Structural basis of the gating mechanism of connexin 26 and related hearing loss

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SUMMARY Connexin 26, as a gap junction channel, directly regulates the cell-to-cell exchange of ion, metabolites, and second messengers. Mutations of connexin 26 can cause severe hearing loss. However, the molecular gating mechanism on the structural basis of connexin 26 is still unclear, especially the closed state and malfunction of connexin 26 mutations. We have developed the native cell membrane nanoparticle (NCMN) system for high-resolution single-particle cryo-EM structure determination and functional studies of membrane proteins within their physiological conditions. We recently solved the cryo-EM structures of connexin 26 in the absence or presence of calcium ions in an open state at 2.3 Å and 2.9 Å associated with about 36 lipid molecules via the NCMN system. However, the close state of connexin 26 with high resolution is still uncovered. We hypothesize that our NCMN system's application to structure determination of Cx26 and Cx26-R75W in the presence and absence of calcium and calmodulin can provide unique structural information. Aim 1: To investigate the CLOSED conformation of the gating mechanism of Cx26. Aim 2: To elucidate the molecular basis of how the single point mutation R75W in Cx26 leads to profound hearing loss. The proposed experimental results will provide insights into detailed structural information and biochemical analysis in understanding the gating mechanism, also provide mechanistic details on abnormal channel gating function detected in this hearing disorder. The structural information may lead to some novel strategy to modulate the malfunction CX26-R75W for hearing loss.