The recent discovery that several recessive mutations in the gene for the gap junction provide connexin26 (also termed beta2) have been associated with non-syndrome hereditary deafness suggests that gap junctions play an important role in cochlear function. A complete understanding of how beta2 [Cx26] mutations alter gap junction channel function will only be realized with high resolution structural information. We recently used electron cryo-microscopy and image analysis to determine a three-dimensional structure at 7.5A resolution of gap junctions formed by a C-terminal truncation mutation of the principal gap junction protein in the heart, alpha1[Cx43]. For this two year proposal the aims are to: (1) Clone and express beta2 [Cx26] (2) Solubilize and purify the recombinant protein (3) Grow 2D crystals suitable for structure analysis] (4) Initiate high resolution electron cryo-crystallography No doubt such a project constitutes a high risk endeavor because membrane proteins tend to be labile when solubilized in detergent, crystallization of polytopic membrane proteins is substantially more difficult than for soluble proteins, and electron cryo-crystallography is an emerging field compared with X-ray crystallography. To date, our collaborators, Dr. Nalin Kumar and Dr. Norton B. Gilula have made substantial progress on the first two aims. It is now timely to proceed with 2D crystallization trials of recombinant, wild-type beta2[CX26]. The long term goal is to use electron cryo-crystallography to elucidate the high resolution structure of gap junctions formed by normal beta2[CX26] and mutant forms associated with hereditary deafness. Detailed structural knowledge of beta2[CX26] gap junction channels will be fundamental for understanding the molecular pathology of some forms of hereditary deafness and may provide a framework for the rational design of therapeutic strategies.
