L. DeHaan
Keyword(s):  
Gap Junction ◽  
Single Channel ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Gap Junction Channel ◽  
Whole Cell Patch Clamp ◽  
Junctional Conductance ◽  
Low Levels ◽  
Active Channels ◽  
Channel Currents

We have recorded single-gap junction-channel currents from pairs of 7-day chick embryo ventricle cells, using the double whole cell patch-clamp technique. Junctional conductance (Gj) was variable from one preparation to the next, ranging from 0.15 to 35.0 nS. Single-channel conductance (gamma j) of the main junctional channel was 166 +/- 51 pS and was independent of Gj; a second conductance level of 60–80 pS was also seen in favorable records. The transition time from the closed to the open state was 285 +/- 153 microseconds, with some slow transitions lasting 1–5 ms. Channels opened and closed stochastically; Gj could be defined by the product of the number of active channels in the junction (N), the mean open-state probability (Po) of the channels, and gamma j. Channel activity was unaffected by cell membrane potential or by transjunctional potential. Po and Gj were reversibly reduced to low levels by 1-octanol or by elevated [Cai], whereas gamma j was unchanged by these agents. The 60–80 pS conductance mechanism was octanol- and Ca-resistant, but it is not clear whether this represents a subconductance level of the main channel or a separate class of smaller channels.

scholarly journals Molecular characterization and functional expression of the human cardiac gap junction channel.

1990 ◽  
Vol 111 (2) ◽  
pp. 589-598 ◽  
Author(s):  
G I Fishman ◽  
D C Spray ◽  
L A Leinwand
Keyword(s):  
Gap Junction ◽  
Dna Analysis ◽  
Single Channel ◽  
Expression System ◽  
Molecular Genetic ◽  
Predicted Amino Acid Sequence ◽  
Gap Junction Channel ◽  
Junction Protein ◽  
Junctional Conductance ◽  
Single Channel Currents

Gap junctions permit the passage of ions and chemical mediators from cell to cell. To identify the molecular genetic basis for this coupling in the human heart, we have isolated clones from a human fetal cardiac cDNA library which encode the full-length human cardiac gap junction (HCGJ) mRNA. The predicted amino acid sequence is homologous to the rat cardiac gap junction protein, connexin43 (Beyer, E. D., D. Paul, and D. A. Goodenough. 1987. J. Cell Biol. 105:2621-2629), differing by 9 of 382 amino acids. HCGJ mRNA is detected as early as fetal week 15 and persists in adult human cardiac samples. Genomic DNA analysis suggests the presence of two highly homologous HCGJ loci, only one of which is functional. Stable transfection of the HCGJ cDNA into SKHep1 cells, a human hepatoma line which is communication deficient, leads to the formation of functional channels. Junctional conductance in pairs of transfectants containing 10 copies of the HCGJ sequence is high (approximately 20 nS). Single channel currents are detectable in this expression system and correspond to conductances of approximately 60 pS. These first measurements of the HCGJ channel are similar to the junctional conductance recorded between pairs of rat or guinea pig cardiocytes.

Heterotypic gap junction channel formation between heteromeric and homomeric Cx40 and Cx43 connexons

2001 ◽  
Vol 281 (5) ◽  
pp. C1559-C1567 ◽  
Author(s):  
G. Trevor Cottrell ◽  
Janis M. Burt
Keyword(s):  
Gap Junction ◽  
Single Channel ◽  
Cell Voltage ◽  
Cell Types ◽  
Channel Formation ◽  
Gap Junction Channel ◽  
Heteromeric Channel ◽  
A7r5 Cells ◽  
Voltage Dependent ◽  
Heterotypic Channels

Recent evidence indicating formation of functional homomeric/heterotypic gap junction channels by connexin40 (Cx40) and connexin43 (Cx43) raises the question of whether data previously interpreted as support for heteromeric channel formation by these connexins might not instead reflect the activity of homomeric/heterotypic channels. To address this question and to further characterize the behavior of these channels, we used dual whole cell voltage-clamp techniques to examine the junctions formed between cells that express only Cx40 (Rin40) or Cx43 (Rin43) and compared the results with those obtained when either of these cell types was paired with cells that naturally express both connexins (A7r5 cells). Rin40/Rin43 cell pairs formed functional gap junctions that displayed a strongly asymmetric voltage-dependent gating response. Single-channel event amplitudes ranged between 34 and 150 pS, with 90- to 130-pS events predominating. A7r5/Rin43 and A7r5/Rin40 cell pairs had voltage-dependent gating responses that varied greatly, with most pairs demonstrating strong asymmetry. These cell pairs exhibited a variety of single-channel events that were not consistent with homomeric/homotypic Cx40 or Cx43 channels or homomeric/heterotypic Cx40/Cx43 channels. These data indicate that Cx40 and Cx43 form homomeric/heterotypic as well as heteromeric/heterotypic channels that display unique gating and conductance properties.

Cx40 and Cx43 expression ratio influences heteromeric/ heterotypic gap junction channel properties

2002 ◽  
Vol 282 (6) ◽  
pp. C1469-C1482 ◽  
Author(s):  
G. Trevor Cottrell ◽  
Yan Wu ◽  
Janis M. Burt
Keyword(s):  
Gap Junction ◽  
Single Channel ◽  
Dye Transfer ◽  
Expression Ratio ◽  
Cx43 Expression ◽  
Electrophysiological Properties ◽  
Gap Junction Channel ◽  
Cellular Environment ◽  
Voltage Dependent ◽  
Dye Permeability

In cells that coexpress connexin (Cx)40 and Cx43, the ratio of expression can vary depending on the cellular environment. We examined the effect of changing Cx40:Cx43 expression ratio on functional gap junction properties. Rin cells transfected with Cx40 or Cx43 (Rin40, Rin43) were cocultured with 6B5n, A7r5, A7r540C1, or A7r540C3 cells for electrophysiological and dye coupling analysis. Cx40:Cx43 expression ratio in 6B5n, A7r5, A7r540C1, and A7r540C3 cells was ∼1:1, 3:1, 5:1, and 10:1, respectively. When Rin43 cells were paired with coexpressing cells, there was an increasing asymmetry of voltage-dependent gating and a shift toward smaller conductance events as Cx40:Cx43 ratio increased in the coexpressing cell. These observations could not be predicted by linear combinations of Cx40 and Cx43 properties in proportion to the expressed ratios of the two Cxs. When Rin40 cells were paired with coexpressing cells, the net voltage gating and single-channel conductance behavior were similar to those of Rin40/Rin40 cell pairs. Dye permeability properties of cell monolayers demonstrated that as Cx40:Cx43 expression ratio increased in coexpressing cells the charge and size selectivity of dye transfer reflected that of Rin40 cells, as would be predicted. These data indicate that the electrophysiological properties of heteromeric/heterotypic channels are not directly related to the proportions of Cx constituents expressed in the cell; however, the dye permeability of these same channels can be predicted by the relative Cx contributions.

scholarly journals Monovalent Cation Permeation through the Connexin40 Gap Junction Channel

1997 ◽  
Vol 109 (4) ◽  
pp. 509-522 ◽  
Author(s):  
Dolores A. Beblo ◽  
Richard D. Veenstra
Keyword(s):  
Gap Junction ◽  
Single Channel ◽  
Ion Selectivity ◽  
Ion Pairs ◽  
Monovalent Cation ◽  
Chloride Salt ◽  
Monovalent Cations ◽  
Gap Junction Channels ◽  
Gap Junction Channel ◽  
Cation Radius

The unitary conductances and permeability sequences of the rat connexin40 (rCx40) gap junction channels to seven monovalent cations and anions were studied in rCx40-transfected neuroblastoma 2A (N2A) cell pairs using the dual whole cell recording technique. Chloride salt cation substitutions (115 mM principal salt) resulted in the following junctional maximal single channel current-voltage relationship slope conductances (γj in pS): CsCl (153), RbCl (148), KCl (142), NaCl (115), LiCl (86), TMACl (71), TEACl (63). Reversible block of the rCx40 channel was observed with TBA. Potassium anion salt γj are: Kglutamate (160), Kacetate (160), Kaspartate (158), KNO3 (157), KF (148), KCl (142), and KBr (132). Ion selectivity was verified by measuring reversal potentials for current in rCx40 gap junction channels with asymmetric salt solutions in the two electrodes and using the Goldman-Hodgkin-Katz equation to calculate relative permeabilities. The permeabilities relative to Li+ are: Cs+ (1.38), Rb+ (1.32), K+ (1.31), Na+ (1.16), TMA+ (0.53), TEA+ (0.45), TBA+ (0.03), Cl− (0.19), glutamate− (0.04), and NO3− (0.14), assuming that the monovalent anions permeate the channel by forming ion pairs with permeant monovalent cations within the pore thereby causing proportionate decreases in the channel conductance. This hypothesis can account for why the predicted increasing conductances with increasing ion mobilities in an essentially aqueous channel were not observed for anions in the rCx40 channel. The rCx40 effective channel radius is estimated to be 6.6 Å from a theoretical fit of the relationship of relative permeability and cation radius.

Sign in / Sign up

Export Citation Format

Share Document