Gating and Regulation of Connexin Hemichannels

The vast majority of the 20 human connexin isoforms have been associated with pathological dysfunctions, suggesting an essential physiological role for these proteins in human health. Although connexin channels are permeable to both atomic ions and small metabolites, they are highly selective to different molecules, which likely determines their distinct biological functions. They are also regulated by various stimuli, including Ca2+, voltage, and pH. Our understanding, however, of the molecular mechanisms underlying permeation and gating is extremely superficial, especially when compared with other classical ion channels. Recently, we made the surprising discovery that connexin hemichannels (and other large-pore channels) are not passive diffusion pores for molecules. Instead, they display saturation suggesting binding sites in the permeation pathway that might determine selectivity in a similar manner to transporters/carriers. Additionally, published and preliminary data from our lab suggested a major role for the N-terminal (NT) domain, which lines the channel pore, in permeation of molecules and pore occlusion. Thus, we hypothesize that the NT in connexin hemichannels serves: (1) as a critical energetic barrier for transport of molecules and (2) as the gate for controlling ionic conduction. In this proposal, we focus on two connexins, Cx26 and Cx30, in which human mutations at the NT are associated with deafness and congenital skin disorders. We will identify whether these NT mutations affect permeability, gating or both, and will provide a mechanistic basis of hemichannel dysfunction. Novel high-resolution structures of the connexin channel have been solved, which will serve as a guide for the proposed molecular dynamics simulations. Furthermore, we will complement computational data with experimental studies addressing key mechanistic questions that remain still unanswered. Our proposal is highly significant because we are testing an innovative hypothesis that will provide better understanding of the mechanisms of permeation and gating of connexin channels. Importantly, this will assist in the rational development and design of drugs or other therapeutic approaches that can specifically correct or compensate for the varied human diseases produced by connexin protein dysfunction, including deafness and skin disorders.