Targeting of PCNA to sites of DNA replication in the mammalian cell nucleus.

We have examined the targeting of proliferating cell nuclear antigen (PCNA), an integral component of the mammalian replicative enzyme DNA polymerase delta, with sites of DNA replication by using confocal microscopy and computer image analysis. Labeling (5 min pulse) of DNA replication sites in normal human diploid fibroblast cells (NHF1) with BrdU was followed by immunostaining with PCNA antibodies. A striking degree of colocalization was seen between PCNA and the characteristic patterns of DNA replication sites of early, middle and late S-phase (Nakayasu and Berezney [1989] J. Cell. Biol. 108:1-11). These observations were confirmed by quantitative computer image analysis which revealed that approximately 90% of the PCNA-stained area overlapped with DNA replication sites in early S-phase. Pulse-chase experiments, involving in vivo labeling for replication followed by PCNA staining at later time points, suggested that PCNA disassembles from previously replicated sites and targets to newly active sites of DNA replication. To further study this phenomenon in living cells, stable GFP-PCNA transfectants under the control of a tetracycline-inducible promoter were created in mouse 3T6 cells. Like the endogenous PCNA, GFP-PCNA targeted to sites of replication (approximately 80% colocalization) and demonstrated similar dynamic changes following pulse-chase experiments in fixed cells. Studies of living cells revealed progressive changes in the GFP-PCNA distribution that mimic the replication patterns observed in fixed cells. We conclude that GFP-PCNA targets to DNA replication sites in living cells and is an effective marker for tracking the spatio-temporal dynamics of DNA replication as cells transverse the S-phase.

Identification of a peptide toxin from Grammostola spatulata spider venom that blocks cation-selective stretch-activated channels.

We have identified a 35 amino acid peptide toxin of the inhibitor cysteine knot family that blocks cationic stretch-activated ion channels. The toxin, denoted GsMTx-4, was isolated from the venom of the spider Grammostola spatulata and has <50% homology to other neuroactive peptides. It was isolated by fractionating whole venom using reverse phase HPLC, and then assaying fractions on stretch-activated channels (SACs) in outside-out patches from adult rat astrocytes. Although the channel gating kinetics were different between cell-attached and outside-out patches, the properties associated with the channel pore, such as selectivity for alkali cations, conductance ( approximately 45 pS at -100 mV) and a mild rectification were unaffected by outside-out formation. GsMTx-4 produced a complete block of SACs in outside-out patches and appeared specific since it had no effect on whole-cell voltage-sensitive currents. The equilibrium dissociation constant of approximately 630 nM was calculated from the ratio of association and dissociation rate constants. In hypotonically swollen astrocytes, GsMTx-4 produces approximately 40% reduction in swelling-activated whole-cell current. Similarly, in isolated ventricular cells from a rabbit dilated cardiomyopathy model, GsMTx-4 produced a near complete block of the volume-sensitive cation-selective current, but did not affect the anion current. In the myopathic heart cells, where the swell-induced current is tonically active, GsMTx-4 also reduced the cell size. This is the first report of a peptide toxin that specifically blocks stretch-activated currents. The toxin affect on swelling-activated whole-cell currents implicates SACs in volume regulation.

Different ionic selectivities for connexins 26 and 32 produce rectifying gap junction channels.

The functional diversity of gap junction intercellular channels arising from the large number of connexin isoforms is significantly increased by heterotypic interactions between members of this family. This is particularly evident in the rectifying behavior of Cx26/Cx32 heterotypic channels (. Proc. Natl. Acad. Sci. USA. 88:8410-8414). The channel properties responsible for producing the rectifying current observed for Cx26/Cx32 heterotypic gap junction channels were determined in transfected mouse neuroblastoma 2A (N2A) cells. Transfectants revealed maximum unitary conductances (gamma(j)) of 135 pS for Cx26 and 53 pS for Cx32 homotypic channels in 120 mM KCl. Anionic substitution of glutamate for Cl indicated that Cx26 channels favored cations by 2.6:1, whereas Cx32 channels were relatively nonselective with respect to charge. In Cx26/Cx32 heterotypic cell pairs, the macroscopic fast rectification of the current-voltage relationship was fully explained at the single-channel level by a rectifying gamma(j) that increased by a factor of 2.9 as the transjunctional voltage (V(j)) changed from -100 to +100 mV with the Cx26 cell as the positive pole. A model of electrodiffusion of ions through the gap junction pore based on Nernst-Planck equations for ion concentrations and the Poisson equation for the electrical potential within the junction is developed. Selectivity characteristics are ascribed to each hemichannel based on either pore features (treated as uniform along the length of the hemichannel) or entrance effects unique to each connexin. Both analytical GHK approximations and full numerical solutions predict rectifying characteristics for Cx32/Cx26 heterotypic channels, although not to the full extent seen empirically. The model predicts that asymmetries in the conductance/permeability properties of the hemichannels (also cast as Donnan potentials) will produce either an accumulation or a depletion of ions within the channel, depending on voltage polarity, that will result in rectification.

Identification of a proline residue as a transduction element involved in voltage gating of gap junctions.

Gap junction channels are structurally distinct from other ion channels in that they comprise two hemichannels which interact head-to-head to form an aqueous channel between cells. Intercellular voltage differences together with increased intracellular concentrations of H+ and Ca2+ cause closure of these normally patent channels. The relative sensitivity to voltage varies with the subunit (connexin) composition of the channels. The third of four transmembrane-spanning regions (M3) in connexins has been proposed to form the channel lining, and a global 'tilting' of the hemichannel subunits has been correlated with channel closure. But specific components involved in transduction of channel gating events have not been identified in either gap junctions or other ion channel classes (however, see model in ref. 5). We have examined a strictly conserved proline centrally located in M2 of connexin proteins. Mutation of this proline (Pro 87) in connexin 26 causes a reversal in the voltage-gating response when the mutant hemichannel is paired with wild-type connexin 26 in the Xenopus oocyte system. This suggests that the unique properties associated with this residue are critical to the transduction of voltage gating in these channels.

Molecular cloning and functional expression of mouse connexin40, a second gap junction gene preferentially expressed in lung.

From a mouse genomic library, a clone has been isolated that codes for a connexin-homologous sequence of 358 amino acids. Because of its theoretical molecular mass of 40.418 kD it is named connexin40 (Cx40). Based on both protein and nucleotide sequence, mouse Cx40 is more closely related to mouse Cx43 (alpha subgroup of connexins) than to mouse Cx32 (beta subgroup). The highest overall homology detected, however, was to chick Cx42 (67% amino acid and 86% nucleotide identity), raising the possibility that Cx40 may be the mouse analogue. The coding region of Cx40 is uninterrupted by introns and is detected as a single copy gene in the mouse genome. High stringency hybridization of Northern blots with the coding sequence of Cx40 identified a single transcript of 3.5 kb that is at least 16-fold more abundant in lung-similar to mouse Cx37-than in other adult tissues (kidney, heart, and skin). In embryonic kidney, skin, and liver the level of the Cx40 transcript is two- to fourfold higher than in the corresponding adult tissues. Microinjection of Cx40 cRNA into Xenopus oocytes induced functional cell-to-cell channels between pairs. These channels show a symmetrical and markedly cooperative closure in response to transjunctional voltage (Boltzmann parameters of Vo = +/- 35 mV; A = 0.32) which is also fast relative to other connexin channels recorded similarly (tau = 580 ms at Vj of +/- 50 mV). Although Cx40-expressing oocytes did not couple efficiently with oocytes expressing endogenous connexins, they did couple well to Cx37-expressing oocytes. The heterotypic channels which formed had voltage-gating properties modified from those of the original homotypic forms. Transfection of mouse Cx40 DNA, under control of the SV-40 early promoter, into coupling-deficient human HeLa or SK-Hep-1 cells resulted in expression of the expected transcript and restoration of fluorescent dye transfer in transfected clones.

Gap junctions formed by connexins 26 and 32 alone and in combination are differently affected by applied voltage.

Gap junctions are formed by a family of homologous proteins termed connexins. Their channels are dodecamers, and homomeric forms differ in their properties with respect to control by voltage and other gating stimuli. We report here the properties of coupling from expression of connexin complementary RNAs (cRNAs; sense to mRNA, antisense to cDNA) in Xenopus oocyte pairs in which endogenous coupling was blocked by injection of DNA oligonucleotides antisense to the mRNA of Cx38, the principal endogenous connexin. We found that a connexin recently sequenced from rat liver, Cx26, formed functional gap junctions whose conductance exhibited voltage dependence with unusual characteristics suggestive of two gating mechanisms. Junctional conductance (gj) was increased to a small degree by depolarization and decreased by hyperpolarization of either cell in a coupled pair, indicating dependence on the potential between the inside and outside of the cells (Vi-o). These changes were fast compared with the resolution of their measurement (ca. 10 ms). On a slower timescale, large transjunctional potentials (Vj) of either sign caused a more substantial decrease in conductance similar to that previously reported for several other gap junctions. Homotypic junctions formed of another connexin, Cx32, exhibited a similar slow dependence on Vj but no dependence on Vi-o. In contrast, heterotypic junctions between an oocyte expressing Cx26 and one expressing Cx32 were electrically asymmetric; they exhibited a greater fast change in gj, which depended, however, on Vj, such that gj increased with relative positivity on the Cx26 side and decreased with relative negativity on the Cx26 side. There was also a large slow decrease in gj in response to Vj for relative positivity on the Cx26 side but not for Vj of the opposite sign. These data indicate that properties of the hemichannels contributed by the two connexins in the heterotypic case were changed from their properties in homotypic junctions. The fast change in gj may involve a mechanism analogous to that at fast rectifying electrical synapses. Experiments in which oocytes expressing Cx32 were paired with oocytes expressing both Cx26 and Cx32 demonstrated that asymmetric junctions would form between oocytes expressing both connexins, thereby confirming their potential relevance in vivo, where the same coupled cells are known to express both proteins.

Mouse connexin37: cloning and functional expression of a gap junction gene highly expressed in lung.

The coding sequence (333 amino acids) of a new connexin protein, designated mouse connexin37 (Cx37 or Cx37.6) due to the deduced theoretical molecular mass of 37.600 kD, has been determined from cDNA and genomic clones. As seen in other connexins, its gene has no introns within the coding region and the deduced amino acid sequence is predicted to have similar topology to other connexins that form intercellular channels. The amino acid sequence of mouse Cx37 is most similar to rat connexin43 (59% identity) and Xenopus connexin38 (66% identity) when compared from the NH2 terminus to the end of the fourth putative transmembrane region. When expressed in Xenopus oocytes Cx37 forms functional intercellular channels that exhibit more sensitive and rapid gating in response to voltage than any previously characterized vertebrate gap junction. Under stringent conditions the Cx37 cDNA hybridizes to an mRNA of 1.7 kb that is found highly abundant in lung and to progressively lesser extents in brain, kidney, skin, spleen, liver, intestine, and heart. Embryonic brain, kidney, and skin express two to fivefold higher levels of the Cx37 transcript than the corresponding adult tissues. Cx37 transcripts were also found to increase two to threefold in response to retinoic acid treatment of cultured embryonic carcinoma F9 cells.