Vicia faba SV channel VfTPC1 is a hyperexcitable variant of plant vacuole Two Pore Channels

  1. Jinping Lu
  2. Ingo Dreyer
  3. Miles Sasha Dickinson
  4. Sabine Panzer
  5. Dawid Jaślan
  6. Carlos Navarro-Retamal
  7. Dietmar Geiger
  8. Ulrich Terpitz
  9. Dirk Becker
  10. Robert M Stroud
  11. Irene Marten
  12. Rainer Hedrich

  • Curated by eLife

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    Plant intracellular ion channels are poorly understood. In this important manuscript, patch-clamp is used to define functional differences between two cation channels present in the vacuole of different plants. The authors find a calcium-biding site whose absence or presence modulate activation at lower voltages and is responsible for increased excitability in the vacuole of the faba bean plant. The experimental evidence presented is convincing and findings have practical implications for the field of plant electrophysiology and channel biophysics.

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Abstract

To fire action-potential-like electrical signals, the vacuole membrane requires the two-pore channel TPC1, formerly called SV channel. The TPC1/SV channel functions as a depolarization-stimulated, non-selective cation channel that is inhibited by luminal Ca 2+ . In our search for species-dependent functional TPC1 channel variants with different luminal Ca 2+ sensitivity, we found in total three acidic residues present in Ca 2+ sensor sites 2 and 3 of the Ca 2+ -sensitive AtTPC1 channel from Arabidopsis thaliana that were neutral in its Vicia faba ortholog and also in those of many other Fabaceae. When expressed in the Arabidopsis AtTPC1-loss-of-function background, wild-type VfTPC1 was hypersensitive to vacuole depolarization and only weakly sensitive to blocking luminal Ca 2+ . When AtTPC1 was mutated for these VfTPC1-homologous polymorphic residues, two neutral substitutions in Ca 2+ sensor site 3 alone were already sufficient for the Arabidopsis At-VfTPC1 channel mutant to gain VfTPC1-like voltage and luminal Ca 2+ sensitivity that together rendered vacuoles hyperexcitable. Thus, natural TPC1 channel variants exist in plant families which may fine-tune vacuole excitability and adapt it to environmental settings of the particular ecological niche.

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  1. Version published to 10.7554/elife.86384 on eLife
  2. eLife

    eLife assessment

    Plant intracellular ion channels are poorly understood. In this important manuscript, patch-clamp is used to define functional differences between two cation channels present in the vacuole of different plants. The authors find a calcium-biding site whose absence or presence modulate activation at lower voltages and is responsible for increased excitability in the vacuole of the faba bean plant. The experimental evidence presented is convincing and findings have practical implications for the field of plant electrophysiology and channel biophysics.

  3. eLife

    Reviewer #1 (Public Review):

    In this manuscript, the authors compare the behavior of the intracellular ion channel TPC1 in two species of plants. TPC1 channels are cation permeable pseudo tetramers which in plants are expressed in the intracellular vacuole and whose function is not well understood.

    It was previously known that calcium ions are modulators of the function of these channels and the authors identify a cluster of negative charges facing the lumen of the vacuole that can regulate the action of calcium and modulate the voltage dependence of the channels. Interestingly, this cluster is not entirely present in the faba bean (Vicia faba) vfTPC1 channel.

    Through electrophysiological recordings of transfected channels into TPC1-lacking vacuoles, the authors show that the vfTPC1 channels activate at more negative voltages, even in the presence of high concentrations of calcium, leading to cation entrance into the vacuole and increased excitability, as assessed by action potential firing.

    Though the findings are interesting and the methods are of high quality, the authors fail to convey the importance of the problem they are tackling and do not frame their findings in the broad physiology of the plant species they study.

    These findings should be of interest to plant physiologists and channel biophysicists interested in TPC1 channels.

  4. eLife

    Reviewer #2 (Public Review):

    This manuscript describes the structural differences between the prototypical TPC1 channel from Arabidopsis thaliana and its ortholog in Vicia faba that are responsible for divergent channel sensitivity to luminal Ca2+ and in turn make the Vf vacuoles hyperexcitable. It is speculated that these differences might be responsible for species-specific adaptation of these plant species to their environments. Luminal Ca2+ inhibits both of these TPCs (to a different extent) by slowing down the voltage activation and shifting the voltage dependence towards positive potentials. Previously, authors demonstrated that site 3 residues (E605, D606 and D607) in AtTPC1 sense inhibitory luminal Ca2+ that is allosterically coupled to the voltage sensor.

    The main strength of the current manuscript is that authors cloned the VfTPC1 channel, and then using a combination of mutagenesis and electrophysiology to demonstrate that only two of the above three residues at site 3 (that are acidic in At but neutral in Vf) are primarily responsible for the luminal Ca2+-sensitivity differences observed between these two species (At and Vf). These modifications make Vf vacuoles hyperexcitable relative to At. The experimental data is robust, and the primary conclusions are mostly justified.

    Although the results are fascinating, they are not entirely surprising based on the data presented in a previous publication from the same group. The exact significance of these findings with respect to species-specific plant physiology is not clear, yet this presents a promising and fertile ground for future research.

  5. eLife

    Reviewer #3 (Public Review):

    In this manuscript by Lu et al., the authors cloned TPC1 from Vicia faba (VfTPC1) and characterized its channel properties by patching the vacuoles isolated from VfRPC1 expressing TPC1-loss-of-function Arabidopsis mutant tpc1-2. They found that VfTPC1 displayed faster kinetics, higher voltage dependence, and less sensitivity to luminal calcium than its Arabidopsis orthologue (AtTPC1). Mutating three luminal residues (E457, E605 and D606) in AtTPC1 to the corresponding ones in VfTPC1 converted the channel into one that resembles VfTPC1: hyperactive and desensitized to luminal Ca2+. By constructing a VfTPC1 model based on the published Ca2+-bound AtTPC1-D454N (fou2) cryo-EM structures, the authors proposed a Ca2+-dependent interaction between the E605/D606 motif and a Ca2+ coordination site at the luminal entrance of the selectivity filter (D269/E637; in VfTPC1, D271/E639). Finally, they showed that vacuoles with VfTPC1 or AtTPC1- triple mutant were hyperexcitable. Overall, this is an interesting study that might have both evolutional and functional implications.

  6. Version published to 10.1101/2021.12.22.473873v2 on bioRxiv
  7. Version published to 10.1101/2021.12.22.473873v1 on bioRxiv