Expressions, Localization And Functional Analysis Of Deafness-Associated Connexin26 Mutants

Hearing impairment is the most common sensory disorder causing communication disturbance.Nearly 50% of autosomal recessive non-syndromic hearing loss is associated with mutations in GJB2. The 35delG mutation showed a high prevalence among Caucasian populations while the 235delC mutation was found predominantly within individuals of Asian decent. Mutations in the connexin26 gene (GJB2) are also associated with autosomal dominant non-syndromic hearing loss and inherited syndromic hearing loss. It's the most common cause of genetic deafness. CX26 (Connexin 26) is a gap junctional protein that is encoded by GJB2. Gap junction channels,formed at the appositional plasma membranes by a family of related proteins named connexins,allow the diffusion of ions and small molecules (molecular weight<1000 Da,such as metabolites and the second messengers) between adjacent cells and provide a mechanism of synchronizing response of groups of cells to environment stimuli. Gap junction intercellular communication (GJIC) plays a key role in cell metabolism,cell proliferation and cell differentiation and homeostasis,Gap junction intercellular communication includes ionic coupling and biochemical coupling.Objective Our objective is to study the effect of various point mutations (S19T,E47K,V84L,V95M,R165W,R32H,R143W,S199F and L214P) in different domains of CX26 as well as mutations (35delG,235delC, 572delT,465 T→A and 631-632delGT) causing truncated proteins of various lenghth on assembly, localization and function of CX26. Among these mutations, R32H, R165W, S199F,465T→A,572delT and 631-632delGT have not been reported to be studied in exogenous expression system.All of the 5 deletion mutations have no carboxyl terminus.The possible effects of the absence of carboxyl terminus on trafficking,localization and function of mutant proteins will be discussed.Our study will improve the knowledge on the mechanism of how GJB2 mutations cause hearing loss and the development of therapeutic methods.Method We constructed expression plasmids of 9 missense mutations each of which is located in a domain of CX26 and 5 mutations causing truncated proteins including c.235delC and c.35delG which are most common in Asia and Caucasian population respectively,c.465 T→A that we discovered previously,c.572del and c.631-632delGT.We used overlap extension PCR and "long-primer PCR" to introduce mutations in CDS of CX26.The CDS with mutations as well as the CDS of wild type CX26 were directionally subcloned into pEGFP-Nl respectively. After successfully constructed,these plasmids were transfected into Hela cells using lipofectamine 2000.The expression of mutants and wild type CX26 was analyzed by Western-blot and the localization were observed under fluorescence microscopy with immunofluorescence technics.The subcellular localization of the mutants which couldn't form gap junctions were identified by analyzing the colocalization of these mutants with Endoplasmic Reticulum (ER) marker or Golgi marker. Biochemical coupling of the mutants which can form gap junctions was tested by calceinAM dye transfer experiments.Result The mutations p.R32H,p.E47K,p.V84L,p.V95M,p.R143W, p.R165W,p.S199F and c.35delG,were introduced by overlap PCR,and the mutations p.S19T,p.L214P,c.235delC,c.465T→A,c.572delT and c.631-632delGT were introduced by long-primer PCR.The expression of all mutant proteins except c.35delG was visible as the results of western blot showed.Mutants p.R32H,p.E47K,p.V84L,p.V95M,p.R143W, p.R165W,p.S199F,p.S19T and p.L214P encoded full-length products while the molecular weights of the mutant proteins c.235delC,c.465T→A,c.572delT and c.631-632delGT were lower than that of wild type CX26. The mutant proteins p.S19T, p.E47K, p.V84L, p.V95M, p.R165W meanly located on cell membrane and could form gap junction plaques.The mutant proteins p.R32H,p.R143W, p.S199F, p.L214P,c.35delG,c.235delC,c.465 T→A, c.572delT,c.631-632delGT displayed cytoplasmic accumulation and couldn't be transported to plasma membrane.Further study showed that,these mutant proteins were colocalized with ER marker but not Golgi maker,indicating that they were accumulated in ER.The dye transfer rate of p.V84L showed no significant difference with wild type CX26,but p.S19T,p.E47K,p.V95M, p.R165W couldn't mediate dye transfer.Conclusion Mutant proteins p.R32H,p.R143W,p.S199F,p.L214P, c.235delC,p.Y155X,c.572delT and c.631-632delGT could not form gap junction and accumulated in ER after synthesis probably demonstrating that these 8 mutant proteins got defect in ER-to-plasma membrane trafficking.4 truncated proteins c.235delC,c.465T→A,c.572delT and c.631-632delGT, also accumulated in ER and couldn't be transport to plasma membrane,indicating that the C-terminal of CX26 contains sequence important for trafficking.p.V84L can form functional gap junction with biochemical coupling while p.S19T, p.E47K, p.V95M, p.R165W couldn't,although they could form gap junction plaques.The mutant c.35delG couldn't express in HeLa cells.The deafness-causing mechanisms of different missense mutations might not be identical and no correlation could be observed between the mutation and the topological domain of the mutant protein.