XB-IMG-150760
Xenbase Image ID: 150760
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Figure 4. Homology Modeling of the DmHKT1 Pore.(A) DmHKT1 channel (ribbon plot in cyan) embedded in an artificial phosphatidylethanolamine (POPE) membrane bilayer (lipid molecules shown as stick representation). The model of DmHKT1 was placed in the bilayer using the ProBML web server (http://compbio.clemson.com/problm_webserver). The thickness of the membrane bilayer and the distance between the potassium ion (magenta sphere) in the selectivity filter and the upper boundary of the membrane were measured with Quanta2008. The insert shows the Km dependency of DmHKT1 S84G on clamped voltage. Data were fitted with an exponential function (Km = Km0 exp[δzFVm/RT]). Since zF/RT = 0.0417 mV−1, the resulting parameters were Km0 = 139.46 ± 11.54 mM and δ = 0.370 ± 0.012 (n = 6). Currents and voltages were recorded with standard bath solution containing indicated K+ concentrations.(B) 3D homology model of the D. muscipula ion channel DmHKT1 shown as ribbon plot with the chain colored from blue (N terminus) to red (C terminus). The pore and metal-binding site are indicated.(C) Magnification of the four pore-lining loops of wild-type DmHKT1 with the carbon atoms colored blue (loop 1, amino acids [aa] Thr81 to Ser86), green (loop 2, aa Ser235 to Thr240), yellow (loop 3, aa Arg359 to Ser364), and red (loop 4, aa Gly464 to Ser469). The hydroxyl group of serine 84 is located inside the pore lumen and participates in the coordination of the sodium ion (magenta sphere). Stippled lines indicate the metal ion coordination by the surrounding carbonyl groups of Val82, Ser236, Val237, His360, Thr361, Asn465, and Val466, and the hydroxyl group of Ser84. The distance between the carbonyl group of Val82 and the metal ion exceeds 3.2 Å, and therefore may not contribute to metal coordination (black stippled line).(D) Magnification of the DmHKT1 variant with a glycine residue occupying position 84 (S84G). The exchange of serine 84 in the first pore-lining loop by glycine results in altered backbone conformation of the preceding amino acid residues Ser83 and Val82 (see also Supplemental Figure 3), thereby reorienting their carbonyl groups toward the metal coordination site in the pore. This conformation is also found in potassium-conducting bacterial ion channels of this channel family, e.g. TrkH and KtrB, suggesting that pore loops containing a central glycine residue can adopt favorable backbone conformations for enhanced potassium binding.(E) Magnification of a mutant ion channel DmHKT1 with alanine at position 84 (S84A). The metal coordination is supposedly similar to wild-type DmHKT1 carrying a serine residue at this position. However, the missing serine hydroxyl group, hence the lack of one metal coordination site, impairs the Na+–K+ discrimination.(F) Steady-state currents (Iss) of DmHKT1 S84G (black) and DmHKT1 S84A expressing oocytes at −140 mV and in 100 mM of the indicated cations (n = 4, mean ± SD). Image published in: Böhm J et al. (2016) © 2016 The Authors. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives license Larger Image Printer Friendly View |