Résumé des résultats (Abstract)
(Anglais)
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MATHEMATICAL MODEL OF VERTEBRATE GAP JUNCTIONS
Over the last few years, we have determined the basic electrical properties of a large number of different vertebrate gap junctions and gap junction channels using pairs of transfected HeLa cells (provided by K. Willecke, Bonn) in conjunction with the dual voltage-clamp method. With these data at hand, we developed a generalized mathematical model which describes the conductive and kinetic properties of homotypic and heterotypic gap junction channels. The model consists of two submodels connected in series. Each submodel simulates a hemichannel and consists of two conductances corresponding to a high (main open state) and low conductance state (residual state) and a switch, which simulates the voltage-dependent channel gating. It has been assumed that the conductances of the high state and low state vary exponentially with the voltage across the hemichannel. The design of the model has been kept flexible. It can be easily expanded to include additional features, such as channel substates or a closed state.
The model turned out to be very useful in interpreting experimental results gained from both homotypic and heterotypic channels. Furthermore, it has been used to design experimental protocols aimed at verifying experimentally some specific channel properties.
ELECTRICAL PROPERTIES OF Cx45 GAP JUNCTION CHANNELS
There is some controversy about the data published on electrophysiological properties of Cx45 gap junction channels. On the one hand, the parameters extracted from total current measurements are in good agreement: Vj,o = 23 mV, gj,min = 0.17, z= 1.5 (Barrio et al., Progr. Cell Res. (1995)4:391); Vj,o = -15/17 mV, gj,min = 0.17/0.17, z = 3.7/3.5 (Bukauskas & Weingart, unpublished). On the other hand, the parameters deduced from unitary current measurements deviate considerably: gj,main = 30 pS (Moreno et al., Progr. Cell Res. (1995)4:409); gj,main = 460 pS, gj,residual = 100 pS (Bukauskas & Weingart, unpublished). This discrepancy may be related to the source of connexin (chicken vs. mouse) or the connexin expression system used (injected Xenopus oocytes vs. transfected human HeLa cells). To solve this problem, we performed experiments on pairs of HeLa cells stably transfected with cDNA coding for chicken Cx45 (provided by K. Willecke, Bonn). This study complemented our earlier work on mouse Cx45 expressed in HeLa cells (Bukauskas & Weingart, unpublished). Examining chicken connexin channels we obtained the following data: Vj,o = -22/20 mV, gj,min = 0.2/0.19, z = 2.6/2.8; gj,main = 48 pS, gi,residual = 18 pS. This suggests the difference in unitary conductance is real and hence must be related to structural differences between mouse and chicken connexin.
Cx32-KNOCK-OUT MOUSE
Hepatocytes were freshly isolated from wild-type and Cx32-deficient mice. Pairs of cells were chosen to study the electrical properties of gap junction channels with the dual voltage-clamp method. Total gap junction currents revealed that Cx32-deficient hepatocytes express one type of connexin (Cx26) while wild-type hepatocytes express two types of connexins (Cx26, Cx32). Unitary gap junction currents indicated that Cx3-defient cells have homotypic channels (Cx26-Cx26) and wild-type cells have homotypic (Cx26-Cx26, Cx32-Cx32) and heterotypic channels (Cx26-Cx32). A comparison of total and unitary current measurements led to the following conclusions: I) Cx32 hemichannels are 'more abundant than Cx26 hemichannels in prenatal (ratio 4:1) and adult wild-type hepatocytes (ratio 23:1) and the total number of gap junction channels is larger in prenatal than in adult cells; ii) the ratio of hemichannels Cx26:Cx32 is variable among hepatocytes of wild-type animals; iii) Cx26 hemichannels are down-regulated in Cx32-deficient cells. The data did not provide evidence for the presence of heteromeric channels. The outcome of this study now serves as basis for a similar investigation on heart cells during re-differentiation.
GATlNG PROPERTIES OF Cx43 GAP JUNCTION CHANNELS
We have pursued this project to elucidate the kinetic properties of Cx43 gap junction channels. Pairs of myocytes from neonatal rat hearts were voltage clamped to determine the voltage- and time-dependent inactivation of lj. The bell-shaped relationships gj,ss = f(Vj) (gj,ss: gj at steady state) depended on the ionic composition of the pipette solution. The amplitudes of ljss and the time constants of lj inactivation,xxi, were sensitive to the pipette solution. lj,ss was smaller for TEA+ aspartate-.than for KCI while ti was smaller for KCI than for TEA+ aspartate-. The modification of lj,ss is readily explained by a change in unitary conductance; the alterations in ti appear to be caused by a change in b (rate constant of main state -> residual state). The pipette solutions may change the kinetics of the channels by altering the conductive or kinetic parameters. Computer simulations argue for the latter possibility.
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