Human Cx26, coded by gene GJB2, belongs to beta family of gap junction. It widely distributes in human, including cochlea, skin, liver, placenta and breast. Cx26 is co-expressed with Cx30 in the inner ear (458,459), forming homo-or hetero-gap junctions in the cochlea. The gap junction channels in the cochlea play critical role in sound generating. During the sound transduction, the K+ current flows through the organ of corti to the perilymph, generating the sound. Then this K+ current is recycled back to the endolymph by gap junction. Mutations in Cx26 and Cx30 are known to be associated with deafness. According to a survey performed by Florida University, about 14% deafness is caused by Cx26 mutations. Now over 100 mutations in Cx26 have been identified to be linked with nonsyndromic sensorineural deafness.
Many of these syndromic deafness mutations localize to the amino-terminal and first extracellular loop (EL) domains. Studies of mutations in the EL using mammalian cells or oocytes revealed altered [Ca2+]o regulation and permeability in Cx26 hemichannels formed by those mutations. It has been reported that G45E transfection to HeLa cells resulted in cell apoptosis and death within 24 hours of transfection (460). G45E transfection into Xenopus laevis oocytes led to huge hemichannel currents and cell lysis (461) (Fig. 4.1). Elevated concentration of [Ca2+]
o in the culture medium could rescue those aberrant hemichannels. It was proposed that Cx26
hemichannels formed by mutation G45E could not be properly regulated by [Ca2+]o and those leaky hemichannel caused cell apoptosis and death. In the EL1, other mutations such as A40V, E47K, and D50N are also reported to show impaired channel permeability, altered [Ca2+]o sensitivity and disturbed regulation by [Ca2+]o (460,462,463).
Figure 4.1 Hemichannel activity-dependent cell death can be rescued by elevated Ca2+.
Wild type Cx26 or its mutant RNA was injected into oocyte for hemichannel expression on surface of oocytes. Both D50N and G45 caused early cell lysis as indicated by red arrow.
Increase the concentration of [Ca2+]
o in bath solution could prevent oocyte “budding” and
successfully rescued oocyte with mutants expression. Water injection was used as negative control.
It is well know that gap junction hemichannels are regulated by [Ca2+]
o. However, the mechanism behind it is unclear. The available crystal Cx26 crystal structure when we started this
project is an open structure without any bound Ca2+ ions. Using our computational algorithm, we predicted one Ca2+ binding site formed by residues from EL1 and EL2. Interestingly, many mutation sites are in our predicted Ca2+-binding site (Table 4.1). It is definitely not a coincidence since several functional studies have already revealed abnormal [Ca2+]o regulation on mutants from the predicted Ca2+-binding site.
Table 4.1 Disease mutations in the predicted Ca2+-binding site.
Calcium-binding
I II
Possible Function Hemichannel protection Plaque formation
Locations EL1 EL2
Involved Residues E42, D46, E47, D50 N170, D179, E187
Deafness Mutations W44C2/S2, G45E4, D46E3,
E47K2, D50Y1/N1,4, N54K5, T55N1, T55G5
P173R1, D179N1, W172R2, P173S5, K168R5
1. Mutations prevent the formation of gap junction channels.
2. Mutations do not affect formation of gap junction channels, but the mutated gap junction channels display null function.
3. Mutations specifically impair the gap junction channel-mediated biochemical coupling. 4. Mutations cause a gain-of-function effect due to abnormal hemichannel opening. 5. Mutations that have not been thoroughly studied in vitro.
Challenges associated with large mammalian membrane protein expression and purification also greatly hampered the study of Ca2+ regulation of gap junction channels. The
dodecamer of Cx26 gap junction is a 312 kDa eukaryotic oligomer on the cell surface. Lower host such as E.coli and yeast may not harbor the capability of handling the post-translational modification, trafficking, folding, and translocation of it. The extraction of this protein form higher host also brings another challenge for purification. How to preserve the native structure of this protein as much as possible during the detergent extraction and how to prevent aggregation after detergent extraction are all problems need to be addressed. Besides, the physiological extracellular Ca2+ concentration remains at mM level, which indicates that the Ca2+-binding site in Cx26 are weak sites with mM Ca2+ binding affinity. How to directly monitor the fast on and off rate Ca2+ binding with Cx26 at molecular level present another big challenge in front of us. We need to establish direct methods which is sensitive enough to capture the interaction between Ca2+ and Cx26 proteins. All these challenges greatly hindered the study to understand the role of Ca2+ in Cx26 regulation and the molecular basis for related diseases.
In this chapter, I will report our effort in obtain purified membrane protein Cx26 using three different methods including animal tissue isolation, E.coli expression, and baculovirus expression system. After establishing high level expression of Cx26 and developing of purification method, the metal-binding properties of Cx26 and conformation change induced by metal-binding were also investigated using various spectroscopic methods. Cx26 hemichannel regulation by [Ca2+]o was also probed using dye uptake assay in mammalian cells and using electrophysiological measurement in oocytes. I will summarize all work I have done on this project, and the advantages and limitations of methods used. Then I will report some new discovery about Ca2+ regulation in this filed recently in the discussion section.