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.

Evidence for heteromeric gap junction channels formed from rat connexin43 and human connexin37

1997 ◽  
Vol 273 (4) ◽  
pp. C1386-C1396 ◽  
Author(s):  
P. R. Brink ◽  
K. Cronin ◽  
K. Banach ◽  
E. Peterson ◽  
E. M. Westphale ◽  
...  
Keyword(s):  
Gap Junction ◽  
Large Range ◽  
Cell Types ◽  
Channel Activity ◽  
Fluorescent Marker ◽  
Gap Junction Channels ◽  
Gap Junction Channel ◽  
Whole Cell Patch Clamp ◽  
A Cell ◽  
Heterotypic Channels

Homomeric gap junction channels are composed solely of one connexin type, whereas heterotypic forms contain two homomeric hemichannels but the six identical connexins of each are different from each other. A heteromeric gap junction channel is one that contains different connexins within either or both hemichannels. The existence of heteromeric forms has been suggested, and many cell types are known to coexpress connexins. To determine if coexpressed connexins would form heteromers, we cotransfected rat connexin43 (rCx43) and human connexin37 (hCx37) into a cell line normally devoid of any connexin expression and used dual whole cell patch clamp to compare the observed gap junction channel activity with that seen in cells transfected only with rCx43 or hCx37. We also cocultured cells transfected with hCx37 or rCx43, in which one population was tagged with a fluorescent marker to monitor heterotypic channel activity. The cotransfected cells possessed channel types unlike the homotypic forms of rCx43 or hCx37 or the heterotypic forms. In addition, the noninstantaneous transjunctional conductance-transjunctional voltage ( G j/ V j) relationship for cotransfected cell pairs showed a large range of variability that was unlike that of the homotypic or heterotypic form. The heterotypic cell pairs displayed asymmetric voltage dependence. The results from the heteromeric cell pairs are inconsistent with summed behavior of two independent homotypic populations or mixed populations of homotypic and heterotypic channels types. The G j/ V jdata imply that the connexin-to-connexin interactions are significantly altered in cotransfected cell pairs relative to the homotypic and heterotypic forms. Heteromeric channels are a population of channels whose characteristics could well impact differently from their homotypic counterparts with regard to multicellular coordinated responses.

Gap junction function in vascular smooth muscle: influence of serotonin

1995 ◽  
Vol 269 (4) ◽  
pp. H1481-H1489 ◽  
Author(s):  
L. K. Moore ◽  
J. M. Burt
Keyword(s):  
Smooth Muscle ◽  
Gap Junction ◽  
Cell Types ◽  
Rat Aorta ◽  
Open Probability ◽  
Gap Junction Channel ◽  
Reduced Frequency ◽  
Rat Mesentery ◽  
A7r5 Cells ◽  
Junctional Conductance

In this study we examined the effects of serotonin (5-hydroxytryptamine, 5-HT) on the function of gap junctions between smooth muscle cells isolated from human and pig coronary and rat mesentery arteries and between A7r5 cells (cell line derived from embryonic rat aorta). Mesentery and pig coronary cells expressed connexin (Cx) 43, and human coronary cells expressed Cx40. Mesentery and pig coronary cells each exhibited a single gap junction channel population with unitary conductances of 75 and 59 pS, respectively. Human coronary cells exhibited two channel populations with unitary conductances of 51 and 107 pS. The A7r5 cells express Cx40 and Cx43 and exhibit three channel populations with unitary conductances of 70, 108, and 141 pS. Under control conditions, junctional conductance between the four cell types ranged from 11 to 20 nS. During maximal stimulation with 5-HT (1-10 microM), junctional conductance increased (29-75%) in all four cell types. The unitary conductance profiles in the rat mesentery and pig coronary cells were unaffected by 5-HT, suggesting that the observed increase in macroscopic conductance reflects an increase in open probability. Unitary conductances were also unaffected in the human coronary and A7r5 cells. However, there was a reduced frequency of the 105-pS channel in the human coronary cells and of the 70- and 141-pS channels in the A7r5 cells. These changes in the relative frequency histograms suggest that the open probabilities of the various channel types are differentially affected by the 5-HT treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Alteration of Cx43:Cx40 expression ratio in A7r5 cells

2001 ◽  
Vol 280 (3) ◽  
pp. C500-C508 ◽  
Author(s):  
Janis M. Burt ◽  
Anna M. Fletcher ◽  
Timothy D. Steele ◽  
Yan Wu ◽  
G. Trevor Cottrell ◽  
...  
Keyword(s):  
Gap Junction ◽  
Banding Pattern ◽  
Electrical Coupling ◽  
Cell Types ◽  
Mrna Levels ◽  
Expression Ratio ◽  
Protein Levels ◽  
Gap Junction Channel ◽  
A7r5 Cells ◽  
Mrna Ratio

Connexins (Cx) 40 and 43 are coexpressed by several cell types at ratios that vary as a function of development, aging, and disease. Because these connexins form heteromeric channels, changes in expression ratio might be expected to significantly alter the connexin composition of the gap junction channel population and, therefore, gap junction function. To examine this possibility, we stably transfected A7r5 cells, which naturally coexpress Cx43 and Cx40, with a vector encoding antisense Cx43. Cx43 mRNA continued to be expressed in the antisense transfected clones, although levels were inversely related to the number of copies of antisense DNA incorporated into the genome. Protein levels, quantified in the clones with the highest and lowest Cx43:Cx40 mRNA ratios, were not well predicted by the mRNA levels, although the trends predicted by the Cx43:Cx40 mRNA ratio were preserved. Electrical coupling did not differ significantly between clones, but the clone with elevated Cx43:Cx40 protein expression ratio and unchanged Cx43 banding pattern was significantly better dye coupled than the parental A7r5 cells. These results suggest that as the Cx43:Cx40 ratio increases, provided alterations of Cx43 banding pattern (phosphorylation) have not occurred, permeability to large molecules increases even though electrical coupling remains nearly constant.

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.

Electrical coupling of retinal horizontal cells mediated by distinct voltage-independent junctions

1999 ◽  
Vol 16 (5) ◽  
pp. 811-818 ◽  
Author(s):  
CHENGBIAO LU ◽  
DAO-QI ZHANG ◽  
DOUGLAS G. McMAHON
Keyword(s):  
Single Channel ◽  
Lucifer Yellow ◽  
Horizontal Cell ◽  
Electrical Coupling ◽  
Cell Voltage ◽  
Horizontal Cells ◽  
Simultaneous Application ◽  
Dependent Inactivation ◽  
Cellular Junctions ◽  
Voltage Dependent

Electrical coupling between H2 horizontal cell pairs isolated from the hybrid bass retina was studied using dual whole-cell, voltage-clamp technique. Voltage-dependent inactivation of junctional currents in response to steps in transjunctional voltage (Vj) over a range of ±100 mV was characterized for 89 cell pairs. Approximately one-quarter of the pairs exhibited strongly voltage-dependent junctions (>50% reduction in junctional current at ±100 mV), another quarter of the pairs exhibited voltage-independent junctional current (<5% reduction at ±100 mV), and the remainder of the pairs exhibited intermediate values for voltage inactivation. We focused on further characterizing the Vj-independent junctions of horizontal cells, which have not been described previously in detail. When Lucifer Yellow dye was included in one recording pipette, pairs exhibiting Vj-independent coupling showed no (9/12), or limited (3/12), passage of dye. Vj-independent coupling was markedly less sensitive to the modulators SNP (100–300 μM, −9% reduction in coupling) and dopamine (100–300 μM, −6%) than were Vj-dependent junctions (−45% and −44%). However, simultaneous application of both SNP and dopamine significantly reduced Vj-independent coupling (−56%). Both Vj-independent and Vj-dependent junctions were blocked by DMSO (1–2%), but Vj-independent junctions were not blocked by heptanol. Single-channel junctional conductances of Vj-independent junctions range from 112–180 pS, versus 50–60 pS for Vj-dependent junctions. The results reveal that Vj-independent coupling in a subpopulation of horizontal cells from the hybrid bass retina is mediated by cellular junctions with physiological and pharmacological characteristics distinct from those previously described in fish horizontal cells.

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 Permeability of homotypic and heterotypic gap junction channels formed of cardiac connexins mCx30.2, Cx40, Cx43, and Cx45

2007 ◽  
Vol 293 (3) ◽  
pp. H1729-H1736 ◽  
Author(s):  
Mindaugas Rackauskas ◽  
Vytas K. Verselis ◽  
Feliksas F. Bukauskas
Keyword(s):  
Molecular Mass ◽  
Gap Junction ◽  
Fluorescent Dyes ◽  
Single Channel ◽  
Lucifer Yellow ◽  
Cell Communication ◽  
Fluorescent Imaging ◽  
Anionic Dyes ◽  
Electrophysiological Recordings ◽  
Heterotypic Channels

We examined the permeabilities of homotypic and heterotypic gap junction (GJ) channels formed of rodent connexins (Cx) 30.2, 40, 43, and 45, which are expressed in the heart and other tissues, using fluorescent dyes differing in net charge and molecular mass. Combining fluorescent imaging and electrophysiological recordings in the same cell pairs, we evaluated the single-channel permeability ( Pγ). All homotypic channels were permeable to the anionic monovalent dye Alexa Fluor-350 (AF350), but mCx30.2 channels exhibited a significantly lower Pγ than the others. The anionic divalent dye Lucifer yellow (LY) remained permeant in Cx40, Cx43, and Cx45 channels, but transfer through mCx30.2 channels was not detected. Heterotypic channels generally exhibited Pγ values that were intermediate to the corresponding homotypic channels. Pγ values of mCx30.2/Cx40, mCx30.2/Cx43, or mCx30.2/Cx45 heterotypic channels for AF350 were similar and approximately twofold higher than Pγ values of mCx30.2 homotypic channels. Permeabilities for cationic dyes were assessed only qualitatively because of their binding to nucleic acids. All homotypic and heterotypic channel configurations were permeable to ethidium bromide and 4,6-diamidino-2-phenylindole. Permeability for propidium iodide was limited only for GJ channels that contain at least one mCx30.2 hemichannel. In summary, we have demonstrated that Cx40, Cx43, and Cx45 are permeant to all examined cationic and anionic dyes, whereas mCx30.2 demonstrates permeation restrictions for molecules with molecular mass over ∼400 Da. The ratio of single-channel conductance to permeability for AF350 was ∼40- to 170-fold higher for mCx30.2 than for Cx40, Cx43, and Cx45, suggesting that mCx30.2 GJs are notably more adapted to perform electrical rather than metabolic cell-cell communication.

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.

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