Calmodulin dependent protein kinase increases conductance at gap junctions formed by the neuronal gap junction protein connexin36

Abstract

The major neuronal gap junction protein connexin36 (Cx36) exhibits the remarkable property of "run-up", in which junctional conductance typically increases by 10-fold or more within 5-10min following cell break-in with patch pipettes. Such conductance "run-up" is a unique property of Cx36, as it has not been seen in cell pairs expressing other connexins. Because of the recent observation describing CaMKII binding and phosphorylation sites in Cx36 and evidence that calmodulin dependent protein kinase II (CaMKII) may potentiate electrical coupling in neurons of teleosts, we have explored whether CaMKII activates mammalian Cx36. Consistent with this hypothesis, certain Cx36 mutants lacking the CaMKII binding and phosphorylation sites or wild type Cx36 treated with certain cognate peptides corresponding to binding or phosphorylation sites blocked or strongly attenuated run-up of junctional conductance. Likewise, KN-93, an inhibitor of CaMKII, blocked run-up, as did a membrane permeable peptide corresponding to the CaMKII autoinhibitory domain. Furthermore, run-up was blocked by phosphatase delivered within the pipette and not affected by treatment with the phosphatase inhibitor okadaic acid. These results imply that phosphorylation by CaMKII strengthens junctional currents of Cx36 channels, thereby conferring functional plasticity on electrical synapses formed of this protein.

Figure 1

Time course of “run-up” in Cx36-GFP transfected Neuro2A cells A. Phase (upper) and fluorescence (lower) micrograph of a pair of Cx36-GFP transfected Neuro2A cells. Arrows indicate junctional plaques between the cells. B. Normalized “run-up” from 18 cell pairs in which long term recording was achieved. Continuous black line represents data fitting with Boltzmann equation with half time of 6.35 min and maximal slope of 0.45 fold/min.

Figure 2

Perforated patch clamp recordings of “run-up”. A. Using amphotericin B, cells were held for 10 min at which time electrical access to cell interior was achieved through insertion of monovalent cation channels. The degree of run-up during the subsequent 10 min period was minimal, whereas run-up was quickly obtained when access to cell interior was obtained with strong suction “Dialyze”, arrow. N=3 cell pairs. B. Run-up was obtained in perforated patch recordings in the presence of calcium ionophore (ionomycin, 1 μM, added at arrow). C. Run-up was largely prevented in dialyzed cells by the Ca buffer BAPTA (25 μM) included in the patch pipette. D. Quantitative comparison of the fold changes during run-up in multiple experiments under perforated patch, dialyzed and perforated patch and Ca ionophore conditions.

Figure 3

Calmodulin inhibitors arrest and block run-up in Cx36 transfectants. A. Application of KN-93 (50 μM) during run-up reversibly blocked the increase in conductance, which resumed after the drug is washed away. B. Pre-treatment (20 min) with 100 μM KN-93 substantially reduced the extent of run-up. C. Treatment with the membrane permeable TAT-CN21 at the beginning of the experiment substantially reduced final junctional conductance. D. Quantitation of effects of KN-93 and the peptide CaMKII inhibitor on runup. *P<0.05

Figure 4

Effects of intracellular peptides on rate and extent of run-up. A. Peptide sequences corresponding to binding and phosphorylation domains of Cx36 cytoplasmic regions. In each panel, control and peptide experiments were done on the same days for comparison. B. CLP peptide had no effect on run-up. C. CLB peptide blocked run-up virtually completely. D. CTB peptide strongly inhibited run-up. E. CTP (CT2) peptide also reduced extent of run-up. Asterisks in C-E indicate p<0.5 compared to control.

Figure 5

Deletions of specific regions of Cx36 reduce the extent of run-up. A. Combined controls for all experiments, performed on the same days as the mutant experiments. Note approximately >5 fold run-up at 13 min. N=9 cell pairs. B. Deletion of region CLB decreased but did not prevent run-up. N=7–9 cell pairs/time point. C. Deletion of region CTB (N=4) or both CTB and CLB (N=6) (D) strongly reduced run-up. E. Normalized plots of each of these data sets illustrating that each mutant (n=4–6 for each) showed strong differences in extent of run-up. Although not graphed, ΔCTB was also significantly different from control. Asterisks indicate p<0.05.

Figure 6

Runup was inhibited by phosphatase treatment and was preserved when cells are pretreated with a phosphatase inhibitor. A. An example of a recording from a cell pair in which each pipette contained alkaline phosphatase (1U/ml). In three experiments of this type, junctional conductance was reduced, although time course of uncoupling varied, as illustrated in B. C. An example of a recording of runup following incubation in the presence of okadaic acid (6 μM for 30–75 min). D. Means and SE of runup in three experiments in the presence of okadaic acid.