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The growth of many types of cancer cells can be controlled by surrounding normal cells. However, mechanisms underlying this phenomenon have not been defined. We used a layered culture system to investigate how nontransformed cells suppress the growth of neighboring transformed cells. Direct physical contact between transformed and nontransformed cells was required for growth suppression of transformed cells in this system; communication by diffusible factors was not sufficient. However, significant gap junctional communication was not required, indicating that other intercellular junctions mediated this growth regulatory response. We also report that the Src kinase activity in transformed cells was not directly inhibited by contact with nontransformed cells. Instead, nontransformed cells increased the expression of serum deprivation-response protein and the transcription factor four and a half LIM domain 1 in tumor cells. In addition, these results suggest mechanisms by which normal cells may block Wnt signaling, inhibit insulin-like growth factor activity, and promote host recognition of neighboring tumor cells.

Water movement in the lacrimal gland is a consequence of ion channel and transporter activity. The production of tears is a consequence of acetylcholine (Ach) stimulation of acinar and duct cells, which produce a transepithelial flux of ions resulting in fluid movement. Ach acts through phospholipase C via Gq/11 and subsequently results in elevation of intracellular IP3 and DAG. The former raises intracellular calcium while the latter activates many PKC isoforms (Walcott, 1998; Dartt et al., 1998). In lacrimal gland K+, chloride and cation channels are activated by elevated intracellular calcium (Marty et al., 1984; Begenisich and Melvin, 1998).

Gap junctions were assayed during re-differentiation of adult rat cardiomyocytes in long-term culture to gain insight into the processes of remodeling. Double immunostaining allowed the localization of connexins Cx40, Cx43, and Cx45 between myocytes and demonstrated co-expression and co-localization in individual cells and gap junction plaques, respectively. Immunoblots showed differential time-dependent changes in connexin expression and phosphorylation. The total amount of connexins and the ratio of phosphorylated/non-phosphorylated isoforms gradually increased during the re-establishment of intercellular communication. Dual voltage-clamp studies showed the involvement of several types of gap junction channels. Multichannel currents yielded diverse spectra of g j,inst=f(V j) and g j,ss=f(V j) relationships (g j,inst: instantaneous gap junction conductance; g j,ss: conductance at steady state; V j: transjunctional voltage), indicative of homotypic and heterotypic channels. Single-channel currents revealed two prominent conductances reflecting γj,main and γj,residual. The histograms of γj,main showed four discrete peaks (41–44, 59–61, 70–76, and 100–107 pS) attributable to a combination of Cx45-Cx45, Cx40-Cx45 and Cx43-Cx45 channels (1st peak), Cx43-Cx43 and Cx40-Cx43 channels (2nd peak), Cx43-Cx43 channels (3rd peak) and Cx40-Cx40 and Cx40-Cx43 channels (4th peak). However, the presence of heteromeric channels cannot be excluded. The data are consistent with an up-regulation of Cx45 and Cx43 during re-differentiation.

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.

Abstract —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. The full text of this article is available at http://www.circresaha.org.

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.

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.

Publisher Summary Mammalian gap junction channels are formed when each cell of an adjacent pair contributes six connexins to form an oligomerized hemichannel. Any two hemichannels can form a link through extracellular loops extending from their respective membrane-spanning domains to form an aqueous intercellular pathway. Gap junction channels are therefore composed of 12 subunit proteins. These subunits are referred to as “connexins” and are named according to their predicted molecular weights. The biophysical characteristics of the gap junction family of proteins have been rigorously studied. Historically, however, most electrophysiological studies of connexins have concentrated on elucidating the macroscopic behavior and unitary conductance of homotypic gap junction channels, that is, gap junction channels formed of 12 identical connexin proteins. However, because many cell types express more than one connexin protein, distinct combinations of these intercellular channels among parenchymal cells within a given tissue are, at the very least, theoretically possible. In particular, it is quite conceivable that gap junction channels in a given tissue cell type can also be formed by the union of hemichannels that are each composed of homologous, but different connexin proteins. Recently, evidence has been advanced consistent with the supposition that the expression of more than one connexin type in a single cell results in the mixing of nonidentical connexins (i.e., heteromeric) in a given hemichannel or connexon. As such, this chapter reviews the extant evidence for heteromeric connexins formed of the two most prominent connexins found in vascular wall cells, that is, connexin43 (Cx43) and connexin37 (Cx37). In addition, the electrophysiological criteria that distinguish heteromeric connexins from their heterotypic and homotypic counterparts are reviewed, as along with the potential physiological relevance of heteromeric channels to tissue function in situ.

Abstract 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 μmol/L), heptanol (3 mmol/L), and arachidonic acid (100 μmol/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 (V j ), the channels revealed multiple conductances, a main open state [γ j (main state)], several substates [γ j (substates)], and a residual state [γ j (residual state)]. On rare occasions, the channels closed completely. The same phenomena were observed in groups 2 and 3. The existence of γ j (residual state) provides an explanation for the incomplete inactivation of the junctional current (I j ) at large values of V j in cell pairs with many gap junction channels. The values of γ j (main state) and γ 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 γ j were dependent on the composition of the pipette solution. Solutions were as follows: (1) KCl solution, γ j (main state)=96 pS and γ j (residual state)=23 pS; (2) Cs + aspartate − solution, γ j (main state)=61 pS and γ j (residual state)=12 pS; and (3) tetraethylammonium + aspartate − solution, γ j (main state)=19 pS and γ j (residual state)=3 pS. The respective γ j (main state)-to-γ 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 I j between open states (main open state, substates, and residual state) were fast (j ; the closed state, as the ground state of chemical gating.

Gap junction channels are composed of connexins, which exhibit specific permeability to the variety of larger solutes including second messengers, polypeptides and siRNAs.Here, we report the permeability of solutes with different size and net charge Lucifer Yellow (443 (no counterion), -2); Carboxyfluorescein (376, -2); AlexaFluor350 (326, -1); Ethidium bromide (314, +1) and NBD-m-TMA (280, +1) through gap junction channels in HeLa cells expressing Cx26, Cx40, Cx43 and Cx45.The channel permeability was determined using simultaneous measurements of junctional conductance and the cell-cell flux of a fluorescent probe.All four connexins transferred negative charged probes: LY, CF and AF350, however, Cx40 and Cx26 exhibited reduced permeability when compared to Cx43. These connexins revealed following permeability ratios for LY/ CF/ AF350, respectively, relative to the ubiquitous cation K+: 0.029/ 0.032/ 0.069 for Cx43; 0.014/ 0.021/ 0.049 for Cx45; 0.0044/ 0.014/ 0.0245 for Cx26 and 0.0026/ 0.0034/ 0.0206 for Cx40.The positive charged NBD and EthBr exhibited the following permeability relative to K+: 0.045 and 0.0125 for Cx43; 0.048 and 0.0036 for Cx45; 0.055 and 0.026 for Cx26; 0.040 and 0.009 for Cx40.In summary, all negative charged species showed a similar permeability order: Cx43>Cx45>Cx26>Cx40. For positively charged species the permeability orders were: Cx26≈Cx43≈Cx40≈Cx45 (NBD) and Cx26≥Cx43≈Cx40>Cx45 (EthBr). Reduced EthBr permeation through Cx45 channels in comparison to other connexins suggests a size-dependent discrimination of the solute. However, the reduced correlation between junctional conductance and positive charged probes flux suggests intracellular binding of the solute. Therefore quantitative comparison of positively charged solutes has to be taken cautiously.These results confirm that channels formed from individual connexins can discriminate for solutes based on size and charge suggesting that channel selectivity may be a key factor in cell signaling.Supported by AHA0335236N, NIHGM55263.