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Protoplasma
2022 May 01;2593:615-626. doi: 10.1007/s00709-021-01677-3.
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The molecular identity of the characean OH- transporter: a candidate related to the SLC4 family of animal pH regulators.
Quade BN
,
Parker MD
,
Hoepflinger MC
,
Phipps S
,
Bisson MA
,
Foissner I
,
Beilby MJ
.
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Characeae are closely related to the ancient algal ancestors of all land plants. The long characean cells display a pH banding pattern to facilitate inorganic carbon import in the acid zones for photosynthetic efficiency. The excess OH-, generated in the cytoplasm after CO2 is taken into the chloroplasts, is disposed of in the alkaline band. To identify the transporter responsible, we searched the Chara australis transcriptome for homologues of mouse Slc4a11, which functions as OH-/H+ transporter. We found a single Slc4-like sequence CL5060.2 (named CaSLOT). When CaSLOT was expressed in Xenopus oocytes, an increase in membrane conductance and hyperpolarization of resting potential difference (PD) was observed with external pH increase to 9.5. These features recall the behavior of Slc4a11 in oocytes and are consistent with the action of a pH-dependent OH-/H+ conductance. The large scatter in the data might reflect intrinsic variability of CaSLOT transporter activation, inefficient expression in the oocyte due to evolutionary distance between ancient algae and frogs, or absence of putative activating factor present in Chara cytoplasm. CaSLOT homologues were found in chlorophyte and charophyte algae, but surprisingly not in related charophytes Zygnematophyceae or Coleochaetophyceae.
Fig. 1. OH− transporter background. a
Chara australis plant (Beilby and Casanova 2014). b Internodal Chara cell in the light shows the banding pattern in pH indicator Bromothymol Blue. c DIC (dissolved inorganic carbon) speciation in water (Pedersen et al. 2013). d The transporters involved in the banding pattern and the OH− transporter response in saline media (Absolonova et al. 2018). In the alkaline band: OH− transporters (blue). In the acid band: H+ ATPase AHA (red), Na+/H+ antiporter NHX (orange), non-selective cation channel HKT (violet), which is thought to let Na+ into the cell in saline media (see Phipps et al. 2021, for characterization of the transporters). CO2 and H2CO3 (black) permeate through the lipid bilayer. For further details see text. The contrast of the e I/V and f G/V characteristics of the proton pump dominated state (black, pH 7.1, statistics: 12 I/V profiles from 5 cells) and OH− transpoter dominated state (blue, pH 11.1, statistics: 12 I/V profiles from the same 5 cells). The data were fitted (lines) by Gradmann-Sanders-Slayman model for the proton pump and E-GHK model for the OH− transporter (see Beilby and Bisson 2012 for model parameters)
Fig. 2. Protein sequence alignment of mouse Slc4a11 and Chara australis CaSLOT. Alignment performed by Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) and manually refined. Colours represent the three major structural domains: cytosolic amino-terminal domain (yellow), membrane-spanning domain (blue, with individual membrane spans in grey), and cytosolic carboxy-terminal domain (red). Conserved residues mutated in disease in human SLC4A11 (Alka and Casey 2018) are shown in pink
Fig. 3. Protein sequence alignment of homologous forms of CaSLOT proteins of various green algae. Bicarbonate transporter family domains (IPR003020) are marked according to InterPro analyses (https://www.ebi.ac.uk/) and denote sequence homology to bicarbonate transporters of the SLC4 protein family rather than demonstrated functionality. Sequences used in the alignment: Chara australis SLOT (CaSLOT, GenBank: MN103545); Chara braunii, hypothetical protein CBR_g23541 (GenBank: GBG77214.1); Klebsormidium nitens (GenBank: GAQ87198.1); Ostreococcus tauri, HCO3− transporter family-domain-containing protein (GenBank: OUS47809.1); Micromonas pusilla (XP_003055831.1)
Fig. 4. Protein sequence alignment of homologous forms of CaSLOT proteins of various land plants. Bicarbonate transporter family domains (IPR003020) are marked according to InterPro analyses (https://www.ebi.ac.uk/) and denote sequence homology to bicarbonate transporters of the SLC4 family rather than functionality. Sequences used in the alignment: Arabidopsis thaliana (GenBank: AAD26598.1); Glycine soja (GenBank: KHN06659.1); Selaginella moellendorffii, hypothetical protein (GenBank: EFJ22813.1); Physcomitrella patens, anion exchange protein 4-like isoform X1 (XP_024395568.1); Marchantia polymorpha, hypothetical protein (GenBank: PTQ38262.1); and Chara australis SLOT (CaSLOT, GenBank: MN103545)
Fig. 5. Representative I-V plots gathered from H2O-injected and CaSLOT-expressing oocytes. a H2O-injected cell and b CaSLOT-cRNA-injected cell. The white data points show the initial relationship between current and voltage with the cells in pH 7.5 bath solution. The black data points show the relationship between current and voltage of the cell membrane after 5 min of exposure to pH 9.5 bath solution. The grey data points in panel b show that the change in the relationship between current and voltage in CaSLOT-expressing cells in response to increased extracellular pH is reversible upon return to pH 7.5 bath solution
Fig. 6. Comparison of conductance values gathered from water-injected controls and CaSLOT-expressing oocytes. The first column (a) shows the response of H2O-injected cells as the bath solution switches from pH 7.5 to pH 9.5. The second column (b) shows changes in conductance of CaSLOT-expressing cells under the same conditions. The third column (c) shows the effect on a subset of the cells from column (b), of restoring bath pH to 7.5. The fourth column (d) shows how the conductance of a subset of the CaSLOT-expressing cells from column (b) responds to a 5 min exposure to 1 mM ZnSO4. Statistics are the result of paired t-tests
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