TEXAS TECH UNIVERSITY HEALTH SCIENCES CENTER
Connexins are the proteins that form the gap-junction channels that mediate cell-to-cell permeation of ions and hydrophilic molecules, hence underlying electrical and chemical coupling between neighboring cells. Connexins are essential for embryonic development and normal function of cells and tissues, and they also participate in pathological processes, both genetic and acquired. Gap-junction channels are formed by end-to-end docking of hemichannels (connexin hexamers), one from each of two adjacent cells. The pore of a gap-junction channel consists of three regions in series: the transmembrane region of one cell, the extracellular space between the two cells (gap), and the transmembrane region of the other cell. The general goal of the project is to understand the structural bases for the permeability properties of the gap-junction channel and hemichannel pore, and our central hypothesis is that transmembrane helices 1 and 3 of the primary sequence line the pore.
Our proposal aims to identify the pore helices in hemichannels in a lipid bilayer environment. The activities of the parent grant that are related to the present supplement request are related to the measurements of inter-helix distances in purified and reconstituted hemichannels using luminescence resonance energy transfer (LRET). The administrative supplement is aimed at accelerating the pace of progress in the completion of Aims 2 and 3 of the parent grant and incorporate validation experiments using genetically-encoded tags for the attachment of optical probes. Experiments of the parent grant have proceeded at a slower pace than anticipated because of the complex nature of the preparation (functional purified hemichannels reconstituted in liposomes) and measurements (LRET), and the fact that not all purified hemichannel preparations yield functional protein. In addition, we realized the need to perform additional experiments for validation of the LRET-based calculated distances and to determine whether there are significant differences between the crystal structure of Cx26 just published and that of Cx26 hemichannels in the lipid bilayer, the relevant biological environment. The work of the new personnel funded by this supplement, and the purchase of a new purification setup that includes the requested equipment, will allow us to obtain and analyze the proteins at a rate 2-3 fold faster than without the supplement.
The relevant Specific Aims of the parent grant are:
Aim 2: To identify the individual Cx43 helices in hemichannels by measuring inter-helix distances. We will assign the Cx43 helices of the cryoelectron-microscopy structure from inter-helices distances. The hemichannels show a symmetric arrangement and the differences in distances between diametrically-opposed homologous helices from facing monomers are large and can be easily determined using LRET. Starting with Cysteine-less Cx43, we will introduce a single cysteine at or near the end of each of the four Cx43 transmembrane helices, separately, and estimate the distance between homologous residues of the same helix in diametrically-opposed hemichannel monomers (e.g., distance between transmembrane helices 1 on the cytoplasmic side). To accomplish this aim, we will employ a newly-developed experimental system where we will use LRET to measure inter-helical distances in purified and reconstituted hemichannels of controlled composition, containing a single donor and known number of acceptors per hemichannel.
Aim 3: To determine the distances between transmembrane helices of Cx26 hemichannels. Cx26 and Cx43 vary with respect to the number of amino acids, C-terminal domain structure and gap-junction channel and hemichannel permeability. Following the Aim 2 approach, we will estimate inter-helical distances in Cx26 hemichannels to determine whether the folding of transmembrane helices within hemichannels formed by different connexin isoforms is the same.