Peracchia C .

             calmodulin binding
             cell communication by chemical coupling

	Figure 1. In oocytes expressing CaMCC (A) before Cx32, Gj is very low (B), but rapidly increases with 180 µM BAPTA superfusion (B,C) as [Ca2+]i, measured with Calcium Green-1, drops (B, F/F0). sensitivity. In CaMCC the NH2-terminal EF-hand pair (res. 9–76) is replaced by the COOH-terminal pair (res. 82–148). Lower [BAPTA] (90 µM) are much less effective (C). Cx32 channels expressed after CaMCC are more sensitive to 100% CO2 than controls (D,E), as Gj rapidly drops to zero (D,E) with CO2 applications for as short as 3 min (D) or 15 min (E; mean ± standard error, SE, n = 3), while in controls it decreases by only ~15% even with CO2 applications as long as 15 min (E; mean ± SE, n = 7). After CO2 washout, Gj/Gjmax remains at 0% but rapidly increases with 180 µM BAPTA superfusion (D). Expression of Cx32 before CaMCC (E; mean ± SE, n = 3) or expression of CaMNN (A) has no effect on gating (E; mean ± SE, n = 3). From [39].
	Figure 2. CT-deleted Cx43 channels (Cx43TR257), expressed after CaMCC, are much more sensitive to CO2 than controls (A and B), as Gj rapidly drops to ~50% (A) and nearly 0% (B) with 3 and 15 min 100% CO2 applications, respectively, while in controls Gj decreases by only ~25% even with 15 min CO2 (B). Curiously, in controls paradoxically Gj increases reversibly with brief applications of CO2 (A); similarly, Gj increases before dropping in control Cx43TR257 (B). The initial Gj rise may result from activation of the CaMKII cascade (see text). After CO2-washout, in some experiments, Gj remains indefinitely at nearly 0% (C,D), but reversibly increases with 180 µM BAPTA superfusion (C,D). An original voltage (V1 = Vj) and current (I2 = Ij) chart record is shown in C. The changes in Gj (µS) of the experiment shown in C are shown in D (Gj = Ij/Vj). These Cx43 data are mine, new and not published elsewhere.
	Figure 3. The junctional conductance (Gj), monitored in Xenopus oocyte pair expressing Cx45 during CO2 application, has a biphasic course: initial rise followed by a rapid drop to full uncoupling (red circles). Since activation of CaMKII increases Gj (see text), the initial Gj rise may result from activation of the CaMKII cascade and opening of dormant, CaM-Cork gated channels. We believe that subsequent rapid Gj drop results from the activation of the Ca-CaM-Cork mechanism. Inhibition of CaM expression greatly reduces the CO2 sensitivity, as Gj reversibly drops (monophasically) by only ~17% (green circles)—the absence of Gj rise suggests that with reducing [CaM]i at rest, most channels are open. From [38].
	Figure 4. Immunofluorescence labeling of CaM (A) and Cx32 (B) in HeLa cells expressing Cx32. CaM and Cx32 colocalize in linear (regions between arrowheads) and punctated areas of cell–cell contact (C). Labeling is also seen in the cytoplasm (A–C); this is likely to correspond to CaM and CaM linked to Cx32 in cytoplasmic organelles. Scale bar 10 μm. From [39].
	Figure 5. Most connexins have three CaM-binding sites. The CaM-binding sites of rat-Cx32: NH2-terminus (NT), second half of the cytoplasmic loop (CL2) and initial domain of the COOH-terminus (CT1) are shown in (A). Most relevant for chemical gating is likely to be the CL2 site of connexins (B) and Innexin-1 (C). The CaM-binding site was identified by a computer program that rate the sites 0–9 [73].
	Figure 6. Sequences of the cytoplasmic loop (CL) of rat Cx32 and Xenopus Cx38 (A). Gj changes induced by 100% CO2, monitored in oocytes expressing Cx32, Cx38, or Cx32/38 chimeras are shown in B. Channels made of Cx32/38CL (Cx32′s CL being replaced by that of Cx38) or Cx32/38CL2 (Cx32′s CL2 being replaced by that of Cx38) reproduce precisely the gating efficiency of Cx38 channels in both magnitude and rate (B), but Gj recovers faster in Cx32/38CL2 channels. Note that CL2 contains a CaM-binding site (see Figure 5A,B). Adapted from [65,66].
	Figure 7. Ij/Vj records of Xenopus oocyte pairs expressing either homotypic Cx32 channels (32–32) or heterotypic tandem-32 channels (A,B). While 32–32 channels manifest a characteristic Ij sensitivity to Vj (B), tandem-32 channels show an unusual behavior (B). With mutant side negative (B, left), initial and final Ij gradually drop to very low values, while with mutant side positive (B, right), Ij gradually increases to high values and only at Vj = 100–120 mV, a more typical Ij behavior appears. With repeated application of 60-mV Vj pulses (tandem side positive), 3 types of Ij behavior are observed: monophasic Ij increase (C, pulses 1–3), biphasic Ij time-course (C, pulses 4–9 and conventional Ij behavior (C, pulses 10–18). Subsequent applications of the Vj protocol (tandem side positive) result in fairly normal Ij behaviors (C, pulses 19–27). The asymmetric Ij-Vj behavior of tandem-32 channels is demonstrated in plots of normalized Gj versus Vj (D). Significantly, the asymmetrical Ij/Vj behavior is not observed following inhibition of CaM expression (E). (B,C) from [77]; (D,E) from [37].
	Figure 8. Response of Gj to steady-state Vj in tandem-32 channels (A,B). In oocytes initially clamped at Vm = 20 mV (Vj = 0), a 40–60 mV Vj step (tandem side positive) exponentially increases Gj in tandem-32 channels (A,B). Upon return to Vj = 0 mV from Vj = 40 mV (A), Gj decreases exponentially to near control values. With Vj reversal to 60 mV (tandem side negative), Gj decreases exponentially below control values (B), indicating that Vj negative at the tandem side actively closes channels. Upon return to Vj = 0 from Vj = 40 (A) or 60 (B) mV (tandem side positive), Gj increases abruptly before dropping (A,B), because the fast Vj-gates of the Cx32 side reopen (Cx32 is a negative gater). Of course, this is not observed with Vj polarity reversal (B) because while the fast Vj-gates open at Cx32wt side (positive Vj side) they close at the tandem side (negative Vj side). From [77].
	Figure 9. Percent Gj change in oocytes expressing Cx32-mutant (Cx32-5R/E) or Cx26-mutant (Cx26/4pos-E) heterotypic channels. Cx26-4pos/E channels behaved as Cx32-5R/E (or tandem-32) channels when subjected to Vj gradients, in spite of the fact that their fast Vj gates are activated by opposite Vj polarities (Cx32 is “negative gater”, Cx26 is “positive gater”). This confirms that the slow Gj change with Vj gradients is a gating behavior based on the activity of the chemical/slow gate, which is clearly distinct from that of the fast Vj gate1]. Adapted from [1].
	Figure 10. Effects of 100% CO2 on single-channel gating behavior in gap junctions of fibroblast pairs subjected to Vj 30 (A) or 55 (B) mV. Gating was monitored before total uncoupling ((A,B) left traces) and at the initial recoupling phase ((A,B) right traces). With Vj = 30 mV (A), each channel closes by a slow Ij transition from open, yj(main state), to closed state ((A) left trace and inset (Aa)) and reopens with a slow transition from closed state to yj(main state)((A) right trace and inset (Ab)). With Vj = 55 mV (B), the channels show two Ij transition: (1) between open and closed state (120 pS, ~10 ms; (B) left trace, arrows and inset (Ba)), and (2) between open and residual state (90 pS, 2 ms; (B) left trace and inset (Ba)). The channels reopen by a slow Ij transition to open state ((B) right trace, arrows and inset (Bb)), followed by fast flickering to residual state. Thus, the chemical gate closes slowly and completely, while the fast Vj gate closes fast and partially. Chemical gate transitions between open and fully closed states (Ca) and vice versa (Cb), often display fluctuations (C, red arrows), suggesting that the gate (CaM’s N-lobe?) may flicker in and out of the channel’s mouth before settling (C, inset). From [79].
	Figure 11. Slow Gj decay in oocytes expressing either Cx32 or its CT-truncated mutant (Cx32-D225) subjected to long −100 mV Vj pulses. (A,B) show junctional current (Ij) tracings recorded from Cx32wt and Cx32-D225, respectively. Note the gradual drop in Ij-Peak (IjPK) and, to a lesser extent, Ij-Steady-State (IjSS; A and B). GjPK drops exponentially by 50–60%, eventually reaching steady state (C). GjSS/GjPK increases by 60% and 93% in Cx32wt and Cx32-D225, respectively (D). The large drop in Ij during the first 8 pulses is likely to result from the closure of both chemical/slow gate and fast Vj gate ((A,B) double-headed red arrows and left drawing). The Ij behavior of the following 6 Vj pulses is likely to reflect the closure of only the fast Vj gate of the remaining open channels ((A,B) double-headed blue arrows and right drawing). From [82].
	Figure 12. Effect of Vj polarity-reversal on junctional conductance (Gj) in oocyte pairs expressing either Cx32 subjected to Vj pulses of -100 mV. The Ij and GjPK records ((A,B) respectively) show that in the first few pulses of the second series peak Ij (IjPK; A, arrow) and consequentially GjPK (B) are substantially greater than that of the last pulse of the first series. This indicates that positive Vj is more effective in opening hemichannels than negative Vj in closing them. Significantly, this phenomenon is more pronounced with Cx32-D225 channels (B). From [82].
	Figure 13. Schematic representation of Ca-CaM-Cork gating. Our hypothesis is that under normal conditions, while most channels are open (A) some are closed (B) by the CaM-Cork mechanism (B). With a small [Ca2+]i rise above resting values, CaMKII becomes activated, possibly resulting in the opening of CaM-Cork gated channels (C; orange connexins). With greater [Ca2+]i rise the Ca2+-activated CaM’s N-lobe interacts hydrophobically and electrostatically with the connexin’s gating site and plugs the channel’s pore (D; red connexins), probably also causing conformational changes in connexins (D).
	Figure 14. Cx32′s CL1 (first half of CL) and CT1 (initial domain of CT) domains in α-helical conformation. Our hypothesis is that in coupled conditions CL1 and CT1 interact electrostatically (negative CL1, positive CT1) and hydrophobically. CL1 and CT1 mutation (bottom panel) are believed to prevent the interaction allowing the negatively charged CaM’s N-lobe to access the positively charged channel’s mouth and plugging it by interaction with it electrostatically (CaM-Cork model). We suggest that with an increase in [Ca2+]i the activated CaM’s N-lobe accesses the gating site and plugs the channel’s mouth by breaking the CL1-CT1 interaction (Ca-CaM-Cork model).
	Figure 15. Schematic representation of CaM-Cork gating. CaM is anchored to connexins by its C-lobe at resting [Ca2+]i (A,B). Certain connexin mutations enable the negatively charged CaM’s N-lobe to access the channel’s pore and plug it by interacting electrostatically with the positively charged channel’s mouth even at resting [Ca2+]i (Aa); the N-lobe can be moved out of the pore with Vj gradients positive at the mutant side (Ab). In wild-type connexins (Cx32-CX32), with the application of large Vj gradients the N-lobe can be reversibly forced to plug the channel’s mouth at the negative side of Vj at resting [Ca2+]i (Bb).
	Figure 16. Both the positively charged channel’s mouth (A,C) and the negatively charged CaM lobes (B) are ~25 × 35 Å in size. Therefore, a CaM lobe could interact and fit well within the positively charged connexon’s mouth (vestibule) (A,C). In (C) the channel is split lengthwise so that the pore diameter (light blue area) is seen throughout the entire channel’s length. Both the CaM and connexon images (B,C) were provided by Drs. Francesco Zonta and Mario Bortolozzi (VIMM, University of Padua, Italy).

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