Peptide NMR Studies of Cx26 Interdomain Interactions and their Regulation by Taurine

Protonated aminosulfonates (AS), notably taurine, directly and reversibly inhibit homomeric and heteromeric channels that contain Cx26, a widely distributed connexin, but not homomeric Cx32 channels. Our previous biophysical studies identify the carboxyl-terminal domain of Cx26 (Cx26CT) as a key component of the channel modulation, suggesting a mechanism by which AS disrupts a pH-dependent association between the CT and cytoplasmic loop of Cx26 (Cx26CL), leading to occlusion of the pore [1,2]. Using recombinantly expressed Cx26CL and a Cx26CT peptide, NMR showed that taurine binds to the CL and not the CT, and that the CT and CL directly interact [3]. Our next goal to further characterize this mechanism is to identify the Cx26CL residues that directly interact with the Cx26CT and taurine. The Cx26CL construct used in the prior study (residues 98-139) contained a number of hydrophobic residues on the C-terminus, likely membrane associated, which decreased solubility below levels needed for detailed structural studies. Therefore, we created a shorter Cx26CL construct (residues 98-134) and were able to achieve millimolar soluble concentrations. Using this construct, we demonstrated by NMR a substantial interaction between the Cx26CT and the Cx26CL, which was blocked by taurine. Ongoing studies will determine (i) the binding affinities between the molecular partners, (ii) identify the Cx26CL residues involved in these regulatory interactions, and (iii) explore the potential effects of the Cx32CL and CT domains on these interactions, as may occur in Cx26-Cx32 heteromeric channels. Support: GM072631 & GM101950.References1. Bevans, C.G. & Harris, A.L. (1999) J. Biol. Chem. 274:3711.2. Tao, L. & Harris, A.L. (2004) J. Biol. Chem. 279:38544.3. Locke, D. et al. (2011) J. Gen. Physiol. 138:321.