Faculty Bibliography

pH-induced closure of connexin43 (Cx43) channels involves interaction of the Cx43 carboxyl-terminal (Cx43CT) with a separate 'receptor' domain. The receptor location and structure and whether the interaction is directly intramolecular are unknown. Here we show resonant mirror technology, enzyme-linked sorbent assays, and nuclear magnetic resonance (NMR) experiments demonstrating pH-dependent binding of Cx43CT to region 119-144 of Cx43 (Cx43L2), which we propose is the receptor. NMR showed that acidification induced alpha-helical order in Cx43L2, whereas only a minor modification in Cx43CT structure was detected. These data provide the first demonstration of chemically induced structural order and binding between cytoplasmic connexin domains

Gap junctions are the morphological correlates of direct cell-cell communication and are formed of hexameric assemblies of gap junction proteins (connexins) into hemichannels (or connexons) provided by each coupled cell. Gap junction channels formed by each of the connexin subtypes (of which there are as many as 20) display different properties, which have been attributed to differences in amino acid sequences of gating domains of the connexins. Recent studies additionally indicate that connexin proteins interact with other cellular components to form a protein complex termed the Nexus. This review summarizes current knowledge regarding the protein-protein interactions involving of connexin proteins and proposes hypothesized functions for these interactions

Previous studies show that chemical regulation of connexin43 (Cx43) gap junction channels depends on the integrity of the carboxyl terminal (CT) domain. Experiments using Xenopus oocytes show that truncation of the CT domain alters the time course for current inactivation; however, correlation with the behavior of single Cx43 channels has been lacking. Furthermore, whereas chemical gating is associated with a 'ball-and-chain' mechanism, there is no evidence whether transjunctional voltage regulation for Cx43 follows a similar model. We provide data on the properties of transjunctional currents from voltage-clamped pairs of mammalian tumor cells expressing either wild-type Cx43 or a mutant of Cx43 lacking the carboxyl terminal domain (Cx43M257). Cx43 transjunctional currents showed bi-exponential decay and a residual steady-state conductance of approximately 35% maximum. Transjunctional currents recorded from Cx43M257 channels displayed a single, slower exponential decay. Long transjunctional voltage pulses caused virtual disappearance of the residual current at steady state. Single channel data revealed disappearance of the residual state, increase in the mean open time, and slowing of the transition times between open and closed states. Coexpression of CxM257 with Cx43CT in a separate fragment restored the lower conductance state. We propose that Cx43CT is an effector of fast voltage gating. Truncation of Cx43CT limits channel transitions to those occurring across the higher energy barrier that separates open and closed states. We further propose that a ball-and-chain interaction provides the fast component of voltage-dependent gating between CT domain and a receptor affiliated with the pore

Connexin43 (Cx43) is the principal connexin isoform in the mouse ventricle, where it is thought to provide electrical coupling between cells. Knocking out this gene results in anatomic malformations that nevertheless allow for survival through early neonatal life. We examined electrical wave propagation in the left (LV) and right (RV) ventricles of isolated Cx43 null mutated (Cx43(-/-)), heterozygous (Cx43(+/)(-)), and wild-type (WT) embryos using high-resolution mapping of voltage-sensitive dye fluorescence. Consistent with the compensating presence of the other connexins, no reduction in propagation velocity was seen in Cx43(-/-) ventricles at postcoital day (dpc) 12.5 compared with WT or Cx43(+/)(-) ventricles. A gross reduction in conduction velocity was seen in the RV at 15.5 dpc (in cm/second, mean [1 SE confidence interval], WT 9.9 [8.7 to 11.2], Cx43(+/)(-) 9.9 [9.0 to 10.9], and Cx43(-/-) 2.2 [1.8 to 2.7; P<0.005]) and in both ventricles at 17.5 dpc (in RV, WT 8.4 [7.6 to 9.3], Cx43(+/)(-) 8.7 [8.1 to 9.3], and Cx43(-/-) 1.1 [0.1 to 1.3; P<0.005]; in LV, WT 10.1 [9.4 to 10.7], Cx43(+/)(-) 8.3 [7.8 to 8.9], and Cx43(-/-) 1.7 [1.3 to 2.1; P<0.005]) corresponding with the downregulation of Cx40. Cx40 and Cx45 mRNAs were detectable in ventricular homogenates even at 17.5 dpc, probably accounting for the residual conduction function. Neonatal knockout hearts were arrhythmic in vivo as well as ex vivo. This study demonstrates the contribution of Cx43 to the electrical function of the developing mouse heart and the essential role of this gene in maintaining heart rhythm in postnatal life

Chemical regulation of connexin (Cx) 40 and Cx43 follows a ball-and-chain model, in which the carboxyl terminal (CT) domain acts as a gating particle that binds to a receptor affiliated with the pore. Moreover, Cx40 channels can be closed by a heterodomain interaction with the CT domain of Cx43 and vice versa. Here, we report similar interactions in the establishment of the unitary conductance and voltage-dependent profile of Cx40 in N2A cells. Two mean unitary conductance values ('lower conductance' and 'main') were detected in wild-type Cx40. Truncation of the CT domain at amino acid 248 (Cx40tr248) caused the disappearance of the lower-conductance state. Coexpression of Cx40tr248 with the CT fragment of either Cx40 (homodomain interactions) or Cx43 (heterodomain interactions) rescued the unitary conductance profile of Cx40. In the N2A cells, the time course of macroscopic junctional current relaxation was best described by a biexponential function in the wild-type Cx40 channels, but it was reduced to a single-exponential function after truncation. However, macroscopic junctional currents recorded in the oocyte expression system were not significantly different between the wild-type and mutant channels. Concatenation of the CT domain of Cx43 to amino acids 1 to 248 of Cx40 yielded a chimeric channel with unitary conductance and voltage-gating profile indistinguishable from that of wild-type Cx40. We conclude that residence of Cx40 channels in the lower-conductance state involves a ball-and-chain type of interaction between the CT domain and the pore-forming region. This interaction can be either homologous (Cx40 truncation with Cx40CT) or heterologous (with the Cx43CT)

Surface plasmon resonance (SPR) allows examination of protein-protein interactions in real time, from which both binding affinities and kinetics can be directly determined. We have used the SPR technique to search for proteins in heart tissue that would be candidate binding partners for the cardiac gap junction protein, connexin43 (Cx43). Heart lysate showed a strong, pH-dependent binding to the carboxyl terminus (CT) of Cx43 (amino acids 254-382) covalently linked to an SPR cuvette. Binding was inhibited by the presence of v-src transfected 3T3 cell lysate, suggesting that binding partners in these two lysates may compete for overlapping epitopes on Cx43CT. The combined application of proteomic and functional studies is expected to identify which proteins within heart tissue interact with Cx43 and what roles they may play in gap junction function

Gap junctions are formed by oligomerization of a protein called connexin. Most cells express more than one connexin isotype. Atrial myocytes, for example, coexpress connexin (Cx) 40 and Cx43. The consequence of connexin coexpression on the regulation of gap junctions is not well understood. In the present study, we show that cells coexpressing Cx40 and Cx43 are more susceptible to acidification-induced uncoupling than those cells expressing only one connexin isotype. Xenopus oocytes were injected with mRNA for Cx40, Cx43, or a combination of both. Intracellular pH and junctional conductance were simultaneously measured while cells were progressively acidified by superfusion with a bicarbonate-buffered solution gassed with increasing concentrations of carbon dioxide. The data show that the pKa (ie, the pH at which junctional conductance decreased to 50% from maximum) shifted from approximately 6.7 when cells expressed only Cx40 or only Cx43 to approximately 7.0 when one of the oocytes was coexpressing both connexins. Truncation of the carboxyl terminal domains of the connexins caused the loss of pH sensitivity even after coexpression. The data are interpreted on the basis of previous studies from our laboratory that demonstrated heterodomain interactions in the regulation of Cx40 and Cx43 gap junctions. The possible implications of these findings on the regulation of native gap junctions that express both connexins remain to be determined. The full text of this article is available at http://www.circresaha.org. Web Site Feature The full-length article can be found on the World Wide Web at http://www.circresaha.org Key Words: connexin gap junctions pH(i)

Gap junctions are formed by oligomerization of a protein called connexin. Most cells express more than one connexin isotype. Atrial myocytes, for example, coexpress connexin (Cx) 40 and Cx43. The consequence of connexin coexpression on the regulation of gap junctions is not well understood. In the present study, we show that cells coexpressing Cx40 and Cx43 are more susceptible to acidification-induced uncoupling than those cells expressing only one connexin isotype. Xenopus oocytes were injected with mRNA for Cx40, Cx43, or a combination of both. Intracellular pH and junctional conductance were simultaneously measured while cells were progressively acidified by superfusion with a bicarbonate-buffered solution gassed with increasing concentrations of carbon dioxide. The data show that the pKa (ie, the pH at which junctional conductance decreased to 50% from maximum) shifted from approximately 6.7 when cells expressed only Cx40 or only Cx43 to approximately 7.0 when one of the oocytes was coexpressing both connexins. Truncation of the carboxyl terminal domains of the connexins caused the loss of pH sensitivity even after coexpression. The data are interpreted on the basis of previous studies from our laboratory that demonstrated heterodomain interactions in the regulation of Cx40 and Cx43 gap junctions. The possible implications of these findings on the regulation of native gap junctions that express both connexins remain to be determined