A Particle-Receptor Model for the Insulin-Induced Closure of Connexin43 Channels

From the SUNY Health Science Center (N.H., J.L.A., K.S., S.M.T., M.D.), Syracuse, NY, and the Cancer Research Center (A.F.L.), University of Hawaii at Manoa, Honolulu.

Correspondence to Mario Delmar, MD, PhD, Department of Pharmacology, SUNY Health Science Center, 750 E Adams St, Syracuse, NY 13210. E-mail delmarm{at}vax.cs.hscsyr.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract—Connexin43(Cx43) channels can be regulated by a variety of factors, including low pH_i. Structure/function studies from this laboratory have demonstrated that pH gating follows a particle-receptor mechanism, similar to the "ball-and-chain" model of voltage-dependent inactivation of ion channels. The question whether the particle-receptor model is applicable only to pH gating or to other forms of Cx43 regulation as well remains. To address this question, we looked at the uncoupling effects of insulin and of insulin-like growth factor-1 (IGF) on Cx43 channels expressed in Xenopus oocytes. These agonists do not induce changes in pH_i. Junctional conductance (Gj) was measured by the dual 2-electrode voltage-clamp technique. Control studies showed that relative Gj did not change spontaneously as a function of time. Continuous exposure of Cx43-expressing oocytes to insulin (10 µ/L) led to a decrease in Gj. After 80 minutes, Gj was 54±5% from control (n=12). Exposure of oocytes to IGF (10 nmol/L) caused an even more pronounced change in Gj (37±4% of control, n=6). The time course of the IGF-induced uncoupling was similar to that observed after insulin exposure. The effect of insulin was abolished by truncation of the carboxyl-terminal domain of Cx43 at amino acid 257 (M257). Interestingly, as in the case of pH gating, coexpression of the carboxyl-terminal domain (amino acids 258 to 282) together with M257 rescued the ability of insulin to reduce coupling (Gj, 39±12% from control; n=6). Structure/function experiments using various deletion mutants of the carboxyl-terminal domain showed that insulin treatment does not modify Gj if amino acids 261 to 280 are missing from the Cx43 sequence. Our results suggest that a particle-receptor (or ball-and-chain) mechanism, similar to that described for pH gating, also applies to chemical regulation of Cx43 by other factors.

Key Words: connexin43 • gap junction conductance • insulin • phosphorylation • Xenopus oocyte

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Gap junctions allow for the synchronization of electrical and biochemical activity between cells in a tissue. Studies on transgenic animals show that intercellular communication through gap junctions is critical for a variety of biological functions, such as female fertility,¹ control of cell proliferation and growth,² embryonic development,² lens transparency,^{3 4} cardiogenesis,^{5 6} and normal cardiac electrophysiology.² Moreover, medical genetic studies show that mutations of connexin genes are linked to hereditary diseases such as Charcot-Marie-Tooth,^{7 8} nonsyndromic sensorineural deafness,⁹ and some (though not all; see References 10 and 11^{10 11} ) cases of a severe cardiac malformation called visceroatrial heterotaxia.¹²

In vertebrate systems, gap junctions are formed by oligomerization of an integral membrane protein called connexin. At least 13 different connexin genes have been identified. Our recent studies have focused on the regulation of Cx43. This 43-kD 382–amino acid connexin is expressed in a variety of tissues, including heart, ovary, and beta pancreatic cells (see References 8, 13, and 14^{8 13 14} for review).

We have previously shown that truncation of the CT of Cx43 impaired pH gating.¹⁵ More recently, we showed that the CT of Cx43 can modulate acidification-induced uncoupling even if it is expressed separately from the rest of the channel.¹⁶ Our studies led to a particle-receptor model for pH gating, similar to the "ball-and-chain" model of voltage-dependent gating of nonjunctional channels.¹⁷ In its simplest version, we propose that the CT of Cx43 constitutes a gating particle and that a separate domain of Cx43, related to the pore-forming region, is a specific receptor for such a particle. On intracellular acidification, the particle would bind noncovalently to the receptor, thus causing channel closure.

Acidification of the intracellular space is only one of the several ways by which the intracellular and extracellular environments modulate cell-to-cell communication. Therefore, we asked whether the central premise of the particle-receptor model applies to the chemical regulation of connexins by factors other than pH. That is, we aimed to determine whether the regulatory domain, even if expressed separately from the rest of the protein, can recognize its receptor and interact with it to modify the channel function. To address this question, we developed an experimental model of insulin-induced uncoupling in Cx43-expressing oocytes. Our results show that the insulin-induced regulation of Cx43 follows the particle-receptor paradigm. The data suggest that this mechanism is a common path for the chemical regulation of intercellular communication in Cx43-expressing cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Oocyte Preparation, Electrophysiological Recordings, and pH_i Measurement
Experiments were conducted in Xenopus laevis oocytes at stages V to VI of development. Procedures for oocytes preparation and injection have been previously described.^{15 16 18} Briefly, cells were defolliculated and injected with 50 ng of antisense against the endogenous Cx38. After 2 to 5 days of incubation, the vitelline layer was mechanically removed, and the cells were injected with 8 to 20 ng of mRNA for rat Cx43 or its mutants. Cells were paired 24 hours after mRNA injection and tested 12 to 24 hours after pairing.

Oocytes were kept in culture medium (0.5x L-15) before electrophysiological analysis. Approximately 80 to 120 minutes before data collection, L-15 was substituted by a saline (Barth's) solution. During that time, the cells were impaled, and the electrophysiological recordings were allowed to stabilize. Data acquisition started 10 minutes before insulin was added to the bath, but the cells had already been in Barth's solution for 1.5 to 2 hours and impaled for at least 30 minutes.

Gj (ie, the electrical macroscopic conductance between the oocyte pair) was measured by conventional dual 2-electrode voltage clamp.¹⁹ The voltage-clamp protocol and the hardware for data acquisition were the same as previously described.^{15 16 18} Antisense-injected oocytes were not electrically coupled. Nevertheless, oocyte pairs showing Gj values <0.8 µS were not included in the study. This criterion was set to avoid recording from oocyte pairs that showed small levels of Cx43-induced endogenous coupling.²⁰ Furthermore, to ensure good voltage control over the preparation, oocyte pairs expressing Gj values 10 µS were discarded. Experiments were conducted in a sodium acetate–buffered saline (Barth's) solution (pH 7.4) of the following composition (mmol/L): sodium acetate 130, KCl 1, NaHCO₃ 2.4, MgSO₄ 0.82, CaCl₂ 0.74, HEPES 15, and NaCl 20.^{15 18} pH_i was measured by detecting the light emission of the proton-sensitive fluorophore SNARF (dextran form), as previously described.¹⁶

cDNA and mRNA Preparation
Rat cardiac Cx43 cDNA was subcloned into pBluescript IISK⁺ (Stratagene). A fragment encoding a large fraction of the CT domain of Cx43 (amino acids 259 to 382) was generated by conventional polymerase chain reaction of rat Cx43 and subcloned into pBluescript SK^- (Stratagene). cDNAs for all mutants were produced by oligonucleotide-directed mutagenesis,²¹ as previously described.²² Deletion mutants are identified by the symbol , followed by 2 numbers, the first and the last amino acid that is missing from the sequence (eg, 261–280 is a deletion mutant of Cx43 lacking amino acids 261 to 280). Truncation mutants are identified by the letter M and the number of the last amino acid in the sequence (eg, M361 is a truncation mutant of Cx43 at amino acid 361).

Chemicals
Insulin and IGF were purchased commercially from Sigma Chemical Co. The concentrations used (10 µmol/L and 10 nmol/L, respectively) were based on previous studies in which these agonists were used to activate the IGF-R in Xenopus oocytes.²³ Insulin was dissolved in modified Barth's solution^{15 18} alkalinized with a small amount of 1N NaOH. Once insulin was dissolved, the pH was adjusted to 7.4 with 1N HCl. IGF was dissolved directly in modified Barth's solution. In both cases, agonists were added to the bath and maintained throughout the duration of the experiment.

Data Analysis
Unless otherwise stated, Gj values were normalized to the Gj recorded immediately before the onset of agonist exposure (ie, Gj/Gj control). Results are reported as mean±SEM. Statistical comparisons by ANOVA, followed by a Bonferroni test, were conducted to establish whether the effect of insulin (or IGF) on the Gj of oocytes expressing a particular construct was different from that induced by insulin in Cx43-expressing oocytes. Statistical significance set at P<0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Insulin and IGF Induce a Reduction of Gj
Our experiments required prolonged continuous recording of Gj. Thus, as a control, we determined whether spontaneous changes in the Gj of Cx43-expressing oocytes could be detected with time. Figure 1A shows average measurements of Gj (relative to the control Gj of each individual experiment) during continuous recording of Cx43-expressing oocyte pairs maintained in a normal saline solution. In this case, the value of Gj measured 10 minutes after the onset of recording was used as Gj control. Time zero corresponds to the beginning of data acquisition. The results show that no significant spontaneous changes in Gj were observed for up to 90 minutes of continuous recording. Gj/Gj control after 90 minutes was 0.99±0.05 (n=14).

View larger version (15K): [in this window] [in a new window]   Figure 1. Insulin and IGF induce uncoupling of Cx43 gap junctions. A, Stability of Gj with time. Gj was continuously recorded from a pair of Cx43-expressing oocytes maintained in normal saline. Gj control (Gj cont) was defined as the Gj value recorded 10 minutes after the onset of data acquisition. All measurements were standardized relative to Gj cont. Bars indicate SEM. The value of Gj/Gj cont after 90 minutes of recording was 0.99±0.05 (mean±SEM, n=14). B, Insulin-induced uncoupling in Cx43-expressing oocytes. The horizontal dotted line and the arrow indicate the onset of 10 µmol/L insulin exposure. Gj cont was defined as the Gj value recorded immediately before the addition of insulin to the superfusate. For each experiment, Gj measurements were normalized to their own Gj cont. Values of Gj/Gj cont at each point in time from several experiments were then averaged. The plot shows the averaged values, and the bars represent the SEM for the indicated data point. For the sake of clarity in the diagram, only 1 of every 10 error bars is displayed. Insulin caused a reduction in Gj. After 80 minutes of insulin exposure, Gj/Gj cont was 0.54±0.05 (n=12). C, IGF (10 nmol/L) induces partial uncoupling of Cx43-expressing Xenopus oocytes. IGF was applied at the time indicated by the broken line and maintained throughout. Gj/Gj cont dropped to 0.37±0.04 after 70 minutes of IGF exposure. Bars indicate SEM (n=6).

Figure 1B shows the time course of changes in Gj that result from continuously exposing the oocytes to 10 µmol/L of insulin. After a delay of 10 to 15 minutes, the Gj of insulin-treated oocytes progressively decreased, until reaching an asymptotic value 70 minutes after the onset of insulin exposure. The Gj/Gj control value at 80 minutes after the onset of insulin was 0.54±0.05 (n=12). These results show that insulin leads to a reduction of Gj in Cx43-expressing oocytes. In the present study, we use the term "insulin-induced uncoupling" to refer to this reduction on Gj caused by insulin exposure

Insulin is known to activate the IGF-R in oocytes.^{23 24} As a first approach to determine whether the effect of insulin could be mediated by IGF-R activation, Cx43-expressing oocyte pairs were exposed to 10 nmol/L of IGF. As shown in Figure 1C, IGF led to a reduction of Gj similar to that observed when a 1000-fold greater concentration of insulin was used (Gj/Gj control, 0.37±0.04; n=6).

The Insulin Effect Is Not Mediated by a Reduction in pH_i
Because acidification-induced closure is a common property of gap junctions, we conducted control experiments to eliminate the possibility that the observed insulin-induced uncoupling resulted indirectly from intracellular acidification. Figure 2 shows the magnitude of pH_i (measured by the fluorescence emission of the pH-sensitive fluorophore SNARF; see Reference 16¹⁶ ) recorded from a Xenopus oocyte as a function of time. Switching the bathing solution from L-15 to modified sodium acetate–buffered Barth's solution led to a drop in pH_i from 7.86 to 7.78. pH_i slowly recovered to more alkaline levels, reaching 7.85 after 130 minutes. Insulin exposure (10 µmol/L) was initiated at that time and maintained for the rest of the recording period. Clearly, addition of insulin to the bath did not reduce pH_i over the ensuing 80 minutes. Similar results were obtained in 3 additional experiments. As noted in Materials and Methods, data acquisition for the electrophysiological experiments presented in the present study started 1.5 to 2 hours after the culture medium (L-15) was substituted for saline (Barth's) solution. Thus, relative to the onset of Barth's superfusion, the timing of insulin addition in the experiment shown in Figure 2 is similar to that used for the electrophysiological experiments. The results show that (1) pH_i had reached a steady state by the time data acquisition started and (2) insulin addition did not modify pH_i. Thus, the effects of insulin on Gj cannot be ascribed to changes in pH_i.

View larger version (18K): [in this window] [in a new window]   Figure 2. Insulin does not induce intracellular acidification in Xenopus oocytes. The plot shows the magnitude of pHi as a function of time after the solution is switched from the culture medium (L15) to the acetate-containing saline solution. Addition of insulin to the bath is noted by the vertical broken line.

Insulin-Induced Uncoupling of Cx43 Channels Requires the CT Domain of Cx43
Structure-function studies have shown that most of the regulatory functions of Cx43 involve the CT domain.^{13 25} In particular, truncation of the CT domain at amino acid 257 of Cx43 (mutant M257) prevented pH gating.^{15 16} Therefore, we tested the effect of insulin exposure on the Gj of M257-expressing oocytes. As shown in Figure 3 (open circles) truncation of the Cx43 CT domain caused the loss of insulin sensitivity. The effect of insulin on wild-type Cx43 channels is displayed for comparison (solid circles). The Gj recorded from M257-expressing oocytes after 80 minutes of insulin exposure was not significantly different from the untreated control (Gj/Gj control, 1.06±0.12; n=8).

View larger version (34K): [in this window] [in a new window]   Figure 3. The CT fragment of Cx43 rescues the insulin sensitivity of M257 channels. Data from 3 different groups of experiments are depicted: wild-type Cx43 (solid circles, same data as in Figure 1B), M257 (open circles), and a third group (stippled circles) in which oocytes were injected with 2 separate mRNAs: M257 and another mRNA coding exclusively for the CT fragment (amino acids 258 to 382 of Cx43). In contrast to the effect of insulin on the Gj/Gj control (Gj/Gj cont) values of wild-type channels, the values recorded from M257-expressing oocytes 80 minutes after the onset of insulin exposure were not different from control (Gj/Gj cont=1.06±0.12, n=8). Coexpression of the CT fragment provided the M257 channels with insulin sensitivity (Gj/Gj cont for M257+CT=0.39±0.12, n=6).

Previous studies from our laboratory have shown that acidification-induced uncoupling follows a particle-receptor model of gating.¹⁶ Therefore, we tested whether the CT fragment could also act as an independent domain during insulin-induced uncoupling. Each oocyte was injected with 2 separate mRNAs: one coding for the insulin-insensitive Cx43 channel (M257) and the other one coding exclusively for the CT region of Cx43 (amino acids 258 to 382). Oocyte pairs coexpressing these constructs were exposed to insulin following the protocol described above. Figure 3 shows that coexpression of the separate Cx43 CT fragment rescued the insulin sensitivity of the otherwise insulin-insensitive (M257) channel (stippled circles). The results demonstrate that the regulatory domain (the CT fragment) can specifically recognize and noncovalently interact with the rest of the channel protein to switch the channel from a conductive to a nonconductive state. In this regard, the particle-receptor mechanism is not unique to acidification-induced uncoupling but common to other forms of chemical regulation of Cx43.

Structure-Function Relation of Insulin-Induced Uncoupling
Insulin and IGF are known to activate the IGF-R in oocytes, thus triggering a cascade that involves activation of MAPK.²⁶ Separate in vitro studies have shown that MAPK can phosphorylate Cx43 at serines 255, 279, and 282.²⁷ As a first approach to characterize the structural bases for insulin-induced uncoupling, we tested the effect of insulin on the Gj of oocyte pairs expressing a variety of deletion and truncation mutants of the Cx43 CT domain. Figures 4 and 5 show the results. In all panels, the open circles show the data obtained from a mutant channel. Data obtained from wild-type Cx43 (same data as in Figures 1B and 3, solid circles) are shown for comparison.

View larger version (18K): [in this window] [in a new window]   Figure 4. Deletion of amino acids 261 to 280 in Cx43 prevented insulin-induced uncoupling. In all panels, solid circles depict the effect of insulin (10 µmol/L) on the Gj of Cx43-expressing oocytes. The dotted line represents the onset of continuous exposure to insulin. Open circles represent data obtained from mutants of Cx43 in which either amino acids 241 to 260 (241-260, panel B), 261 to 280 (261-280, panel A), or 281 to 300 (281-300, panel C) were deleted from the Cx43 sequence. In all 3 cases, the values of Gj/Gj control (Gj/Gj cont) recorded after 80 minutes of insulin exposure were compared with those obtained from the wild-type channel. Only deletion 261-280 eliminated the sensitivity of Cx43 to insulin (statistical analysis by ANOVA corrected by Bonferroni test, P<0.01). Values from the other 2 mutants were not statistically different from those of the wild-type channel. Gj/Gj cont values were 0.54±0.05, 0.63±0.04, 0.98±0.03, and 0.30±0.09 for Cx43, 241-260, 261-280, and 281-300, respectively.

View larger version (39K): [in this window] [in a new window]   Figure 5. Effect of insulin on the Gj of Cx43 mutants. In all panels, solid circles depict the effect of insulin (10 µmol/L) on the Gj of Cx43-expressing oocytes. The dotted line represents the onset of continuous exposure to insulin. Open circles represent data obtained from mutants of Cx43 in which either amino acids 301 to 320 (301-320, panel A), 321 to 340 (321-340, panel B), 341 to 360 (341-360, panel C), or 361 to 382 (truncation mutant M361, panel D) were deleted from the sequence. All asymptotic Gj/Gj control (Gj/Gj cont) values recorded from the mutant channels were compared against those measured from wild-type Cx43. Although a tendency toward an enhanced uncoupling effect of insulin was observed for all mutants, only data from deletion-mutant 301-320 were statistically different from the wild-type channel (ANOVA followed by Bonferroni test). Gj/Gj cont values after insulin exposure were 0.54±0.05, 0.10±0.01, 0.30±0.09, 0.29±0.8, and 0.20±0.06 for Cx43, 301-320, 321-340, 341-360, and M361, respectively.

Figure 4 shows results obtained from deleting 20 amino acid segments that included the known MAPK consensus sites.²⁷ The results indicated that deletion of amino acids 261 to 280 was sufficient to prevent the insulin effect (Figure 4A). Indeed, after 80 minutes of insulin exposure, Gj between oocyte pairs expressing mutant 261-280 was not different from its own Gj control. Moreover, the Gj/Gj control value recorded from mutant 261-280 at the end of insulin exposure was statistically different from that recorded from Cx43-expressing channels. On the other hand, the 241-260 mutant was virtually indistinguishable from the wild-type channel (Figure 4B). The 281-300 mutant showed a slightly increased sensitivity to insulin-induced changes (Figure 4C). These results showed that preservation of region 261 to 280 in Cx43 is essential for the insulin-induced uncoupling.

Figure 5 shows results obtained from oocytes expressing mutants 301-320, 321-340, 341-360, and M361 (ie, truncation of Cx43 at amino acid 361). Clearly, all of these mutants were insulin sensitive. In fact, ANOVA tests show that insulin-induced uncoupling was facilitated by deletion of amino acids 301 to 320 (Figure 5A). The reason for the facilitated effect of the 301-320 deletion is unclear, but it may be related to modifications in the secondary structure of the CT domain consequent to deletion. Comparison of the insulin sensitivity of the other deletion mutants with that of the wild-type channel revealed no statistically significant difference (although they all showed a similar trend of enhanced sensitivity).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The experiments presented here show the following: (1) Exposure of Cx43-expressing oocytes to insulin, or to IGF, leads to a reduction in Gj. (2) The effect of insulin on Gj requires preservation of the CT region, particularly at amino acids 261 to 280. (3) Insulin-induced gating of Cx43 follows a particle-receptor (or ball-and-chain) model.

Possible Mechanisms of Insulin-Induced Uncoupling
Previous studies have shown that insulin and IGF, both at the same concentrations used in the present study, activate IGF-R in Xenopus oocytes and trigger a complex intracellular signaling cascade.^{23 26} We thus speculate that the effect of insulin on Cx43 may be triggered via activation of IGF-R. Yet, the possibility that insulin activates an additional membrane receptor (including the insulin receptor) cannot be completely discarded.

The IGF-R–dependent cascade in oocytes has been investigated by others.²⁶ The results show that one of the kinases that is activated in this manner is MAPK. Interestingly, this kinase is known to phosphorylate Cx43 at 3 separate serines: 255, 279, and 282.²⁷ Our data indicate that deletion 261-280 prevented the insulin effect. A working hypothesis, untested at this point, is that MAPK-mediated phosphorylation of serine 279 may be involved in the insulin-induced uncoupling process. However, it is also possible that other structural features of this region may be important for the insulin effect. For example, deletion 261-280 disrupts the integrity of the proline-rich region (amino acids 274 to 285), which may act as an SH3 binding domain. Whether the effect of insulin on Gj is mediated, at least in part, by an SH3 domain–containing protein remains to be determined.

The central purpose of the present study was to determine whether other chemically induced forms of gap junction closure would follow the model developed for pH gating. It was important to use an agonist that would not modify pH_i. The results shown in Figure 2 demonstrate that pH_i is not affected by insulin exposure. The values of pH_i shown in the figure are on the high end of what we have observed from oocytes maintained in bicarbonate-buffered solutions.¹⁶ These values, however, are not uncommon in oocytes maintained in a sodium acetate buffer.¹⁵ The sodium acetate buffer was chosen for these experiments because it does not require continuous gassing.

The choice of insulin for the present experiments was based on the biochemical effects known for this hormone in Xenopus oocytes. These results, by themselves, should not be interpreted as demonstrative of an insulin-mediated modulation of gap junctions in mammalian systems, nor do we ascribe at the moment a role to insulin on Xenopus development. However, it is tempting to speculate that changes in intercellular communication may be part of the cellular processes related to the effect of IGF on cardiac and vascular tissue.^{28 29} Although perhaps such modulatory mechanisms exist, their demonstration and analysis go beyond the goals of the present project.

A delay was observed between the onset of insulin exposure and the development of uncoupling. It is unlikely that such a delay resulted simply from the time required for the solution exchange in the recording chamber. Indeed, the delay was not observed in some of the mutants (see Figure 5). Furthermore, the time course of insulin-induced uncoupling of Cx43 was similar to that observed for insulin-induced phosphorylation of S6 kinase (one of the target proteins of MAPK) in Xenopus oocytes.³⁰ The MAPK-mediated effect of epidermal growth factor receptor activation on Cx43 was also observed 5 to 10 minutes after epidermal growth factor exposure.³¹ Although these observations are consistent with our results, the nature of the rate-limiting step between insulin exposure and Cx43 uncoupling remains to be determined.

Insulin-Induced Uncoupling and the Particle-Receptor Hypothesis
The ball-and-chain model has been applied to the fast inactivation of Shaker B potassium channels.¹⁷ A key element of the ball-and-chain model is that the gating particle acts as an independent domain that, even when expressed as a separate protein, can identify and react with a receptor, thus eliciting a change in channel function.¹⁷ We recently demonstrated that this condition applies to the acidification-induced uncoupling of Cx43.¹⁶ A remaining question, though, was whether the ball-and-chain model was unique to pH gating or common to other forms of connexin regulation. The present results show that, as in the case of pH gating, insulin-induced closure follows the ball-and-chain model. The data thus indicate that both manners of chemical regulation converge into a common mechanism. Results from the laboratory of Bruce Nicholson (Zhou and Nicholson,³² presented in abstract form) suggest that a ball-and-chain model also applies to the src-induced closure of gap junctions. The term "chemical gating" is therefore loosely justified from these results. However, it should not be interpreted to indicate that the effect of insulin is necessarily (or exclusively) mediated by a conformational change in a preexisting channel that switches the oligomer from an open to a closed state. Given the time course of insulin-induced uncoupling, it may also be possible that insulin induces a reduction in cell-cell communication by, for example, the removal and/or degradation of connexins from the membrane. The latter does not modify the central premise of the present study, ie, that noncovalent intramolecular interactions between the CT domain and its receptor are essential for the regulation of intercellular communication by either pH or other membrane agonists.

Structure-function studies show that regions of the CT domain necessary for pH gating are not required for insulin-induced uncoupling. In particular, our previous studies have shown that deletion 281–300, as well as truncation at amino acid 361, interferes with acidification-induced closure.²² Those same deletions do not affect the sensitivity of Cx43 to insulin. This difference suggests that these 2 chemically induced mechanisms of gating are not mediated by a common intermediary. More likely, intracellular acidification and insulin exposure trigger 2 different intermediary cascades that have different structural requirements of the CT region and different time course, both leading to uncoupling by a particle-receptor type of mechanism.

The concept of particle-receptor interaction opens the possibility that the regulation of gap junctions can be modified by small analogous molecules that compete for the receptor without modifying its function (ie, competitive inhibition). This strategy has been successfully implemented by our laboratory to prevent pH gating of Cx43 in Xenopus oocytes.³³ The latter approach could be used as a tool to study the role of Cx43 in the cellular changes induced by IGF and, perhaps, other cytokines in the cardiovascular system.³⁴

Spray and Burt³⁵ originally proposed that pH gating of Cx43 may involve protonation of histidine residues in the cytoplasmic loop. Our studies¹⁸ have supported this hypothesis; we have further suggested that such histidines may be part of the receptor for the gating particle. Future studies will address the question whether the histidines in the cytoplasmic loop are also a possible part of the receptor for insulin-induced gating.

In conclusion, the present study shows that the particle-receptor (or ball-and-chain) model is a common mechanism of chemical gating of Cx43. Understanding the molecular intricacies of chemical gating may allow for the development of new approaches to interfere with gap junction channel closure in native systems. The latter would have implications in the study of gap junction function both in health and disease.

	Selected Abbreviations and Acronyms

 

CT = carboxyl-terminal region of Cx43 Cx43 = connexin43 Gj = junctional conductance IGF-R = insulin-like growth factor receptor IGF = insulin-like growth factor-1 M257 = mutant of Cx43 truncated at amino acid 257 MAPK = mitogen-activated protein kinase SNARF = seminaphthorhodafluor

	Acknowledgments

 
This study was supported by grant PO1 HL-39707 (Drs Delmar and Taffet) from the National Institutes of Health, Heart Lung and Blood Institute; by grant 52098 (Dr Lau) from the National Cancer Institute; and by a fellowship grant from the American Heart Association, New York State Affiliate (Dr Homma). Dr Delmar is an Established Investigator of the American Heart Association.

Received January 8, 1998; accepted March 17, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Simon AM, Goodenough DA, Li E, Paul DL. Female infertility in mice lacking connexin 37. Nature. 1997;385:525–529.[Medline] [Order article via Infotrieve]

2. Willecke K, Buchmann A, Butzler C, Gabriel H-D, Hagendorff A, Jung D, Kirchhoff S, Kruger O, Nelles E, Schwarz M, Temme A, Traub O, Winterhager E. Biological functions of gap junctions revealed by targeted inactivation of mouse connexin32, -26 and -40 genes. In: Werner R, ed. Gap Junctions. Amsterdam, Netherlands: IOS Press; 1998:304–308.

3. Gong XH, Li E, Klier G, Huang QL, Wu Y, Lei H, Kumar NM, Horwitz J, Gilula NB. Disruption of alpha(3) connexin gene leads to proteolysis and cataractogenesis in mice. Cell. 1997;91:833–843.[Medline] [Order article via Infotrieve]

4. White TW, Goodenough DA, Paul DL. Ocular abnormalities in connexin50 knockout mice [abstract]. Mol Biol Cell. 1997;8:93a.

5. Reaume AG, de Sousa PA, Kulkarni S, Langille BL, Zhu D, Davies TC, Juneja SC, Kidder GM, Rossant J. Cardiac malformation in neonatal mice lacking connexin43. Science. 1995;267:1831–1834.[Abstract/Free Full Text]

6. Ewart JL, Cohen MF, Meyer RA, Huang GY, Wessels A, Gourdie RG, Chin AJ, Park SJ, Lazatin BO, Villabon S, Lo CW. Heart and neural tube defects in transgenic mice overexpressing the Cx43 gap junction gene. Development. 1997;124:1281–1292.[Abstract]

7. Bergoffen J, Scherer SS, Wang S, Oronzi Scott M, Bone LJ, Paul DL, Chen K, Lensch MW, Chance PF, Fischbeck KH. Connexin mutations in X-linked Charcot-Marie-Tooth disease. Science. 1993;262:2039–2042.[Abstract/Free Full Text]

8. Bruzzone R, White TW, Paul DL. Connections with connexins: the molecular basis of direct intercellular signaling. Eur J Biochem. 1996;238:1–27.[Medline] [Order article via Infotrieve]

9. Kelsell DP, Dunlop J, Stevens HP, Lench NJ, Liang JN, Parry G, Mueller RF, Leigh IM. Connexin26 mutations in hereditary non-syndromic sensorineural deafness. Nature. 1997;387:80–83.[Medline] [Order article via Infotrieve]

10. Gebbia M, Towbin JA, Casey B. Failure to detect connexin43 mutations in 38 cases of sporadic and familial heterotaxy. Circulation. 1996;94:1909–1912.[Abstract/Free Full Text]

11. Splitt MP, Tsai MY, Burn J, Goodship JA. Absence of mutations in the regulatory domain of the gap junction protein connexin 43 in patients with visceroatrial heterotaxy. Heart. 1997;77:369–370.[Abstract/Free Full Text]

12. Britz-Cunningham SH, Shah MM, Zuppan CW, Fletcher WH. Mutations of the connexin43 gap junction gene in patients with heart malformations and defects of laterality. N Engl J Med. 1995;332:1323–1329.[Abstract/Free Full Text]

13. Yeager M, Nicholson BJ. Structure of gap junction intercellular channels. Cur Opinion Struct Biol. 1996;6:183–192.

14. Goodenough DA, Goliger JA, Paul DL. Connexins, connexons, and intercellular communication. Annu Rev Biochem. 1996;65:475–502.[Medline] [Order article via Infotrieve]

15. Liu S, Taffet S, Stoner L, Delmar M, Vallano ML, Jalife J. A structural basis for the unequal sensitivity of the major cardiac and liver gap junctions to intracellular acidification: the carboxyl tail length. Biophys J. 1993;64:1422–1433.[Medline] [Order article via Infotrieve]

16. Morley GE, Taffet SM, Delmar M. Intramolecular interactions mediate pH regulation of connexin43 channels. Biophys J. 1996;70:1294–1302.[Medline] [Order article via Infotrieve]

17. Hoshi T, Zagotta WN, Aldrich RW. Biophysical and molecular mechanisms of Shaker potassium channel inactivation. Science. 1990;250:533–538.[Abstract/Free Full Text]

18. Ek JF, Delmar M, Perzova R, Taffet SM. Role of histidine 95 on pH gating of the cardiac gap junction protein connexin43. Circ Res. 1994;74:1058–1064.[Abstract/Free Full Text]

19. Spray DC, Harris AL, Bennett MVL. Equilibrium properties of a voltage-dependent junctional conductance. J Gen Physiol. 1981;77:77–93.[Abstract/Free Full Text]

20. White TW, Paul DL, Goodenough DA, Bruzzone R. Functional analysis of selective interactions among rodent connexins. Mol Biol Cell. 1995;6:459–470.[Abstract]

21. Vandeyar MA, Weiner MP, Hutton CJ, Batt CA. A simple and rapid method for the selection of oligodeoxynucleotide-dictated mutants. Gene. 1988;65:129–133.[Medline] [Order article via Infotrieve]

22. EK-Vitorin JF, Calero G, Morley GE, Coombs W, Taffet SM, Delmar M. pH regulation of connexin43: molecular analysis of the gating particle. Biophys J. 1996;71:1273–1284.[Medline] [Order article via Infotrieve]

23. Chuang LM, Myers MG Jr, Seidner GA, Birnbaum MJ, White MF, Kahn CR. Insulin receptor substrate 1 mediates insulin and insulin-like growth factor 1- stimulated maturation of Xenopus oocytes. Proc Natl Acad Sci U S A.. 1993;90:5172–5175.[Abstract/Free Full Text]

24. Janicot M, Flores-Riveros JR, Lane MD. The insulin-like growth factor 1 (IGF-1) receptor is responsible for mediating the effects of insulin, IGF-1, and IGF-2 in Xenopus laevis oocytes. J Biol Chem. 1991;266:9382–9391.[Abstract/Free Full Text]

25. Lau AF, Kurata WE, Kanemitsu MY, Loo LW, Warn-Cramer BJ, Eckert W, Lampe PD. Regulation of connexin43 function by activated tyrosine protein kinase. J Bioenerg Biomembr. 1996;28:359–368.[Medline] [Order article via Infotrieve]

26. Grigorescu F, Baccara MT, Rouard M, Renard E. Insulin and IGF-1 signaling in oocyte maturation. Horm Res. 1994;42:55–61.[Medline] [Order article via Infotrieve]

27. Warn-Cramer BJ, Lampe PD, Kurata WE, Kanemitsu MY, Loo LW, Eckert W, Lau AF. Characterization of the mitogen-activated protein kinase phosphorylation sites on the connexin-43 gap junction protein. J Biol Chem. 1996;271:3779–3786.[Abstract/Free Full Text]

28. Duerr RL, McKirnan MD, Gim RD, Clark RG, Chien KR, Ross J Jr. Cardiovascular effects of insulin-like growth factor-1 and growth hormone in chronic left ventricular failure in the rat. Circulation. 1996;93:2188–2196.[Abstract/Free Full Text]

29. Delafontaine P. Insulin-like growth factor-1 and its binding proteins in the cardiovascular system. Cardiovasc Res. 1995;30:825–834.[Medline] [Order article via Infotrieve]

30. Stefanovic D, Erikson E, Pike LJ, Maller JL. Activation of a ribosomal protein S6 kinase in Xenopus oocytes by insulin and insulin-receptor kinase. EMBO J. 1986;5:157–160.[Medline] [Order article via Infotrieve]

31. Lau AF, Kanemitsu MY, Kurata WE, Danesh S, Boynton AL. Epidermal growth factor disrupts gap-junctional communication and induces phosphorylation of connexin43 on serine. Mol Biol Cell. 1992;3:865–874.[Abstract]

32. Zhou L, Nicholson BJ. A "ball-and-chain" mechanism for the gating of Cx43 gap junction channels by v-src [abstract]. Mol Biol Cell. 1997;8:93a.

33. Calero G, Kanemitsu M, Taffet SM, Lau AF, Delmar M. A 17mer peptide interferes with acidification-induced uncoupling of connexin43. Circ Res.. 1998;82:929–935.[Abstract/Free Full Text]

34. Doble BW, Chen Y, Bosc DG, Litchfield DW, Kardami E. Fibroblast growth factor-2 decreases metabolic coupling and stimulates phosphorylation as well as masking of connexin43 epitopes in cardiac myocytes. Circ Res. 1996;79:647–658.[Abstract/Free Full Text]

35. Spray DC, Burt JM. Structure-activity relations of the cardiac gap junction channel. Am J Physiol. 1990;258:C195–C205.[Abstract/Free Full Text]

This article has been cited by other articles:

				N. S. Heyman, D. T. Kurjiaka, J. F. Ek Vitorin, and J. M. Burt Regulation of gap junctional charge selectivity in cells coexpressing connexin 40 and connexin 43 Am J Physiol Heart Circ Physiol, July 1, 2009; 297(1): H450 - H459. [Abstract] [Full Text] [PDF]

				J. Kang, N. Kang, D. Lovatt, A. Torres, Z. Zhao, J. Lin, and M. Nedergaard Connexin 43 Hemichannels Are Permeable to ATP J. Neurosci., April 30, 2008; 28(18): 4702 - 4711. [Abstract] [Full Text] [PDF]

				M. H. de Pina-Benabou, V. Szostak, A. Kyrozis, D. Rempe, D. Uziel, M. Urban-Maldonado, S. Benabou, D. C. Spray, H. J. Federoff, P. K. Stanton, et al. Blockade of Gap Junctions In Vivo Provides Neuroprotection After Perinatal Global Ischemia Stroke, October 1, 2005; 36(10): 2232 - 2237. [Abstract] [Full Text] [PDF]

				A. F. Lau c-Src: Bridging the Gap Between Phosphorylation- and Acidification-Induced Gap Junction Channel Closure Sci. Signal., July 5, 2005; 2005(291): pe33 - pe33. [Abstract] [Full Text] [PDF]

				P. L. Sorgen, H. S. Duffy, P. Sahoo, W. Coombs, M. Delmar, and D. C. Spray Structural Changes in the Carboxyl Terminus of the Gap Junction Protein Connexin43 Indicates Signaling between Binding Domains for c-Src and Zonula Occludens-1 J. Biol. Chem., December 24, 2004; 279(52): 54695 - 54701. [Abstract] [Full Text] [PDF]

				K. Maass, A. Ghanem, J.-S. Kim, M. Saathoff, S. Urschel, G. Kirfel, R. Grummer, M. Kretz, T. Lewalter, K. Tiemann, et al. Defective Epidermal Barrier in Neonatal Mice Lacking the C-Terminal Region of Connexin43 Mol. Biol. Cell, October 1, 2004; 15(10): 4597 - 4608. [Abstract] [Full Text] [PDF]

				M. Delmar, W. Coombs, P. Sorgen, H. S Duffy, and S. M Taffet Structural bases for the chemical regulation of Connexin43 channels Cardiovasc Res, May 1, 2004; 62(2): 268 - 275. [Abstract] [Full Text] [PDF]

				A. P Moreno Biophysical properties of homomeric and heteromultimeric channels formed by cardiac connexins Cardiovasc Res, May 1, 2004; 62(2): 276 - 286. [Abstract] [Full Text] [PDF]

				G. Olbina and W. Eckhart Mutations in the Second Extracellular Region of Connexin 43 Prevent Localization to the Plasma Membrane, but Do Not Affect Its Ability to Suppress Cell Growth Mol. Cancer Res., July 1, 2003; 1(9): 690 - 700. [Abstract] [Full Text] [PDF]

				D. Lin, D. L. Boyle, and D. J. Takemoto IGF-I-Induced Phosphorylation of Connexin 43 by PKC{gamma}: Regulation of Gap Junctions in Rabbit Lens Epithelial Cells Invest. Ophthalmol. Vis. Sci., March 1, 2003; 44(3): 1160 - 1168. [Abstract] [Full Text] [PDF]

				A. P. Moreno, M. Chanson, J. Anumonwo, I. Scerri, H. Gu, S. M. Taffet, and M. Delmar Role of the Carboxyl Terminal of Connexin43 in Transjunctional Fast Voltage Gating Circ. Res., March 8, 2002; 90(4): 450 - 457. [Abstract] [Full Text] [PDF]

				L. I. Plotkin, S. C. Manolagas, and T. Bellido Transduction of Cell Survival Signals by Connexin-43 Hemichannels J. Biol. Chem., March 1, 2002; 277(10): 8648 - 8657. [Abstract] [Full Text] [PDF]

				A.-C. N. Le and L. S. Musil A novel role for FGF and extracellular signal-regulated kinase in gap junction-mediated intercellular communication in the lens J. Cell Biol., July 9, 2001; 154(1): 197 - 216. [Abstract] [Full Text] [PDF]

				X. Xu, W.E.I. Li, G.Y. Huang, R. Meyer, T. Chen, Y. Luo, M.P. Thomas, G.L. Radice, and C.W. Lo Modulation of mouse neural crest cell motility by N-cadherin and connexin 43 gap junctions J. Cell Biol., July 9, 2001; 154(1): 217 - 230. [Abstract] [Full Text] [PDF]

				H. Gu, J. F. Ek-Vitorin, S. M. Taffet, and M. Delmar Coexpression of Connexins 40 and 43 Enhances the pH Sensitivity of Gap Junctions : A Model for Synergistic Interactions Among Connexins Circ. Res., May 26, 2000; 86 (10): e98 - e103. [Abstract] [Full Text] [PDF]

				B. W. Doble, P. Ping, and E. Kardami The {epsilon} Subtype of Protein Kinase C Is Required for Cardiomyocyte Connexin-43 Phosphorylation Circ. Res., February 18, 2000; 86(3): 293 - 301. [Abstract] [Full Text] [PDF]

				Y. Omori and H. Yamasaki Gap junction proteins connexin32 and connexin43 partially acquire growth-suppressive function in HeLa cells by deletion of their C-terminal tails Carcinogenesis, October 1, 1999; 20(10): 1913 - 1918. [Abstract] [Full Text] [PDF]

				K. Stergiopoulos, J. L. Alvarado, M. Mastroianni, J. F. Ek-Vitorin, S. M. Taffet, and M. Delmar Hetero-Domain Interactions as a Mechanism for the Regulation of Connexin Channels Circ. Res., May 28, 1999; 84(10): 1144 - 1155. [Abstract] [Full Text] [PDF]

				L. Zhou, E. M. Kasperek, and B. J. Nicholson Dissection of the Molecular Basis of pp60v-src Induced Gating of Connexin 43 Gap Junction Channels J. Cell Biol., March 8, 1999; 144(5): 1033 - 1045. [Abstract] [Full Text] [PDF]

				A. P. Moreno, M. Chanson, J. Anumonwo, I. Scerri, H. Gu, S. M. Taffet, and M. Delmar Role of the Carboxyl Terminal of Connexin43 in Transjunctional Fast Voltage Gating Circ. Res., March 8, 2002; 90(4): 450 - 457. [Abstract] [Full Text] [PDF]

				J. M. B. Anumonwo, S. M. Taffet, H. Gu, M. Chanson, A. P. Moreno, and M. Delmar The Carboxyl Terminal Domain Regulates the Unitary Conductance and Voltage Dependence of Connexin40 Gap Junction Channels Circ. Res., April 13, 2001; 88(7): 666 - 673. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Homma, N. Articles by Delmar, M. Search for Related Content PubMed PubMed Citation Articles by Homma, N. Articles by Delmar, M.