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

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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.