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.

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.

scholarly journals Monovalent Ion Selectivity Sequences of the Rat Connexin43 Gap Junction Channel

1997 ◽  
Vol 109 (4) ◽  
pp. 491-507 ◽  
Author(s):  
Hong-Zhan Wang ◽  
Richard D. Veenstra
Keyword(s):  
Gap Junction ◽  
Relative Permeability ◽  
Connexin 43 ◽  
Pore Radius ◽  
Single Channel ◽  
Ion Selectivity ◽  
Channel Conductance ◽  
Patch Clamp Recording ◽  
Channel Pore ◽  
Gap Junction Channel

The relative permeability sequences of the rat connexin 43 (rCx43) gap junction channel to seven cations and chloride were examined by double whole cell patch clamp recording of single gap junction channel currents in rCx43 transfected neuroblastoma 2A (N2A) cell pairs. The measured maximal single channel slope conductances (γj, in pS) of the junctional current-voltage relationships in 115 mM XCl were RbCl (103) ≥ CsCl (102) > KCl (97) > NaCl (79) ≥ LiCl (78) > TMACl (65) > TEACl (53) and for 115 mM KY were KBr (105) > KCl (97) > Kacetate (77) > Kglutamate (61). The single channel conductance-aqueous mobility relationships for the test cations and anions were linear. However, the predicted minimum anionic and cationic conductances of these plots did not accurately predict the rCx43 channel conductance in 115 mM KCl. Instead, the conductance of the rCx43 channel in 115 mM KCl was accurately predicted from cationic and anionic conductance-mobility plots by applying a mobility scaling factor Dx/Do, which depends upon the relative radii of the permeant ions to an estimated pore radius. Relative permeabilities were determined for all of the monovalent cations and anions tested from asymmetric salt reversal potential measurements and the Goldman-Hodgkin-Katz voltage equation. These experiments estimate the relative chloride to potassium permeability to be 0.13. The relationship between the relative cation permeability and hydrated radius was modeled using the hydrodynamic equation assuming a pore radius of 6.3 ± 0.4 Å. Our data quantitatively demonstrate that the rCx43 gap junction channel is permeable to monovalent atomic and organic cations and anions and the relative permeability sequences are consistent with an Eisenman sequence II or I, respectively. These predictions about the rCx43 channel pore provide a useful basis for future investigations into the structural determinants of the conductance and permeability properties of the connexin channel pore.

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.

Slow intercellular Ca2+ signaling in wild-type and Cx43-null neonatal mouse cardiac myocytes

2000 ◽  
Vol 279 (6) ◽  
pp. H3076-H3088 ◽  
Author(s):  
Sylvia O. Suadicani ◽  
Monique J. Vink ◽  
David C. Spray
Keyword(s):  
Gap Junction ◽  
Cardiac Myocytes ◽  
Channel Blocker ◽  
Atp Release ◽  
Neonatal Mouse ◽  
Wild Type ◽  
Gap Junction Channels ◽  
Gap Junction Channel ◽  
Main Pathway ◽  
Stimulation Of

Focal mechanical stimulation of single neonatal mouse cardiac myocytes in culture induced intercellular Ca2+ waves that propagated with mean velocities of ∼14 μm/s, reaching ∼80% of the cells in the field. Deletion of connexin43 (Cx43), the main cardiac gap junction channel protein, did not prevent communication of mechanically induced Ca2+ waves, although the velocity and number of cells communicated by the Ca2+ signal were significantly reduced. Similar effects were observed in wild-type cardiac myocytes treated with heptanol, a gap junction channel blocker. Fewer cells were involved in intercellular Ca2+ signaling in both wild-type and Cx43-null cultures in the presence of suramin, a P2-receptor blocker; blockage was more effective in Cx43-null than in wild-type cells. Thus gap junction channels provide the main pathway for communication of slow intercellular Ca2+ signals in wild-type neonatal mouse cardiac myocytes. Activation of P2-receptors induced by ATP release contributes a secondary, extracellular pathway for transmission of Ca2+ signals. The importance of such ATP-mediated Ca2+ signaling would be expected to be enhanced under ischemic conditions, when release of ATP is increased and gap junction channels conductance is significantly reduced.

scholarly journals Role of Gap Junctions and Hemichannels in Parasitic Infections

2013 ◽  
Vol 2013 ◽  
pp. 1-17 ◽  
Author(s):  
José Luis Vega ◽  
Mario Subiabre ◽  
Felipe Figueroa ◽  
Kurt Alex Schalper ◽  
Luis Osorio ◽  
...  
Keyword(s):  
Gap Junction ◽  
Viral Infections ◽  
Cellular Communication ◽  
Parasitic Infections ◽  
Pathological State ◽  
Channel Changes ◽  
Gap Junction Channels ◽  
Gap Junction Channel ◽  
Relevant Role

In vertebrates, connexins (Cxs) and pannexins (Panxs) are proteins that form gap junction channels and/or hemichannels located at cell-cell interfaces and cell surface, respectively. Similar channel types are formed by innexins in invertebrate cells. These channels serve as pathways for cellular communication that coordinate diverse physiologic processes. However, it is known that many acquired and inherited diseases deregulate Cx and/or Panx channels, condition that frequently worsens the pathological state of vertebrates. Recent evidences suggest that Cx and/or Panx hemichannels play a relevant role in bacterial and viral infections. Nonetheless, little is known about the role of Cx- and Panx-based channels in parasitic infections of vertebrates. In this review, available data on changes in Cx and gap junction channel changes induced by parasitic infections are summarized. Additionally, we describe recent findings that suggest possible roles of hemichannels in parasitic infections. Finally, the possibility of new therapeutic designs based on hemichannel blokers is presented.

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.

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.

Differences in gap junction channels between cardiac myocytes, fibroblasts, and heterologous pairs

1992 ◽  
Vol 263 (5) ◽  
pp. C959-C977 ◽  
Author(s):  
M. B. Rook ◽  
A. C. van Ginneken ◽  
B. de Jonge ◽  
A. el Aoumari ◽  
D. Gros ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Rat Heart ◽  
Neonatal Rat ◽  
Heart Cells ◽  
Channel Conductance ◽  
Open Probability ◽  
Gap Junction Channels ◽  
Gap Junction Channel ◽  
Junction Protein

Cultures of neonatal rat heart cells contain predominantly myocytes and fibroblastic cells. Most abundant are groups of synchronously contracting myocytes, which are electrically well coupled through large gap junctions. Cardiac fibroblasts may be electrically coupled to each other and to adjacent myocytes, be it with low intercellular conductances. Nevertheless, synchronously beating myocytes interconnected via a fibroblast were present, demonstrating that nonexcitable cardiac cells are capable of passive impulse conduction. In fibroblast pairs as well as in myocyte-fibroblast cell pairs, no sensitivity to junctional voltage could be detected when transjunctional conductance was > 1-2 nS. However, in pairs coupled by a conductance of < 1 nS, complex voltage-dependent gating was evident; gap junction channel open probability decreased with increasing junctional voltage but a nongated residual conductance remained at all voltages tested. Single gap junction channel conductance between fibroblasts was approximately 21 pS, very similar to an approximately 18-pS channel conductance that was found between myocytes next to the major conductance of 43 pS. Single-channel conductance in heterologous myocyte-fibroblast gap junctions was approximately 32 pS, which matches the theoretical value of 29 pS for gap junction channels composed of a fibroblast connexon and the major myocyte connexon. A site-directed antibody against rat heart gap junction protein connexin43 recognized gap junctions between neonatal cardiomyocytes, as demonstrated by immunocytochemical labeling. In contrast, junctions between fibroblasts showed no labeling, while in myocyte-fibroblast junctions labeling occasionally was present. Our results suggest the existence of two gap junction proteins between neonatal rat cardiocytes, connexin43 and another yet unidentified connexin. An alternative explanation (cell-specific regulation of the conductance of connexin43 channels) is discussed.

Modulation of gap-junction channel gating at zebrafish retinal electrical synapses

1994 ◽  
Vol 72 (5) ◽  
pp. 2257-2268 ◽  
Author(s):  
D. G. McMahon ◽  
D. R. Brown
Keyword(s):  
Gap Junction ◽  
Horizontal Cell ◽  
Channel Gating ◽  
Horizontal Cells ◽  
General Mechanism ◽  
Spatial Integration ◽  
Open Probability ◽  
Electrical Synapses ◽  
Gap Junction Channels ◽  
Gap Junction Channel

1. Transmission at electrical synapses is modulated by a variety of physiological signals, and this modulation is a potentially general mechanism for regulating signal integration in neural circuits and networks. In the outer plexiform layer of the retina, modulation of horizontal-cell electrical coupling by dopamine alters the extent of spatial integration in the horizontal-cell network. By analyzing the activity of individual gap-junction channels in low-conductance electrical synapses of zebrafish retinal horizontal cells, we have defined the properties of these synaptic ion channels and characterized the functional changes in them during modulation of horizontal-cell electrical synapses. 2. Zebrafish horizontal-cell gap-junction channels have a unitary conductance of 50–60 pS and exhibit open times of several tens of milliseconds. The kinetic process of channel closure is best described by the sum of two rate constants. 3. Dopamine, and its agonist, (+/-)-6,7-dihydroxy-2-amino-tetralin (ADTN), modulates electrical synaptic transmission between horizontal cells predominantly by affecting channel-gating kinetics. These agents reduced the open probability of gap-junction channels two- to threefold by reducing both the duration and frequency of channel openings. Both time constants for channel open duration were reduced, whereas the duration of shut periods was increased. Similar changes in open-time kinetics were observed in power spectra of higher conductance gap junctions. 4. These results provide a description of rapid electrical synaptic modulation at the single channel level. The description should be useful in understanding the mechanisms of plasticity at these synapses throughout the vertebrate central nervous system.

Biophysical characterization of zebrafish connexin35 hemichannels

2004 ◽  
Vol 287 (6) ◽  
pp. C1596-C1604 ◽  
Author(s):  
Virginijus Valiunas ◽  
Rickie Mui ◽  
Elizabeth McLachlan ◽  
Gunnar Valdimarsson ◽  
Peter R. Brink ◽  
...  
Keyword(s):  
Gap Junction ◽  
Single Channel ◽  
Biophysical Characterization ◽  
Whole Cell ◽  
Gap Junction Channels ◽  
N2a Cells ◽  
Unitary Conductance ◽  
Cell Depolarization

A subset of connexins can form unopposed hemichannels in expression systems, providing an opportunity for comparison of hemichannel gating properties with those of intact gap junction channels. Zebrafish connexin35 (Cx35) is a member of the Cx35/Cx36 subgroup of connexins highly expressed in the retina and brain. In the present study, we have shown that Cx35 expression in Xenopus oocytes and N2A cells produced large outward whole cell currents on cell depolarization. Using whole cell, cell-attached, and excised patch configurations, we obtained multichannel and single-channel current recordings attributable to the Cx35 hemichannels ( Ihc) that were activated and increased by stepwise depolarization of membrane potential ( Vm) and deactivated by hyperpolarization. The currents were not detected in untransfected N2A cells or in control oocytes injected with antisense Cx38. However, water-injected oocytes that were not treated with antisense showed activities attributable to Cx38 hemichannels that were easily distinguishable from Cx35 hemichannels by a significantly larger unitary conductance ( γhc: 250–320 pS). The γhc of Cx35 hemichannels exhibited a pronounced Vm dependence; i.e., γhc increased/decreased with relative hyperpolarization/depolarization ( γhc was 72 pS at Vm = −100 mV and 35 pS at Vm = 100 mV). Extrapolation to Vm = 0 mV predicted a γhc of 48 pS, suggesting a unitary conductance of intact Cx35 gap junction channels of ∼24 pS. Channel gating was also Vm dependent: open time declined with negative Vm and increased with positive Vm. The ability to break down the complex gating of intact intercellular channels into component hemichannels in vitro will help to evaluate putative physiological roles for hemichannels in vivo.

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