1.2 Gap Junction
1.2.4 The regulation of gap junction by Ca 2+
Since communication between neighboring cells is regulated at multiple levels, the gap junction gating displays several different ways, like voltage gating and chemical gating, which are the faster regulations involved in changing the unitary conductance of single channels or altering their permeability of opening. The slower regulation is achieved through slowing the synthesis and assembly rates, thus reducing the number of gap junction present in the membrane. The slow and rapid regulation may overlap, for example, the rate of trafficking the connexon to the membrane
may be slowed down or degradation is accelerated, meanwhile, the conductance of gap junction channels is altered. In this dissertation, I will focus on the chemical gating, especially Ca2+ and CaM regulation of GAP JUNCTION channels.
The discovery of Ca2+ in gap junction regulation can be traced back to 1970s when a plentiful of articles reported that extracellular calcium was required for the heart muscle “healing- over” (72-76). When the cardiac muscle is damaged in injury, the resting membrane potential first depolarizes, followed by a recovery to normal. This process is called “healing-over”, which is initiated as a self-protection mechanism to prevent the spread of damage to neighboring cells. It is concluded that extracellular Ca2+ ions in the bath solution is essential for the healing over of mammalian heart muscle and cell uncoupling mediated through gap junction (77-83). Later on, structural changes induced by [Ca2+]o were observed in gap junctions isolated from calf lens fiber (84). As shown in figure 1.10, loosely packed junctional regions were captured in Freeze-fracture experiment when calf lens were incubated in EDTA solution in which Ca2+ concentration was buffered within a range of 10-8 to 10-6 M. The random center to center distance between each particles were ranging from 8 to 11 nm. In the contrast, gap junction particles were induced to form condensed crystalline array with a center-to-center distance of 6.5-7 nm by Ca2+.
Figure 1.10 Comparison of gap junction particles isolated from calf lens fibers in the presence of EDTA (a) or subsequently incubated in the presence of CaCl2 (b).
Images were obtained from freeze-fracture replica experiments. The distribution pattern of gap junction particles in EDTA buffer is random and loose, on the contrary, condensed
crystalline pattern were detected when gap junction particles were immersed in Ca2+ buffer.
E: E face; P: P face.
In 1980, Dr. Unwin proposed a Ca2+ gating model (Fig. 1.11): the helices of gap junction protein twist to close the channel in the presence of Ca2+ (85). Large conformation changes are required in this proposed model. But until now, no evidence support this model.
Figure 1.11 Ca2+ regulation model from Dr. Unwin.
Six cylinders stand for the six gap junction protein connexin. The helices of six cylinders twist a little to close the channel in the presence of Ca2+ (85).
The rapid development in the atomic force microscopy facilitated the progress of visualization of conformations of gap junction channels. Back to 2002, Daniel imaged the Ca2+
induced conformational change of gap junction channel (Fig. 1.12) using AFM (86). Before the injection of Ca2+ solution, gap junction channels display 1.5 nm diameter entrances, while the injection of 0.5 mM Ca2+ reduces the diameter of entrance to 0.6 nm. Besides, the plaque heights also increased from 0.6 nm to 18 nm, which suggests that Ca2+ not only alters the conformation of the surface of connexon (hemi-channel), but also affects the structure of intact channel.
Figure 1.12 Extracellular connexin surface recorded by AFM in a Ca2+-free buffer (left) and
a buffer with 0.5 mM Ca2+ (right).
A is the AFM image showing single connexons. Circles in the image pointed out some connexons with defects. B is the average of all connexons captured in image A. The connexin arrangement in connexon are depicted, as well as the side view of the channel pore. C is the average standard deviation map calculated from connexons in image A.
Recently, Julian et al. studied the effect of different divalent cations on the pore size of Cx43 (Fig. 1.13) (87). When there was no Ca2+ in the buffer, the addition of Mg2+ or Ni3+ could not close the pores. While 1 mM Ca2+ triggered the decrease of pore size and 1.4 mM Ca2+ could
even make some channels close. At 1.8 mM Ca2+, the pores completely closed. Figure 1.14H and I clearly showed the extracellular surface of the channel in Ca2+ free buffer and 1.8 mM Ca2+ buffer. The size distribution of Cx43 hemichannel pore analysis also clearly indicated that the average pore inner diameter of Cx43 hemichannels in 1.8 mM Ca2+ buffer was approximately 1nm smaller than that in Ca2+ free buffer. With the increase of Ca2+ concentration in the buffer, the population of open pores dramatically reduced from 100% to less than 5%.
Figure 1.13 The diameter change of reconstituted Cx43 hemichannel pores in different cations. A-G shows the surface of the pore mouth in different cation concentrations. H and I are two zoomed connexons presented as open (H) and closed (I) hemichannels. Connexin monomers were numbered as 1 to 6. J is the inner pore diameter distribution graph in the absence of
of open channels in different concentration of bath Ca2+ concentrations are plotted in the figure K.
All the above results suggests that Ca2+ plays a crucial role in channel gating. But the exact mechanism is still unknown. What is the Ca2+ binding sequence? Does it directly bind with the gap junction or not? How does the Ca2+ regulate the opening of gap junction channel? Until this year, the first Ca2+-loaded crystal structure of Cx26 was published, uncovering the binding site of Ca2+ and its mechanism of channel closure induction (88). In this dodecamer structure, 12 Ca2+ ions were coordinated by 5 oxygens from residues E47, G45, and E42. Among the three binding ligand, G45 and E47 are from one monomer, while E42 is provided by another adjacent monomer (Fig. 1.14). The Ca2+-bound and Ca2+-free structures are similar. The threshold diameter of both pores are around 15 Å. Those two structures directly revised the old model in which Ca2+-binding induces major rearrangement of six helices to close the gap junction channel proposed by Unwin
et al earlier. The comparison between Ca2+-bound and Ca2+-free structures revealed that the
arrangement of six helices are nearly identical. The bound Ca2+ was still exposed to the aqueous environment and no occlusion was formed in the Ca2+-bound structure. Even though no major difference between Ca2+-bound and Ca2+-free structures were seen, minor deviations were identified. In the absence of Ca2+, the side chains of both E42 and E47 rotated ≥ 90o and formed intrasubunit hydrogen bond with R75 and K188, respectively. The carbonyl of Cx45 moved closer to E42 to result in additional interactions between subunits when Ca2+ is absent.
Figure 1.14 Ca2+-binding site in Cx26 gap junction channel crystal structure.
The helices arrangement are very similar in Ca2+-bound (a, cyan) and Ca2+-free structures
(e, orange). In the Ca2+-bound structure , Ca2+ is positioned at the interface between two adjacent monomers , coordinated by five oxygens from residues E42 (from monomer F), G45
(from monomer A), and E47 (from monomer A) (b) In the Ca2+-free structure, the side chain
of three Ca2+-binding ligands rotated and build interactions with other surrounding residues.
Side view of Ca2+-bound hemichannel (c) or Ca2+-free hemichannel (g) with three monomers
close to the viewer been removed. (d) Side view of the Ca2+ coordination sites in Ca2+-bound
structure (d) or Ca2+-free structure (h).
The new Ca2+-bound structure did not form steric occlusion of the pore, but it did change the electrostatic potential of the pore. Molecular dynamics (MD) simulations of the Ca2+-free and Ca2+-bound structures found that K+ permeabilization was severely impeded by Ca2+-binding. A positive pore surface potential is created which determines the pore selectivity when Ca2+ binds. Ca2+-free gap junction channels with a negative surface potential prefers the passage of anions or
molecule with negative charges. Based on the above evidence, Bennett et al. proposed that an electrostatic barrier of the pore is formed when the Ca2+ binding sites are occupied, resulting in significantly reduced permeability of positively charged molecules and cations.