Molecular cloning and functional expression of the mouse gap junction gene connexin-57 in human HeLa cells.

A new mouse connexin gene has been isolated that codes for a connexin protein of 505 amino acid residues. Based on the predicted molecular mass of 57.115 kDa, it has been designated connexin-57. Similar to most other mouse connexin genes, the coding region of connexin-57 is not interrupted by introns and exists in the mouse genome as a single-copy gene. Within the connexin family, this new gene shows highest sequence identity to porcine connexin-60 in the alpha group of connexins. The connexin-57 gene was mapped to a position on mouse chromosome 4, 30 centimorgans proximal to a cluster of previously mapped connexin genes. Low levels of connexin-57 mRNA were detected in skin, heart, kidney, testis, ovary, intestine, and in the mouse embryo after 8 days post coitum, but expression was not detected in brain, sciatic nerve or liver. In order to analyze gene function, the connexin-57 coding region was expressed by transfection in human HeLa cells, where it restored homotypic intercellular transfer of microinjected neurobiotin. Heterotypic transfer was observed between HeLa connexin-57 transfectants and HeLa cells, expressing murine connexin-43, -37, or -30.3. Double whole-cell voltage clamp analyses revealed that HeLa-connexin-57 transfectants expressed about 10 times more channels than parental HeLa cells. Voltage gating by transjunctional and transmembrane voltages as well as unitary conductance ( approximately 27 picosiemens) were different from intrinsic connexin channels in parental HeLa cells.     

Temperature dependence of embryonic cardiac gap junction conductance and channel kinetics

We have investigated the effects of temperature on the conductance and voltage-dependent kinetics of cardiac gap junction channels between pairs of seven-day embryonic chick ventricle myocytes over the range of 14–26°C. Records of junctional conductance (G _j ) and steady-state unit junctional channel activity were made using the whole-cell double patch-clamp technique while the bath temperature was steadily changed at a rate of about 4°C/min. The decrease inG _j upon cooling was biphasic with a distinct break at 21°C. In 12 cell pairs,Q ₁₀ was 2.2 from 26 to 21°C, while between 21 and 14°C it was 6.5. The meanG _j at 22°C (G _j22 ) was 3.0±2.1 nS, ranging in different preparations from 0.24 to 6.4 nS. At room temperature, embryonic cardiac gap junctions contain channels with conductance states near 240, 200, 160, 120, 80 and 40 pS. In the present study, we demonstrate that cooling decreases the frequency of channel openings at all conductance levels, and at temperatures below 20°C shifts the prevalence of openings from higher to lower conductance states: all 240 pS openings disappear below 20°C; 200 pS openings are suppressed at 17°C; below 16°C 160 and 120 pS events disappear and only 80 and 40 pS states are seen. Temperature also affected the voltage-dependent kinetics of the channels. Application of a 6 sec, 80 mV voltage step across the junction (V _j80 ) caused a biexponential decay in junctional conductance. Decay was faster at lower temperatures, whereas the rate of recovery ofG _j after returning toV _j0 was slowed. Cooling reduced the fast decay time constant, increased both recovery time constants, and decreased the magnitude of GitG_j decay, thus leaving a 10–16% larger residual conductance (G _ss/G _init,±80 mVV _j ) at 18 than at 22°C. From these results we propose that embryonic chick cardiac gap junctions contain at least two classes of channels with different conductances and temperature sensitivities.     

Molecular and functional characterization of Hv1 proton channel in human granulocytes

Voltage-gated proton current (I(Hv)) has been characterized in several cell types, but the majority of the data was collected in phagocytes, especially in human granulocytes. The prevailing view about the role of I(Hv) in phagocytes is that it is an essential supporter of the intense and sustained activity of Nox2 (the core enzyme of the phagocyte NADPH oxidase complex) during respiratory burst. Recently H(v)1, a voltage-gated proton channel, was cloned, and leukocytes from H(v)1 knockout mice display impaired respiratory burst. On the other hand, hardly anything is known about H(v)1 in human granulocytes. Using qPCR and a self made antibody, we detected a significant amount of H(v)1 in human eosinophil and neutrophil granulocytes and in PLB-985 leukemia cells. Using different crosslinking agents and detergents in reducing and non-reducing PAGE, significant expression of H(v)1 homodimers, but not that of higher-order multimers, could be detected in granulocytes. Results of subcellular fractionation and confocal imaging indicate that H(v)1 is resident in both plasmalemmal and granular membrane compartments of resting neutrophils. Furthermore, it is also demonstrated that H(v)1 accumulates in phagosome wall during zymosan engulfment together with, but independently of Nox2. During granulocytic differentiation early and parallel upregulation of H(v)1 and Nox2 expression was observed in PLB-985 cells. The upregulation of H(v)1 or Nox2 expression did not require the normal expression of the other molecule. Using RNA interference, we obtained strong correlation between H(v)1 expression and I(Hv) density in PLB-985 cells. It is also demonstrated that a massive reduction in H(v)1 expression can limit the Nox2 mediated superoxide production of PLB-985 granulocytes. In summary, beside monomers native H(v)1 forms stable proton channel dimer in resting and activated human granulocytes. The expression pattern of H(v)1 in granulocytes is optimized to support intense NADPH oxidase activity.     

Regulation of cardiac gap junction channel permeability and conductance by several phosphorylating conditions

Short term (15 min) effects of activators of protein kinase A (PKA), PKC and PKG on cardiac macroscopic (g_j) and single channel (_j) gap junctional conductances were studied in pairs of neonatal rat cardiomyocytes. Under dual whole-cell voltage-clamp, PKC activation by 100 nM TPA increased g_j by 16 ± 2% (mean ± S.E.M, n=9), 1.5 mM of the PKG activator 8-bromo-cGMP (8Br-cGMP) decreased g_j by 26 ± 2% (n=4), whereas 1.5 mM of the PKA activator 8Br-cAMP did not affect g_j (1 ± 5%, n=11). Single cardiac gap junction channel events, resolved in the presence of heptanol, indicated two _j sizes of 20 pS and 40–45 pS. Under control conditions, the larger events were most frequently observed. Whereas 8Br-cAMP did not change this distribution, TPA or 8Br-cGMP shifted the _j distribution to the lower sizes. Diffusion of 6-carboxyfluorescein (6-CF), a gap junction permeant tracer, from the injected cell to neighboring cells was studied on small clusters of neonatal rat cardiomyocytes. Under control conditions, 6-CF labeled 8.4 ± 0.4 cells (mean ± S.E.M, n=31). Whereas 8Br-cAMP did not change the extent of dye transfer (8.1 ± 0.5 cells, n=10), TPA restricted the diffusion of 6-CF to 2.2 ± 0.2 cells (n=30) and 8Br-cGMP to 3.5 ± 0.3 cells (n=10). This suggests that permeability and single channel conductance of Cx43 gap junction channels are parallel related. Altogether, these results point to the differential modulation of electrical and metabolic coupling of cardiac cells by various phosphorylating conditions.     

Developmental expression and molecular characterization of two gap junction channel proteins expressed during embryogenesis in the grasshopper Schistocerca americana

Gap junctions are membrane channels that directly connect the cytoplasm of neighboring cells, allowing the exchange of ions and small molecules. Two analogous families of proteins, the connexins and innexins, are the channel-forming molecules in vertebrates and invertebrates, respectively. In order to study the role of gap junctions in the embryonic development of the nervous system, we searched for innexins in the grasshopper Schistocerca americana. Here we present the molecular cloning and sequence analysis of two novel innexins, G-Inx(1) and G-Inx(2), expressed during grasshopper embryonic development. The analysis of G-Inx(1) and G-Inx(2) proteins suggests they bear four transmembrane domains, which show strong conservation in members of the innexin family. The study of the phylogenetic relationships between members of the innexin family and the new grasshopper proteins suggests that G-Inx(1) is orthologous to the Drosophila 1(1)-ogre. However, G-Inx(2) seems to be a member of a new group of insect innexins. We used in situ hybridization with the G-Inx(1) and G-Inx(2) cDNA clones, and two polyclonal sera raised against different regions of G-Inx(1) to study the mRNA and protein expression patterns and the subcellular localization of the grasshopper innexins. G-Inx(1) is primarily expressed in the embryonic nervous system, in neural precursors and glial cells. In addition, a restricted stripe of epithelial cells in the developing limb, involved in the guidance of sensory growth cones, expresses G-Inx(1). G-Inx(2) expression is more widespread in the grasshopper embryo, but a restricted expression is found in a subset of neural precursors. The generally different but partially overlapping expression patterns of G-Inx(1) and G-Inx(2) supports the combinatorial character of gap junction formation in invertebrates, an essential property to generate specificity in this form of cell-cell communication.     

Reconstitution of cardiac gap junction channeling activity into liposomes: a functional assay for gap junctions

Cardiac gap junctions were reconstituted into liposomes. To determine if reconstitution resulted in membrane channel formation, we developed an assay for channel function that used a liposome-entrapped peroxidase to detect entry of a substrate into the liposome. The data demonstrate, for the first time, that reconstituted gap junctions from heart are capable of channel-forming activity in artificial membranes.     

Molecular cloning and developmental expression of two chick embryo gap junction proteins

The connexins are a family of related gap junction proteins which contain conserved transmembrane and extracellular domains but unique cytoplasmic regions. To identify connexins with potential roles in development, a chick embryo cDNA library was screened by hybridization at low stringency with a cDNA for rat connexin-43. cDNA clones for two previously undescribed connexins were isolated. Chick connexin-45 has a predicted molecular mass of 45,376 daltons; connexin-42 has a predicted molecular mass of 41,748 daltons. Both of these predicted connexin proteins share the homologous regions noted in other members of this family, and each has its own unique regions. Southern blots of chicken genomic DNA suggest that each connexin is encoded by a distinct single copy gene. RNA blots demonstrate that while chick connexin-43, -42, and -45 are each expressed in a number of chick organs, they each have a unique tissue distribution. Each connexin mRNA is present in heart. Blots of total RNA isolated from hearts of chick embryos of different ages demonstrate that the abundance of connexin-42 and -43 mRNAs varies no more than 2-fold between the embryo and the adult. However, connexin-45 mRNA shows a dramatic change, falling 10-fold from the 6-day embryonic heart to the adult. These multiple connexins are likely to have different physiological properties and may account for the multiple physiologically distinct gap junction channels which have been observed in cardiac myocytes. They may provide a mechanism for the formation of communication compartments in the developing myocardium.     

Molecular and functional characterization of the melastatin-related cation channel TRPM3

Proteins of the mammalian TRP (transient receptor potential) family form a heterogenous group of cation channels important for cellular Ca2+ signaling and homeostasis. Here we present the full-length sequence of TRPM3, a member of the melastatin-like subfamily (TRPM) of TRP channels. TRPM3 expression was found in human kidney and brain. HEK293 cells transiently transfected with TRPM3 showed a constitutive Ca2+ and Mn2+ entry. Whole-cell patch clamp experiments confirmed the spontaneous activity of TRPM3 and revealed permeability ratios PCa/PNa of 1.57 and PNa/PCs of 0.75. In cell-attached patches, spontaneous inward and outward currents were observed. At negative membrane potentials and in the presence of either 140 mm Cs+, 140 mm Na+, or 100 mm Ca2+ in the pipette solution, the single channel conductance levels were 133, 83, and 65 pS, respectively. The Ca2+ entry in TRPM3-expressing HEK293 cells increased during treatment with hypotonic extracellular solution. The reduction of extracellular osmolarity was accompanied by cell swelling, suggesting volume-regulated activity of TRPM3. From its function and expression in human kidney, we propose a role of TRPM3 in renal Ca2+ homeostasis.     

Two gap junction channel (innexin) genes of the Bombyx mori and their expression

Gap junctions are clusters of intercellular channels that are associated with embryonic development and neural signaling. Innexins, invertebrate gap junction proteins, have been identified in Drosophila and Caenorhabditis. Here, we report the isolation and characterization of two novel members of the insect innexin family, Bm inx2 and Bm inx4, from embryos of the silkworm, Bombyx mori, during the germ-band formation stage. Bm inx2 is a single copy gene with one exon, while Bm inx4 is a single copy gene with four exons and three introns. The predicted proteins show structural similarities with other innexin family members, including four transmembrane (TM) domains, two extracellular loops (ELs), one cytoplasmic loop (CL), and typical conserved amino acids. Bm inx2 is phylogenetically orthologous to the other insect inx2 genes, but Bm inx4 is not orthologous to any known innexin including Dm inx4. Interestingly, Northern blotting and in situ hybridization showed that Bm inx2 was variously expressed across all developmental stages and in various tissues, with high expression seen in the nervous system at the time of embryogenesis. In contrast, Bm inx4 was transiently expressed at the germ-band formation stage of embryogenesis, and was specifically expressed in the ovary and testis during the larval and pupal stages. The isolation and characterization of these novel genes should form the basis for further study of the functional events that occur during development and neuronal communication in B. mori.     

Identification and functional expression of HCx31.9, a novel gap junction gene

By combining in silico and bench molecular biology methods we have identified a novel human gap junction gene that encodes a protein designated HCx31.9. We have determined its human chromosomal location and gene structure, and we have identified a putative mouse ortholog, mCx30.2. We have observed the presence of HCx31.9 in human cerebral cortex, liver, heart, spleen, lung, and kidney and the presence of mCx30.2 in mouse cerebral cortex, liver and lung. Moreover, preliminary data on the electrophysiological properties of HCx31.9 have been obtained by functional expression in paired Xenopus oocytes and in transfected N2A cells.     

Molecular cloning and functional expression of a potassium channel cDNA isolated from a rat cardiac library

A full-length K+ channel cDNA (RHK1) was isolated from a rat cardiac library using the polymerase chain reaction (PCR) method and degenerate oligonucleotide primers derived from K+ channel sequences conserved between Drosophila Shaker H4 and mouse brain MBK1. Although RHK1 was isolated from heart, its expression was found in both heart and brain. The RHK1-encoded protein, when expressed in Xenopus oocytes, gated a 4-aminopyridine (4-AP)-sensitive transient outward current. This current is similar to the transient outward current measured in rat ventricular myocytes with respect to voltage-dependence of activation and inactivation, time course of activation and inactivation, and pharmacology.     

Magnesium gating of cardiac gap junction channels

We aimed to study kinetics of modulation by intracellular Mg²⁺ of cardiac gap junction (Mg²⁺ gate). Paired myocytes of guinea-pig ventricle were superfused with solutions containing various concentrations of Mg²⁺. In order to rapidly apply Mg²⁺ to one aspect of the gap junction, the non-junctional membrane of one of the pair was perforated at nearly the connecting site by pulses of nitrogen laser beam. The gap junction conductance (G_j) was measured by clamping the membrane potential of the other cell using two-electrode voltage clamp method. The laser perforation immediately increased G_j, followed by slow G_j change with time constant of 3.5 s at 10 mM Mg²⁺. Mg²⁺ more than 1.0 mM attenuated dose-dependently the gap junction conductance and lower Mg²⁺ (0.6 mM) increased G_j with a Hill coefficient of 3.4 and a half-maximum effective concentration of 0.6 mM. The time course of G_j changes was fitted by single exponential function, and the relationship between the reciprocal of time constant and Mg²⁺ concentration was almost linear. Based on the experimental data, a mathematical model of Mg²⁺ gate with one open state and three closed states well reproduced experimental results. One-dimensional cable model of thirty ventricular myocytes connected to the Mg²⁺ gate model suggested a pivotal role of the Mg²⁺ gate of gap junction under pathological conditions.