Regulatory Conformational Coupling between CLC Anion Channel Membrane and Cytoplasmic Domains  Toshiki Yamada, Kevin Strange  Biophysical Journal  Volume 111, Issue 9, Pages 1887-1896 (November 2016) DOI: 10.1016/j.bpj.2016.09.037 Copyright © 2016 Biophysical Society Terms and Conditions

Figure 1 Homology models showing arrangement of membrane helices forming the CLH-3b pore and helices H and I. (A) Orientation of pore-forming helices D, F, N, and R are shown. Red circles denote Cl- ions and approximate location of pore. (B) Orientation of pore-forming helices D, F, N, and R and helices H and I are shown. Labeled amino acids are discussed in the text. Models are based on the crystallographic coordinates for CmCLC (PDB id: 3ORG). To see this figure in color, go online. Biophysical Journal 2016 111, 1887-1896DOI: (10.1016/j.bpj.2016.09.037) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 2 Alignment of CLH-3b, CLC-0, and human CLC H and I helices and H-I loops. CLH-3b V228 and Y232 are shown in red. H-I loop is shown by dashed line. CLH-3b, and CLC-0, -1, -2, -Ka, and -Kb function as anion channels. CLCs 3–7 are Cl-/H+ exchangers. To see this figure in color, go online. Biophysical Journal 2016 111, 1887-1896DOI: (10.1016/j.bpj.2016.09.037) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 3 Effect of GCK-3 phosphorylation or phosphorylation mimic by mutation of S742 and S747 to glutamate (EE mutant) in (A) wild-type (WT), (B) V228L, and (C) Y529A mutant channels. Values are mean ± SE (n = 6–9). KD represents kinase dead. ∗P < 0.02, ∗∗P < 0.01, †P < 0.009. Biophysical Journal 2016 111, 1887-1896DOI: (10.1016/j.bpj.2016.09.037) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 4 CLH-3b homology model showing location of extracellular R256C and C505 cysteine residues. Pore-forming helices D, F, N, and R and helices H, I, P, and Q, which form the CLC subunit interface are shown. Model is based on the crystallographic coordinates for CmCLC (PDB id: 3ORG). To see this figure in color, go online. Biophysical Journal 2016 111, 1887-1896DOI: (10.1016/j.bpj.2016.09.037) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 5 Effect of GCK-3 on the activity of (A) WT-WT, (B) V228L-WT, and (C) Y529A-WT CLH-3b concatamers. Values are mean ± SE (n = 4–5). WT represents wild-type; KD represents kinase dead. ∗P < 0.05, ∗∗P < 0.0001, †P < 0.005. Biophysical Journal 2016 111, 1887-1896DOI: (10.1016/j.bpj.2016.09.037) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 6 Effect of GCK-3 on the activity of Y232A-Y232A (A) and Y232A-WT (B) CLH-3b concatamers. Values are mean ± SE (n = 4–6). WT represents wild-type; KD represents kinase dead. ∗P < 0.03, ∗∗P < 0.0001. Biophysical Journal 2016 111, 1887-1896DOI: (10.1016/j.bpj.2016.09.037) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 7 Effect of extracellular Zn2+ on MTSET reactivity of intracellular cysteine residues S809C and G822C in (A and B) wild-type and (B) V228L and Y232A mutant backgrounds. Values are mean ± SE (n = 5–9). ∗P < 0.001, ∗∗P < 0.002 compared with 0 mM Zn2+. Biophysical Journal 2016 111, 1887-1896DOI: (10.1016/j.bpj.2016.09.037) Copyright © 2016 Biophysical Society Terms and Conditions