Reiner Eckert

Characterization of the gap junction protein connexin37 in murine endothelium, respiratory epithelium, and after transfection in human HeLa cells.

Affinity-purified antibodies to oligopeptides derived from two different regions of the carboxyterminus and cytoplasmic loop or to the last 103 C-terminal amino acids of mouse connexin37 (Cx37) were used to characterize expression of this gap junctional protein in endothelium of several murine tissues. Cx37 was expressed in endothelium of large blood vessels in brain, liver, kidney, spleen, heart, and lung, but not in capillaries. In addition, weak Cx37 immuno-signals were observed in lung respiratory epithelium of small bronchi and in alveolar epithelial cells of bronchioli. The ratios of Cx37 protein to Cx37 mRNA in adult and embryonic kidney as well as skin were 29-303-fold larger than in lung, suggesting that Cx37 mRNA was translated at different efficiencies in kidney and skin versus lung. Cx37 protein was more abundant in embryonic kidney and lung than in the corresponding adult tissues. After differential centrifugation of plasma membrane fractions in sucrose gradients, we found that Cx37-containing gap junctions in lung were much smaller than Cx32 and Cx26 aggregates from liver. HeLa cells were transfected with mouse Cx37 cDNA. In these cells, mouse Cx37 protein was phosphorylated mainly at serine, less at tyrosine, and very little at threonine residues. Three conductance states were resolved at 110, 240, and 315 pS.

Biophysical characterization of gap-junction channels in HeLa cells

HeLa cells seem not to be junctionally coupled when probed with techniques such as Lucifer yellow spreading and/or ionic coupling measured with three inserted microelectrodes. When investigated with double whole-cell patch-clamp measurements, HeLa cells in monolayer cultures were electrically coupled in 39% of the cases with very low transjunctional conductances (average one to five open channels). These gapjunction channels had a single-channel conductance γ= 26±6 pS and were voltage-gated with an equivalent gating charge z=3.1±1.5 for a voltage of half-maximal inactivation Uo=49±10 mV. The voltage-dependent component represents only 31±8% of the total junctional conductance. The voltage-insensitive conductance is characterized by a residual open probability po(∞)=0.34±0.12, which corresponds to a ratio Gmin/Gmax=0.50±0.12. Dissociation of monolayer cells into cell pairs yielded about 58% coupled cell pairs with no notably altered single-channel properties.

Functional rescue of defective mutant connexons by pairing with wild-type connexons

Abstract. We have compared the functional and structural integrity of gap junction channels assembled from a Cx45 truncation mutant with those of gap junction channels assembled from wild-type (wt) Cx45 and Cx43. These channel-forming proteins are constitutively expressed in HeLa cells. The truncation mutant lacks the last 26 amino acids of the COOH-terminus, including nine serine phosphorylation sites that are associated with regulatory processes of these channels. We determined the presence of gap junction plaques in these cells with the immunogold freeze fracture technique, which showed that plaque formation is similar in all the clones investigated. Junctional permeability was probed with calcein transfer and flow cytometry analyses and junctional conductance was measured in cell pairs with double whole-cell patch-clamp techniques. For homotypic pairing only the truncated mutant did not form permeable channels. However, coupling was restored for heterotypic channels (pairing wtCx45- or wtCx43- with mutant-connexons), whose junctional communication was not different from that of the homotypic channels. Our results indicate that the presence of gap junction plaques does not warrant functional coupling and that heterotypic trCx45/wtCx45 channels can be regulated by the intact wtCx45 connexons. This dominant-positive effect is also operative when wtCx43 are paired with trCx45 connexons.

Interactions of mitochondrial presequence peptides with the mitochondrial outer membrane preprotein translocase TOM.

TOM protein-conducting channels serve as the main entry sites into mitochondria for virtually all mitochondrial proteins. When incorporated into lipid bilayers, they form large, relatively nonspecific ion channels that are blocked by peptides derived from mitochondrial precursor proteins. Using single-channel electrical recordings, we analyzed the interactions of mitochondrial presequence peptides with single TOM pores. The largest conductance state of the translocon represents the likely protein-conducting conformation of the channel. The frequency (but not the duration) of the polypeptide-induced blockage is strongly modulated by the substrate concentration. Structural differences between substrates are reflected in characteristic blockage frequencies and duration of blockage. To our knowledge, this study provides first quantitative data regarding the kinetics of polypeptide interaction with the mitochondrial TOM machinery.

High-resolution measurements of gap-junctional conductance during perfusion with anti-connexin antibodies in pairs of cultured mammalian cells

Antibodies against the main proteins in hepatic gap junctions — connexin-26 and connexin-32 — have been used in conjunction with high-resolution patch-clamp techniques to investigate whether a relation exists between connexin type and conductance of single gap-junctional channels. Two different cell lines, BRL cells, derived from rat liver, and FL cells a human amniotic cell line exhibited the same single-channel conductances in double whole-cell recordings, but reacted differently upon dialysis with antibodies. Preliminary results indicate that both cell lines express mainly connexin-43. Thus, in spite of the inhibitory action of anti-cx26 and anti-cx32 antibodies observed, the data question the reliability of these antibodies for the functional characterization of gap-junction proteins in electrophysiological experiments.

Single Channel Properties of Cell-Cell Channels in Transfected and Non-Transfected Cultured Human Cell Lines

HeLa cells were considered not to be junctionally coupled when probed with traditional techniques such as Lucifer yellow spreading. However, with double whole-cell recording in monolayer cultures and isolated cell pairs we found about 40%–60% of the measured cells are electrically coupled, though only few channels were active with a single channel conductance γ ≈ 26 pS. The channels were voltage gated with Boltzmann parameters estimated from current recordings of voltage ramps as U 0 ≈ 49 mV and z ≈ 3.1. When transfected with genes for different connexins dye coupling could be restored and the cells had cell-to-cell conductances ranging from 10 to 100 nS when measured in monolayers. The spreading of Lucifer yellow, however, is not directly related to the total electrical conductance of cells with different connexin types. Single channel properties were different for each connexin type. Channels from mouse Cx40 were characterized by γ ≈ 121 pS and 153 pS, U 0 ≈ 44 mV, and z ≈ 4.0 whereas for rat Cx43 channels γ ≈ 40 pS and 60 pS, U 0 ≈ 73 mV, and z ≈ 3.7 were measured. These data were used to identify the connexin type of gap-junction channels in PLC-cells, a human hepatoma cell.

Diffusion Behavior of Gap Junction Hemichannels in Living Cells

Due to its non-invasive character, fluorescence correlation spectroscopy (FCS) is particularly suited for the investigation of diffusion behavior of proteins in living cells. In this study we have investigated the diffusion properties of CFP-labeled gap junction hemichannels in the plasma membrane of living HeLa cells. Gap junction hemichannels or connexons are the precursors for the cell-cell- or gap junction channels that form large plaques at the contact areas between two adjacent cells. It has been proposed that new channels are recruited into a gap junction structure from a pool of hemichannels that can freely diffuse over the entire plasma membrane. The statistical approach shows that the geometry of the membrane within the focus is the most important property for the form of the autocorrelation curve and in turn for the determination of the diffusion coefficient. On the other hand binding-unbinding events which lead to anomalous diffusion have only a minor effect to the position and shape of the correlation curve compared to the geometry of the membrane.