![Figure 6. pH-dependent binding of Cx26CT to Cx26CL is modified by taurine. Interaction between the CT peptide and peptides based on the CL domain at different pHs, and the effect of taurine (absence [−] or presence [+]) on the interaction was assessed by a modified ELISA technique. A peptide corresponding to the entire Cx26CT was immobilized and used as bait. (A) pH-dependant interaction between Cx26CT and the second half of the Cx26CL (CL3) occurred at acidic pH and was largely reversed by taurine. In contrast, CL2 (peptide corresponding to the middle 20 amino acids of the Cx26CL) showed no significant interaction with the CT peptide, and although CL1 (peptide corresponding to the first 20 amino acids of the Cx26CL) showed some interaction at acidic low pH, this was unaffected by taurine. (Bottom) Schematic of the nonmembrane residues of rat Cx26, the positions of the three biotCx26CL peptides used for the ELISA, and homology comparisons (black, neutral; red, negative; blue, positive) between biotCL3 and the corresponding segments in rat Cx32 and human Cx43. The CL-TM3 boundary of human Cx26 as shown by crystallography (Maeda et al., 2009) is identified by apposing arrows. The helical wheel representation of CL3 indicates an amphipathic character, with a strongly hydrophobic stripe (I, L, I), if it is α helical (the CL was unresolved in the crystal structure). (B) Fractional binding is plotted against the concentration of biotCx26CL residues 120–139 (CL3) at pH 6.5 (filled squares) and pH 7.4 (open circles). Dissociation constants (Kds) were determined from single-exponential fits to the data. (C) Fractional binding is plotted as a function of the log taurine concentration at pH 6.5 (filled squares) and pH 7.4 (open circles). Binding at 0 mM taurine is normalized to unity for each pH condition. Each data point is the mean of three independent experiments at 50 and 100 ng/µl of immobilized Cx26CT bait. In A and C, CL peptide concentration was 10 µM. In A, taurine concentration was 10 mM, and filled bars are nonspecific interaction with BSA. No significant binding of any biotCx26CL peptide was detected when BSA (1% wt/vol) was used as bait or ligand, when biotCx26CL was added to uncoated plates, or plates were coated with 1% wt/vol BSA. Error bars are standard errors of the mean.](https://cdn.rupress.org/rup/content_public/journal/jgp/138/3/10.1085_jgp.201110634/4/jgp_201110634_rgb_fig6.jpeg?Expires=1767764378&Signature=BSzZqyZMuleV9stJjImTlwvCngkNUgo3vv72DwGIC~5Cfd456uBe1GfNWj5X1ng3JendE3o6cWx0hxf~ISbdV~zdmrQIzBJEjFODTHp-zC-NYEnw2U8NsAFwrvzBtVAMAsqy8Th2QhPNhqVR7N8puKYztx3PgVIHxhMvzKfob8EFJQDnNkF9xBUOVmRcfEaQsNlZwZsgErtBr3HGf73YXooVIHysORB6h4n2P~MWV2SD0QjkHqs66b~PplH4W8Vfk3RgWcaUDLSITKazTX6QrxrpRuBuUZq0BWSGsHqshxAPPNRgpGGVe7KtnNL-NO36HjJECvEggWm8rc4hpREnzw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
pH-dependent binding of Cx26CT to Cx26CL is modified by taurine. Interaction between the CT peptide and peptides based on the CL domain at different pHs, and the effect of taurine (absence [−] or presence [+]) on the interaction was assessed by a modified ELISA technique. A peptide corresponding to the entire Cx26CT was immobilized and used as bait. (A) pH-dependant interaction between Cx26CT and the second half of the Cx26CL (CL3) occurred at acidic pH and was largely reversed by taurine. In contrast, CL2 (peptide corresponding to the middle 20 amino acids of the Cx26CL) showed no significant interaction with the CT peptide, and although CL1 (peptide corresponding to the first 20 amino acids of the Cx26CL) showed some interaction at acidic low pH, this was unaffected by taurine. (Bottom) Schematic of the nonmembrane residues of rat Cx26, the positions of the three biotCx26CL peptides used for the ELISA, and homology comparisons (black, neutral; red, negative; blue, positive) between biotCL3 and the corresponding segments in rat Cx32 and human Cx43. The CL-TM3 boundary of human Cx26 as shown by crystallography (Maeda et al., 2009) is identified by apposing arrows. The helical wheel representation of CL3 indicates an amphipathic character, with a strongly hydrophobic stripe (I, L, I), if it is α helical (the CL was unresolved in the crystal structure). (B) Fractional binding is plotted against the concentration of biotCx26CL residues 120–139 (CL3) at pH 6.5 (filled squares) and pH 7.4 (open circles). Dissociation constants (Kds) were determined from single-exponential fits to the data. (C) Fractional binding is plotted as a function of the log taurine concentration at pH 6.5 (filled squares) and pH 7.4 (open circles). Binding at 0 mM taurine is normalized to unity for each pH condition. Each data point is the mean of three independent experiments at 50 and 100 ng/µl of immobilized Cx26CT bait. In A and C, CL peptide concentration was 10 µM. In A, taurine concentration was 10 mM, and filled bars are nonspecific interaction with BSA. No significant binding of any biotCx26CL peptide was detected when BSA (1% wt/vol) was used as bait or ligand, when biotCx26CL was added to uncoated plates, or plates were coated with 1% wt/vol BSA. Error bars are standard errors of the mean.
