Analysis of the structure and function of the voltage sensor of connexins 26 and 32

Abstract

Our lab has previously ascertained that the N-terminal region of Cx32 and Cx26 comprises part if not the entire voltage sensor which detects differences in transjunctional voltage between the cytoplasms of the two connected cells and initiates voltage dependent gating. Only one subunit is sufficient to initiate voltage dependent gating. This thesis presents data which shows that residues up to and including residue 10 in the N-terminus of Cx32, when mutated to a negatively charged residue, can change the polarity of gating from closing on relative Vj negativity to closing on relative Vj positivity. We present here a convincing structural argument for the N-terminal portion of type I connexins, in particular, Cx26 and Cx32. We analyze a peptide comprised of the first 15 amino acids of Cx26 using 1H 2D NMR and show that two distinct structures exist within the peptide, separated by a flexible hinge region. This flexible region contains a glycine, G12, which is conserved among all type I connexin proteins. The hinge region forms a turn, which is able to place the relevant residues in the pore where they can sense the transjunctional electric field. When the conserved G12 is mutated to proline, the connexin maintains function, whereas residues with bulky side groups cause no expression of the channel in oocytes. A model is presented which consists of the NMR structure of the N-terminus of Cx26 connected to a previously modeled TM1. A hexamer is constructed to show that, using one of the low energy structures obtained through NMR analysis, we can see that it is possible to place the N-terminus within the channel pore. This thesis brings structure and function together and correlates the biophysical data with the observed NMR data.