2. Cytosolic Calcium and Gap Junction Channel Gating
Nearly a century after Engelmann’s discovery
[10], Jean Délèze reported that in cut cardiac fibers “healing over” only occurred in the presence of external Ca
2+ [16], suggesting that the rise in intracellular calcium concentration ([Ca
2+i]) caused by Ca
2+ influx played a role in the regulation of gap-junctional communication. Soon after, the Ca
2+-role in cell uncoupling was confirmed by evidence that cell–cell communication was lost in cells subjected to treatments that increase the [Ca
2+]
i [9][15][17][18][19][20][21][22][23][24][25].
The [Ca
2+]
i effect on gating has been questioned for more than four decades. Early studies suggested that a [Ca
2+]
i as high as 40–400 µM is needed
[26][27]. In contrast, numerous more recent reports indicate that a much lower [Ca
2+]
i, ranging from ~100 nM to low µM is effective. Data for the effectiveness of nanomolar [Ca
2+]
i were first reported in a study
[28][29] in which [Ca
2+]
i of 251 nM (at pH 7.4), ~400 nM (at pH 7.0) and 2.5 µM (at pH = 6.5) were proven effective in uncoupling cardiac cell pairs, one of which was mechanically perforated to allow the influx of extracellular solutions well buffered for Ca
2+ and H
+. More recently, a treatment with ionomycin and gramicidin of arterially perfused rabbit papillary muscle uncoupled the cells at ~685 nM or greater [Ca
2+]
i [30]; these data were confirmed in cells subjected to ischemia/reperfusion
[30]. Low [Ca
2+]
i proved effective on gating in many other cells, such as crayfish giant axons
[31][32], rat lacrimal cells
[33], Novikoff hepatoma cells
[34][35], astrocytes
[36][37][38], lens cultured cells
[39], pancreatic β-cells
[40], human fibroblasts
[41], and Cx43-expressing cultured cells
[42]. Mammalian pancreatic and lacrimal gland cells briefly uncoupled when secretion was stimulated by the application of acetylcholine or other secretagogues at concentrations below those required for maximal secretion
[43][44][45], as well as by depolarization or cyclic nucleotide load
[46][47]. In pancreatic acinar cells, even the treatment with secretagogues at concentrations capable of stimulating maximal secretory activity increases [Ca
2+]
i only from 180 nM to 860 nM
[48], further supporting the effectiveness on channel gating of nanomolar [Ca
2+]
i.
ResWe
archers have studied, with Ca
2+- and pH-sensitive microelectrodes, the relationship between junctional electrical resistance (Rj), [Ca
2+]
i and [H
+]
i in crayfish septate axons uncoupled by low pH
i [32]. Cytosolic acidification (pH
i = 6.3), caused by the application of Na
+-acetate, increased [Ca
2+]
i by approximately one order of magnitude, from resting values of 100–300 nM, and greatly increased Rj, indicating that that crayfish gap junctions are sensitive to low µM [Ca
2+]
i [32]. The time course of Rj and [Ca
2+]
i matched well, while that of Rj and [H
+]
i did not
[32].
In order to more accurately determine the [Ca
2+]
i effective on gating,
reswe
archers studied Novikoff hepatoma cell pairs by double whole-cell clamp
[34][35]. In these Cx43-expressing cells, Ca
2+i-gating sensitivity was tested by monitoring the decay of junctional conductance (Gj) at different [Ca
2+]
i (buffered with BAPA), at pH
i = 7.2 or 6.1 (buffered with HEPES and MES, respectively). Channel gating was activated by [Ca
2+]
i ranging from 500 nM to 1 µM, irrespective of pH
i [34]. With [Ca
2+]
i = 0.5–1.0 µM, the Gj dropped to ~25% of the initial values with mean τ’s of 5.9 and 6.2 min, at pH
i = 6.1 and 7.2, respectively. With [Ca
2+]
i = 3 µM, the cells uncoupled in <1 min (τ = ~20 s)
[34]. The effectiveness of high nanomolar [Ca
2+]
i on gating was confirmed in the same cells with brief (20 s) exposures to 20 µM arachidonic acid
[35].
Similarly, a [Ca
2+]
I < 0.5–1 µM blocked the cell–cell diffusion of Lucifer Yellow in chicken-lens-cultured cells
[39], and nanomolar [Ca
2+]
i drastically reduced the Gj in pancreatic β-cells, with a temperature drop from 37° to 30° and an external [Ca
2+]
o rise from 2.56 mM to 7.56 mM
[40]. Gating sensitivity to nM [Ca
2+]
i was also reported in astrocytes injected with Lucifer Yellow and Ca
2+ [36]. In these cells, nanomolar [Ca
2+]
i prevented cell–cell dye transfer independently of pH
i; the dye transfer was blocked by [Ca
2+]
i ranging from 150–600 nM
[36]. Consistent with these findings is a report that the addition of 20 mM of BAPTA to the patch pipette solutions substantially improves coupling between astrocytes
[37], which indicates that gating may even be sensitive to basal [Ca
2+]
i. Dye coupling was also blocked in cultured astrocytes treated with ionomycin, which increased the [Ca
2+]
i to 500 nM
[38], and similar values were reported in lens-cultured cells
[49]. In murine Neuro-2a cells (N2a) expressing Cx43, ionomycin treatment increased the Ca
2+-influx and reduced the Gj by 95%
[42], as the [Ca
2+]
i increased from ~80 to ~250 nM. All of these data confirm the idea that Ca
2+i is a fine modulator of gap-junctional coupling.
3. Evidence for Calmodulin Role in Gap Junction Channel Gating
As gap junction proteins do not have highly sensitive intracellular Ca
2+-binding sites, the data described in the previous chapter strongly suggest that Ca
2+i affects gating via an intermediate component. Indeed, since the early eighties
rwe
searchers have proposed calmodulin (CaM) as the intermediate of the Ca
2+ gating effect; rev. in:
[50][51][52].
In 1981, Johnston and Ramón reported that crayfish giant axons lose their cell–cell gating sensitivity to increased Ca
2+i and/or decreased pH
i when they are internally perfused
[53]. Their data, confirmed by Arellano and coworkers
[54], induced them to suggest that a soluble intermediate mediates the Ca
2+/H
+-induced cell uncoupling
[53]. In the same year,
reswe
archers first suggested CaM as the soluble intermediate of Ca
2+i-induced gating
[55][56]. Our idea was also supported by evidence for CaM binding to the gap junction protein connexin32 (Cx32) and to gap junction fragments from crayfish hepatopancreas
[57][58].
In 1988, Arellano and coworkers provided convincing evidence that CaM is in fact the soluble intermediate that had been washed out by the internal perfusion of crayfish axons
[54] because when the lateral giant axons were internally perfused with Ca-CaM (pCa 5.5; CaM + SIS-B), the Rj increased from the control values of ~60 kΩ to 500–600 kΩ in ~60 min (
Figure 1). In contrast, the axons perfused either with CaM in low Ca
2+ solutions (pCa > 7; CaM + SIS-A), with CaM-free high Ca
2+ solutions (pCa 5.5; SIS-B) or with Ca-free solutions (SIS), maintained the Rj at control levels during the 60 min perfusion time (
Figure 1).
Figure 2 schematically summarizes the results of Arellano and coworkers
[54][59]. The same results were reported with either only one axonal segment perfused (
Figure 2) or with both segments perfused. Significantly, while 20 min of the internal perfusion of one axon segment with 1 mM Ca
2+ in the absence of CaM did not change the Rj, the subsequent addition of Na
+-acetate to the external solution, while maintaining the same internal solution, increased the Rj to ~400 kΩ
[54]. This is very significant for two reasons: first, it proves that the septum was not damaged by the SIS perfusion; second, it proves that the uncoupling effect of acetate on the intact axon segment is not just due to an increase in [H
+]
i [59], but rather to an acetate-induced rise in [Ca
2+]
i resulting from the drop in pH
i (
Figure 2), as also reported by us with different methods
[32] (see in the previous).
Figure 1. Changes in Junctional Resistance (Rj) in crayfish lateral giant axons in which one axon of the coupled pair was internally perfused with either of the following Standard Internal solutions (SIS): SIS (no added Ca
2+, 0.1 mM EGTA, pH 7.1); SIS-A (no added Ca
2+, 10 mM EGTA, pH 7.1, pCa > 7); SIS-B (1 mM CaCl
2, 0.1 mM EGTA, pH 7.1, pCa 5.5); CaM + SIS-A or SIS-B (10 μM CaM, pH 7.1). Rj does not increase in the absence of CaM, either in the absence of Ca
2+ (SIS) or with 1 mM Ca
2+ (SIS-B), but does so greatly in the presence of Ca
2+ + 10 μM CaM (CaM+SIS-B). In the authors’ words: “All data points were included in this figure, since the trend illustrated was observed in five other experiments with prolonged perfusion of calmodulin and high calcium”. Reproduced with permission from ref.
[54].
Figure 2. Summary of data from refs.
[53][54][59]. While internal perfusion of crayfish lateral giant axons with Standard Internal Solution (SIS) with high [Ca
2+] and/or [H
+] does not induce channel gating (
A), addition of 10 μM CaM to internal solutions with high [Ca
2+] does (
B). Axons internally perfused with high [Ca
2+] and/or high [H
+] without CaM uncouple with extracellular perfusion of Standard Extracellular Solution (SES) containing 205 mM Na-acetate (
C), as acetate increases [Ca
2+]
i by increasing [H
+]
i in the un-perfused axon segment. (Red circle: CaM’s C-lobe; Green circle: CaM’s N-lobe).
Crayfish express innexins rather that connexins, but innexins are very similar to connexins and contain CaM-binding sites. In crayfish giant axons both innexin-1 and innexin-2 are expressed
[60]. Innexin-1 and -2 contain CaM-binding sites at the CT and CL2 domains (
Figure 3). The CaM-binding prediction to the CT and CL2 domains of these innexins were identified by means of a computer program developed at the University of Toronto (
http://calcium.uhnres.utoronto.ca/ctdb/ctdb/sequence.html, accessed on 28 November 2021. Copyright © 2021 Ikura Lab, Ontario Cancer Institute. All Rights Reserved).
Figure 3. Innexins’ CaM-binding sites at CL2 and CT domain.
In 1986, Arellano and coworkers also confirmed their earlier data of gating insensitivity to H
+i [59]. In this study, a glass capillary was inserted into one of the axons and one side of the junction was perfused with solutions of pH 7 or 6 (
Figure 2A), while monitoring the Rj. Significantly, the Rj remained unchanged when the pH of the perfusate was lowered from 7.1 to 6.0
[59].
RWe
searchers have confirmed the absence of a direct effect of low pH
i on gating in crayfish axons
[32],
Xenopus oocyte pairs
[61], and Novikoff hepatoma cell pairs
[34]. In the Novikoff cells,
reswe
archers monitored the Gj at different pCa (9, 6.9, 6.3, 6, and 5.5; buffered with BAPTA) and pH
i (7.2 or 6.1; buffered with HEPES an MES, respectively). No significant difference in the Gj was observed between pH
i 7.2 and 6.1 as long as the [Ca
2+]
i was carefully buffered with BAPTA
[34].
In the four decades that followed our reports of the early eighties
[55][56][62], CaM participation in channel gating has been confirmed by multiple data generated by a variety of experimental procedures that include: treatment with CaM blockers
[42][49][55][56][62][63][64][65][66][67], inhibition of CaM expression
[68][69][70], overexpression of a CaM mutant (CaMCC) with higher Ca
2+ sensitivity
[71][72], colocalization of CaM and gap junctions by immune-fluorescence microscopy
[71][72] (
Figure 4), intracellular perfusion of crayfish axons with CaM-containing solutions
[54], and in vitro testing of CaM binding to connexins
[57][58][71][72] and synthetic connexin peptides mimicking CaM-binding sites of various connexins
[42][52][73][74][75][76][77][78][79][80]; rev. in:
[50][51][52].
Figure 4. Immuno-fluorescence labeling of CaM (
A) and Cx32 (
B) in HeLa cells. The overlay of (
A,
B) is shown in (
C). Cx32 and CaM colocalize at three punctuated areas of cell–cell contact. From ref.
[72].
After our early evidence for a CaM role in gap junction channel function
[55][62][63][81], numerous other membrane channels have been found to directly involve CaM in their gating mechanisms. Indeed, there are an increasing number of channels regulated by CaM. In addition to connexins, they include: voltage-gated calcium (VGCC, CaV) channels, sodium (VGSC, NaV) channels, potassium channels (VGPC, KV), small conductance calcium-activated K
+ channels (SK), inwardly rectifying potassium channels (Kir, IRK), cyclic nucleotide-gated channels (CNG), ryanodine receptors (RyR), and transient receptor potential channels (TRP), rev. in:
[82][83], as well as the water channel aquaporin-0 AQP0), also known as the eye lens protein MIP26
[84][85][86][87][88][89][90].
CaM is an acidic protein of 148 amino acids, whose sequence is very well preserved from plants to mammals. It is a dumbbell shaped protein, ~65 Å long, made of two fairly spherical lobes of ~35 × 25 Å in size, called the N-lobe and the C-lobe
[91]. A short NH
2-terminus is followed by the N-lobe, which is linked to the C-lobe by a flexible amino acid chain. Each of the two lobes has two domains, known as EF-hands
[92], which bind Ca
2+ with nanomolar affinity. The Ca
2+ affinity of the C-lobe is greater than that of the N-lobe by approximately one order of magnitude. Ca
2+-binding to Ca
2+-free CaM (apo-CaM) induces conformational changes that unmask a hydrophobic pocket in each lobe. Ca
2+-CaM (holo-CaM) interacts with a receptor domain, usually made of a basic amphiphilic alpha-helix, by binding to it hydrophobically and electrostatically.