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Int J Mol Sci
2024 Dec 02;2523:. doi: 10.3390/ijms252312955.
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Protein Structural Modeling and Transport Thermodynamics Reveal That Plant Cation-Chloride Cotransporters Mediate Potassium-Chloride Symport.
Henderson SW
,
Nourmohammadi S
,
Hrmova M
.
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Plant cation-chloride cotransporters (CCCs) are proposed to be Na+-K+-2Cl- transporting membrane proteins, although evolutionarily, they associate more closely with K+-Cl- cotransporters (KCCs). Here, we investigated grapevine (Vitis vinifera L.) VvCCC using 3D protein modeling, bioinformatics, and electrophysiology with a heterologously expressed protein. The 3D protein modeling revealed that the signatures of ion binding sites in plant CCCs resembled those of animal KCCs, which was supported by phylogenomic analyses and ancestral sequence reconstruction. The conserved features of plant CCCs and animal KCCs included predicted K+ and Cl--binding sites and the absence of a Na+-binding site. Measurements with VvCCC-injected Xenopus laevis oocytes with VvCCC localizing to plasma membranes indicated that the oocytes had depleted intracellular Cl- and net 86Rb fluxes, which agreed with thermodynamic predictions for KCC cotransport. The 86Rb uptake by VvCCC-injected oocytes was Cl--dependent, did not require external Na+, and was partially inhibited by the non-specific CCC-blocker bumetanide, implying that these properties are typical of KCC transporters. A loop diuretic-insensitive Na+ conductance in VvCCC-injected oocytes may account for earlier observations of Na+ uptake by plant CCC proteins expressed in oocytes. Our data suggest plant CCC membrane proteins are likely to function as K+-Cl- cotransporters, which opens the avenues to define their biophysical properties and roles in plant physiology.
Figure 3. Cryo-EM structure of DrNKCC1 and full-length molecular model of VvCCC in-complex with ions. (A) Cartoon representations of superposed DrNKCC1 and VvCCC viewed from the membrane plane (rmsd value 0.94 Å over Cα carbons of 1720 and 1696 residues, respectively). Cartoons illustrate the dispositions of dimers with cylindrical α-helices in chains A or B (pink for DrNKCC1, yellow and cyan for VvCCC) with pore TMDs. The pore of DrNKCC1 accommodates K+ and two Cl− ions (cpk spheres) with Na+ bound near K+, while pores of VvCCC accommodate K+ and two Cl− ions. Membrane boundaries are indicated in black dashed lines. (B) Cartoon representations of TMDs (chains B) of DrNKCC1 (yellow) and VvCCC (cyan), illustrating poses of ions (cpk spheres) in pores. Geometries of pores in TMD regions, calculated by HOLE [35], were visualized through the sets of grey dots distributed continuously across the pore diameters.
Figure 4. Details of TMDs of the DrNKCC1 cryo-EM structure (chain B; (left) panel) and VvCCC molecular model (chain B; (right) panel) in-complex with ions (cpk spheres) illustrate the poses of K+ and docked Na+ in DrNKCC1, and two Cl− ions [Cl− (1) and Cl− (2)] in pores. Residues participating in ion binding are: yellow cpk sticks for Na+ in DrNKCC1, atomic-colored cpk sticks for K+ in VvCCC, and yellow and cyan cpk lines for two Cl− in DrNKCC1 and VvCCC, respectively. Separations in dashed lines between residues and K+ (black), Na+ (yellow) and Cl− (green) are between 2.8 Å and 3.1 Å (K+), 2.1 Å and 3.0 Å (Na+), and 3.1 Å and 3.7 Å (two Cl−) for DrNKCC1, and between 2.8 Å and 3.6 Å (K+), and 3.3 Å and 3.8 Å (2Cl−) for VvCCC.
Figure 1. Plant CCC proteins display phylogenetic hallmarks of KCC cotransporters. (A) Bootstrapped cladogram of representative plant and animal CCCs inferred from 1000 replicates. Scale = substitution per site. (B) Sequence alignments of K+-(cyan), Na+-(magenta), and Cl−-(green) binding sites (s1 and s2). Numbers in italics indicate residue positions in VvCCC.
Figure 2. Ancestral sequence reconstruction with FireProtASR (https://loschmidt.chemi.muni.cz/fireprot, accessed on 25 October 2024) estimates the evolutionary history of CCCs across biological kingdoms. Scale = substitution per site. (A) Three clades of CCCs are indicated, where animal KCC and plant CCCs form one clade. (B) ProMals3D alignment of animal (magenta), bacterial (black; also framed), and plant (green) CCC ancestral sequences A24-A37. Descriptors above alignments indicate residues forming K+-(cyan), Na+-(magenta), and two Cl−-(green) binding sites (SCl (1) and SCl (2)).
Figure 5. Cross-eyed stereo views illustrating dispositions of K+ and bumetanide in TMD pores of VvCCC and hNKCC1. In both panels, interacting residues are indicated in cpk sticks, and separations between residues, K+, and bumetanide is shown at ≤3.6 Å as dashed lines. (A) VvCCC and (B) hNKCC1 complexes illustrate poses of K+ (cpk spheres) and bumetanide (yellow cpk sticks) on the background of selected TMD α-helices.
Figure 6. VvCCC-mediated 86Rb tracer fluxes display thermodynamic hallmarks of KCC cotransporters. (A) Predicted thermodynamic driving forces for KCC and NKCC cotransport into Xenopus oocytes as a function of [Cl−]i in the ND96 solution with 106.6 mM [Cl−]o, 96 mM [Na+]o, and 2 mM [K+]o. Driving forces were calculated assuming 100 mM [K+]i and 10 mM [Na+]i at 25 °C using the following equations: ΔµKCC=RTln[K+]i [K+]o+[Cl−]i[Cl−]o, and ΔµNKCC=RTln[Na+]i[K+]i [Cl−]i2[Na+]o[K+]o[Cl−]o2, where R is the gas constant (8.314 kJ·mol−1·K−1) and T is the absolute temperature. The dashed line represents FRP. (B) Time course of 86Rb uptake in oocytes injected with VvCCC or water in standard ND96 solution. Oocytes were pre-incubated in Cl−-free ND96 overnight before measurements (Cl− replaced with gluconate). Each data point is the mean ± standard deviation (SD, calculated in Microsoft Xcel 2019) of six oocytes (water) or eight oocytes (VvCCC). Asterisks represent significant differences at single time points (one-way ANOVA). (C,D) 86Rb uptake in oocytes injected with VvCCC or water after 1 h in ND96 with 10 mM KCl (C) or 5 mM RbCl (D) in the absence or presence of 100 µM bumetanide. Bars indicate mean ± standard error of the mean (SEM, calculated in Microsoft Xcel 2019). Asterisks represent significant differences between means (unpaired t-test). (E) 86Rb uptake in oocytes injected with VvCCC (blue) or water (black) after 20 min in ND96 without Na+ (left) or without Cl− (right). Bars indicate mean ± SEM. Asterisks represent significant differences between means (unpaired t-test). For all panels, asterisks denote * p < 0.05 ** p < 0.01 *** p = 0.001 **** p < 0.0001.
Figure 7. The driving force for VvCCC-mediated 86Rb uptake is positively correlated with external [K+]. (A) Predicted thermodynamic forces for KCC and NKCC cotransport in Xenopus oocytes as a function of [Cl−]i in ND96 solution with 2 mM [K+]o or 10 mM [K+]o. Calculations were performed as described in Materials and Methods. The flux reversal point is indicated by the intercept with the horizontal dotted line. Changes in [K+]o alter predicted FRP for a KCC mechanism (blue and red lines) but not for an NKCC mechanism (black line). (B) 86Rb uptake in oocytes injected with VvCCC (blue circles) or water (black circles) after 1 h in ND96 with 2 mM KCl (left) or 10 mM KCl (right). Note that increasing [K+]o only changed the magnitude of 86Rb uptake by VvCCC-injected oocytes.
Figure 8. VvCCC expression induces an electrogenic cation conductance in Xenopus oocytes. (A–C) Whole-cell electrophysiology traces showing currents recorded from water-injected control oocytes (A), VvCCC-injected oocytes (B), and VvCCC-injected oocytes pre-incubated for 30 min and recorded in the presence of 100 µM furosemide (C). Dotted red lines denote zero current level. (D) Current-voltage relationships of oocytes injected with VvCCC (blue) or water (black) in ND96 solution. Data are the mean ± SEM of four oocytes. Curves were generated by fitting data with a polynomial function. (E) Resting membrane potential of oocytes injected with VvCCC (blue) or water (black) in the ND96 solution. Data indicate mean ± SEM. Asterisks denote statistically significant differences (unpaired t-test). (F) Whole-cell conductance of oocytes injected with VvCCC (blue) or water (black) pre-incubated for 30 min with or without 100 µM furosemide. Conductance was determined from the slope of the I/V curve close to the reversal potential. Asterisks denote statistically significant differences (one-way ANOVA with Tukey’s multiple comparison test). In all panels, asterisks denote * p < 0.05 **** p < 0.0001.
