Browsing by Author "Eckert, Reiner"

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Open Access
Biophysical characterization of gap-junction channels in HeLa cells
(1993) Eckert, Reiner; Dunina-Barkovskaja, Antonina; Hülser, Dieter F.
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 ζ=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.
Open Access
Characterization of gap junctions by electrophysiological and electronmicroscopical methods
(1990) Hülser, Dieter F.; Paschke, Dietmar; Brümmer, Franz; Eckert, Reiner
Gap junctions are ubiquitous in the animal kingdom from mesozoa to vertebrates. They must be discriminated from desmosomes which anchor cells together to form structural or functional units as well as from tight junctions which seal membranes of epithelial cells to each other so that the paracellular path becomes impermeable to molecules and a polarity of apical and basolateral surface is maintained.
Open Access
High-resolution measurements of gap-junctional conductance during perfusion with anti-connexin antibodies in pairs of cultured mammalian cells
(1992) Paschke, Dietmar; Eckert, Reiner; Hülser, Dieter F.
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.
Open Access
Immunochemical and electrophysiological characterization of murine connexin40 and -43 in mouse tissues and transfected human cells
(1994) Traub, Otto; Eckert, Reiner; Lichtenberg-Fraté, Hella; Elfgang, Claudia; Bastide, Bruno; Scheidtmann, Karl Heinz; Hülser, Dieter F.; Willecke, Klaus
Human HeLa or SkHep1 cells, defective in intercellular communication through gap junctions, were transfected with coding sequences of murine connexin40 (Cx40) and -43. The transfected cells were restored in gap junctional coupling as shown by 100-fold increased electrical conductance. When studied by the double whole-cell patch-clamp technique, Cx40 HeLa transfectans exhibited single channel conductances of γ=121 ± 7 pS and γ=153 ± 5 pS. They were voltage gated with an equivalent gating charge of z=4.0 ± 0.5 for a voltage of half-maximal inactivation U 9= 44 ± 7 mV. The corresponding values or connexin43 (Cx43) HeLa transfectants are: γ=60 ± 4 pS and γ=40 ± 2 pS as well as z=3.7 ± 0.8 and U 0 = 73 ± 7 mV. Transfer of the dye Lucifer Yellow was always considerably lower in Cx4- than in Cx43-transfectants though their total junctional conductance was similar or even higher than for Cx43-transfectants.
Open Access
Patch clamp techniques for the characterization of membrane channels
(1991) Eckert, Reiner; Paschke, Dietmar; Hülser, Dieter F.
The examples demonstrate that by means of high resolution patch clamp recordings, physiological properties of the cell membrane may be elucidated on the molecular level of individual ion channels.
Open Access
Patch-clamp measurements of gap-junction channels in cultured cells
(1992) Hülser, Dieter F.; Eckert, Reiner; Zempel, Günther; Paschke, Dietmar; Dunina-Barkovskaja, Antonina
Direct intercellular communication in most tissues is made possible by proteinaceous pores called gap-junction channels. These channels bridge the extracellular gap between apposed cells and connect their intracellular compartments both electrically and metabolically. The extracellular parts of two hemichannels - the connexons - are linked thus forming a communicating gap-junction channel. A connexon is a hexamer of protein subunits which are members of the connexin family. Since connexin 32 (Cx32) was the first gap-junction channel protein to be sequenced from hepatocytes, it serves as a reference to which all other gap-junction proteins are compared. The individual channel conductance may vary between 25 and 150 pS. Gap-junction channels of some tissues are more voltage sensitive (e.g. liver) than others (e.g. heart). The question whether these differences in electrical properties may be attributed to the different connexins being expressed in these tissues is still unanswered. Several approaches to resolve this problem will be discussed in this contribution, all are based on double whole-cell patch-clamp measurements using isolated cell pairs, as follows: (1) Cells with two different channel conductances perfused with anti connexin antibodies to specifically block one channel species; (2) Cells with only one connexin species selected by immunological characterization; (3) Weakly coupled HeLa cells transfected with specific connexin genes, a method which resulted in better correlations between connexin type and single channel properties.