In 2000, we proposed a CaM-based “cork-type” mechanism of gap junction chemical gating
[11293]. This “cork” model envisions a physical obstruction of the cytoplasmic mouth of the channel by a CaM lobe
[19][50][11293][11394], probably combined with conformational changes in the connexins, caused by Ca
2+-CaM binding to the gating site. The model is based on numerous findings that suggest a direct CaM role in gating; rev. in
[50][51][52][11394]. Experimental evidence indicates that the chemical/slow gate is a sizable, negatively-charged particle, likely to be a CaM lobe
[69][11495].
There are many reasons why
wresearche
rs think that CaM is the most likely gating candidate. [Ca
2+]
i in the high nM to low μM values activates chemical gating; rev. in
[19][52]. In view of the fact that the cytoplasmic domains of connexins do not contain sequences able to bind Ca
2+ at such low concentrations, the effect of Ca
2+i on the channel gating is most likely mediated by a CaM-like protein, most likely CaM itself. Indeed, CaM binds to connexins
[52][57][58][71][72][77][11596] which in fact have CaM-binding sites; most of the sites are at the second half of the cytoplasmic loop (CL2), but some are also at the NH
2-terminus (NT-site) and at the NH
2-end of the COOH-terminus (CT1); rev. in
[19][52][75][11394]. Most relevant for gating are likely to be the CL2 and NT sites
[52][73][74][75][77][10897][10998]. Peptides mimicking the CaM-binding site sequences located at CL2, NT, and CT1 of several connexins bind Ca
2+-CaM with high affinity
[42][52][73][74][75][76][78][79][80][99][103100]. Most important is the binding of CaM to the CL2 domain, which has been experimentally confirmed by Jenny Yang’s team for Cx43
[80], Cx44
[79] Cx50
[78], and Cx45
[77] and by Katalin Török’s team for Cx32, Cx35, Cx45, and Cx57
[73][74]. CaM and connexins co-localize at gap junctions (
Figure 4) and intracellular sites
[71][72][77][104101][116102]. Recently, the direct binding of CaM to Cx45 has been visualized in living cells by Bioluminescence Resonance Energy Transfer (BRET)
[77]; the interaction of CaM and Cx45 was Ca
2+-dependent and prevented by W7; the CL2’s CaM binding site (res. 164–186) was confirmed by a study reporting its high-affinity interaction (K
D = ~5 nM) with a peptide matching the CL2 domain of Cx45’s CL2, tested with a fluorescence-labeled CaM
[77]. On the other hand, however, another study provided evidence for both Ca
2+-dependent and Ca
2+-independent CaM-binding to the CL2 domains of Cx45, Cx32, Cx35, and Cx57
[73][74]. The Ca
2+-independent binding of CaM to the CL2 domain
[73][74] confirms earlier data suggesting that the CaM is anchored to the Cxs at normal [Ca
2+]
i (~50 nM)
[69][71][72][77]. Each of the two negatively-charged CaM lobes is ~25 × 35 Å in size
[91], which is the approximate size of the positively-charged cytoplasmic mouth (vestibule) of the channel
[117103][118104][119105] (
Figure 95). So, a CaM lobe would fit nicely in the mouth (vestibule) of the connexon (
Figure 95B). Evidence from a three-dimensional electron density map of isolated gap junctions, which display crystalline (hexagonal) channel arrays (see in the following), studied by X-ray diffraction, proves that the channels are in a closed state as they are inaccessible to sucrose due to a blocking particle at both channel ends similar in size to a CaM lobe
[120106][121107][122108] (see in the following). Significantly, in a double-whole-cell-clamp (single-channel) study the chemical/slow gate opens and closes fully and very slowly (transition time = ~10 ms)
[123109] and the open-to-closed channel transitions, and vice versa, often displayed fluctuations
[123109]. This further supports the idea that a large particle may transiently flicker in and out of the mouth of the channel before closing the channel completely
[123109].