The functional significance of heteromeric cx26 and cx30 gap junction channels in the inner ear.

Gap junctions are sites of direct communication between adjacent cells. Channels through the membrane of one cell are aligned precisely with channels through the membrane of its neighbour allowing the passage of ions, some nutrients and small messenger molecules from one cell to another. These channels are formed by members of the 'connexin' family of proteins. There are 21connexin types in humans. The different connexins form specific channels that can select what is allowed through. Gap junctions are present in almost all body tissues, but each tissue makes only a few connexins, presumably those with channel properties suited for the functioning of that tissue. Mutations in genes that code for a particular connexin can result in an abnormal protein. That affects the ability of the gap junction to allow intercellular transfer. Mutations in the genes for two particular family members, connexin(cx)26 and cx30, cause deafness. Some of these mutations only cause deafness, even though both connexins are produced in other tissues. Mice which have been 'genetically engineered' to remove either cx26 or cx30 from the cochlea are also deaf, but show no other symptoms. Thus, both cx26 and cx30 must be important for hearing. Our previous work has suggested that in the inner ear cx26 and cx30 can combine together to make a unique kind of gap junction channel ('heteromeric' cx26/cx30 channels). Cx26 and cx30 are not present together in the same cell in any other tissue. The ear of birds contains neither cx26 nor cx30. Instead it possesses another connexin called chicken-(c-)cx31 that is found only in the inner ear. Cx26/cx30 and c-cx31 channels may therefore have particular properties that are essential to hearing. This project will determine some of the characteristics of the gap junctions formed by the connexins present in the cochlea. We will first use cultures of cells that do not normally form gap junctions and force them to produce the connexins in which we are interested. Different fluorescent dyes, whose molecules differ in size and charge, will be injected into a single cell to discover whether, and how efficiently, each one can transfer to adjacent cells. This will tell us about the properties of molecules that the channels normally allow to pass. The transfer of certain naturally occurring signalling ions and molecules will also be tested. We predict that gap junctions with cx26/cx30 channels will have similar characteristics to those that contain c-cx31 but different from those which contain only cx26 or only cx30. We will also use this cell culture system to test whether deafness-causing mutations of cx26 affect heteromeric cx26/cx30 channels. This will further test whether cx26/cx30 channels are likely to be important in the inner ear. We will then examine the properties of gap junctions in their real environment using thin slices of the cochlea of mice. These slices provide access to the cells in the cochlea in a living state with the arrangement of cells undisturbed. Dye transfer and passage of signalling molecules as tested in the cell cultures, will show whether and where gap junctions with the characteristics defined in the cultures exist in the cochlea. The pathways of intercellular communication in the cochlea will be traced using a dye that can pass through almost all types of connexin channel. These normal properties will be contrasted with those of gap junctions in a mouse engineered to display an inherited connexin-related deafness. This will find out how and where the mutation affects intercellular communication. The results will help explain how specific connexins support particular cellular functions, and how gap junctional intercellular communication supports hearing.

Gap junctions are large and numerous in the inner ear. The predominant gap junction proteins expressed in the cochlea are connexin(cx)26 and cx30. They co-localise in the supporting cells of the organ of Corti and in cells of the cochlear lateral wall, including the ion-transporting epithelium the stria vascularis. Findings that numerous mutations in the genes encoding cx26 and cx30 cause hereditary sensorineural deafness has established the significance of gap junctional intercellular communication (GJIC) to cochlear function. Several mutations in Cx26 cause deafness non-syndromically. In mice in which cx26 has been ablated exclusively from the supporting cells, the sensory 'hair' cells die. Hair cell death also occurs in mice expressing a dominant deafness mutation in cx26, and in mice lacking cx30, where the endocochlear potential (EP) is also inhibited. EP provides a driving force for cochlear amplification, and is maintained by activity of the stria vascularis. These results reveal a role for both cx26 and cx30 in the maintenance of cochlear homeostasis, but also show that in the organ of Corti these two connexins cannot compensate for each other. One proposed role for gap junctions in the cochlea is K+ re-circulation. Since both cx26 and cx30 are known to be freely permeable to K+ it is difficult to reconcile the removal of either one entirely to effects on K+ permeability. Our previous work suggests that in the inner ear cx26 forms heteromeric channels with cx30, a combination that maybe unique to the inner ear. In birds there a connexin that is unique to the inner ear, chicken-cx31 (c-cx31), whose distribution matches that of cx26/cx30 neither of which is expressed in the avian cochlea. This suggests that gap junction channels with particular properties (beyond K+ permeability) are required in the cochlea. This project will identify characteristics of gap junctions composed of heteromeric cx26/cx30 channels, and c-cx31, to determine likely physiological roles. We anticipate that heteromeric cx26/cx30 and c-cx31channels will show similar characteristics, but that are different from cx26 only or cx30 only channels. In transfected HeLa cells comparisons will be made between cells co-expressing cx26 and cx30 and those expressing only cx26, cx30 or c-cx31 for their abilities to transfer dyes of different size and charge. The ability of these cells to mediate physiological signalling mechanisms will be investigated by imaging Ca2+ waves under appropriate conditions. Transfected HeLa cells will also be used to determine whether cx26 mutations that cause non-syndromic deafness affect heteromeric cx26/30 channels. Failure of these channels to transfer dyes or signalling molecules will be further investigated by double patch clamp experiments. The characteristics of native gap junctions in the structurally mature cochleae of mice will be examined in cochlear slice preparations. Cochlear slices enable access to cells in hearing animals with the normal tissue architecture preserved. Analyses of dye transfer and of physiological signalling pathways will be made to determine whether and where gap junctions with the characteristics of heteromeric cx26/cx30 channels exist in the organ of Corti and cochlear lateral wall. These characteristics in normal tissue will be compared with those in the cochleae of transgenic animals expressing a dominant deafness-causing mutation in cx26 (R75W) to identify the physiological consequences of the mutation. This project will identify the specific properties of GJIC that enable hearing, and help explain a biological event that causes deafness in millions of people worldwide. It will also better inform ongoing mathematical models of GJIC and lead to a deeper understanding of the role played by specific connexins in tissue homeostasis.