Gap junctions are clusters of transmembrane channels that provide a pathway for the passive diffusion of ions and small molecules between closely juxtaposed cells. Each channel is formed by the interaction of two connexons (hemi-channels), hexameric assemblies of six proteins (connexins) delineating an aqueous pore. Gap junctions play essential roles in the normal functions of the heart, allowing the spread of the electrical impulses within the heart and the synchronised contraction of cardiac cells. The activity of gap junctional channels of cardiac myocytes appears to require ATP and to be regulated by phosphorylating reactions (Verrecchia et al. 1999). In cells, ATP is combined with Mg2+ and when ATP declines (during myocardial ischaemia, for example), intracellular Mg2+ concentration ([Mg2+]i) simultaneously rises. Mg2+ is the major divalent cation present in cytoplasm, but the role of changes in its intracellular concentration in cellular regulations remains poorly understood (Takaya et al. 2000). The aim of the present study was to examine the effect of [Mg2+]i on the degree of cell-to-cell communication in ventricular myocytes in primary culture, obtained from neonatal rats killed by cervical dislocation followed by decapitation.
When ATP was present at a minimal concentration of 2 mM in the solution of patch pipettes, the cell-to-cell communication, studied in the whole-cell version of the dual patch-clamp technique, was well preserved (n = 14). In contrast, when ATP was omitted (n = 9), the amplitude of junctional current declined (rundown). The addition of 10 mM MgCl2 induced an acceleration of the channel rundown in ATP-deprived conditions since complete uncoupling was then achieved within 6 min instead of 18 min (n = 8). This phenomenon was slowed down in the presence of the protein phosphatase inhibitor I2 (n = 5). When 2 mM MgATP was present in the pipette solution, the addition of 2 mM MgCl2 (1.9 mM [free Mg2+]) slightly reduced the junctional conductance, from 58.2 ± 14.9 (mean ± S.E.M.) to 49.2 ± 9.0 nS within 15 min (n = 6), but the addition of 5 mM MgCl2 (4.46 mM [free Mg2+]) lowered the junctional conductance (67.8 ± 10.9 nS) to non-measurable low values (n = 8) within 12 to 15 min.
These results suggest that the interruption of cell-to-cell communication observed in some pathological conditions (e.g. ischaemia) could result from a dual mechanism, a progressive fading of protein kinase activity due to ATP depletion and an activation of a protein phosphatase triggered by the rise in [Mg2+].
This study was supported in part by grants from the European Community RDT action QLG1-CT-1999-00516.
- Takaya, J., Higashino, H. & Kobayashi, Y. (2000). Magnesium Res. 13, 139-143.
Verrecchia, F., Duthe, F., Duval, S., Duchatelle, I., Sarrouilhe, D. & Hervé, J.C. (1999). J. Physiol. 561, 447-459. abstract