The malate-activated ALMT12 anion channel in the grass Brachypodium distachyon is co-activated by Ca 2+/calmodulin

J Biol Chem. 2019 Apr 12;294(15):6142-6156. doi: 10.1074/jbc.RA118.005301. Epub 2019 Feb 15.


In plants, strict regulation of stomatal pores is critical for modulation of CO2 fixation and transpiration. Under certain abiotic and biotic stressors, pore closure is initiated through anionic flux, with calcium (Ca2+) playing a central role. The aluminum-activated malate transporter 12 (ALMT12) is a malate-activated, voltage-dependent member of the aluminum-activated malate transporter family that has been implicated in anionic flux from guard cells controlling the stomatal aperture. Herein, we report the characterization of the regulatory mechanisms mediating channel activities of an ALMT from the grass Brachypodium distachyon (BdALMT12) that has the highest sequence identity to Arabidopsis thaliana ALMT12. Electrophysiological studies in a heterologous cell system confirmed that this channel is malate- and voltage-dependent. However, this was shown to be true only in the presence of Ca2+ Although a general kinase inhibitor increased the current density of BdALMT12, a calmodulin (CaM) inhibitor reduced the Ca2+-dependent channel activation. We investigated the physiological relevance of the CaM-based regulation in planta, where stomatal closure, induced by exogenous Ca2+ ionophore and malate, was shown to be inhibited by exogenous application of a CaM inhibitor. Subsequent analyses revealed that the double substitutions R335A/R338A and R335A/K342A, within a predicted BdALMT12 CaM-binding domain (CBD), also decreased the channels' ability to activate. Using isothermal titration calorimetry and CBD-mimetic peptides, as well as CaM-agarose affinity pulldown of full-length recombinant BdALMT12, we confirmed the physical interaction between the CBD and CaM. Together, these findings support a co-regulatory mechanism of BdALMT12 activation by malate, and Ca2+/CaM, emphasizing that a complex regulatory network modulates BdALMT12 activity.

Keywords: calcium; calmodulin (CaM); ion channel; isothermal titration calorimetry (ITC); patch clamp; plant physiology; protein–protein interaction; stomatal function; structure–function.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Brachypodium* / chemistry
  • Brachypodium* / genetics
  • Brachypodium* / metabolism
  • Calcium* / chemistry
  • Calcium* / metabolism
  • Calmodulin* / chemistry
  • Calmodulin* / genetics
  • Calmodulin* / metabolism
  • Ion Channel Gating / physiology
  • Malates / chemistry
  • Malates / metabolism
  • Mutation, Missense
  • Organic Anion Transporters* / chemistry
  • Organic Anion Transporters* / genetics
  • Organic Anion Transporters* / metabolism
  • Plant Proteins* / chemistry
  • Plant Proteins* / genetics
  • Plant Proteins* / metabolism
  • Plant Stomata* / chemistry
  • Plant Stomata* / genetics
  • Plant Stomata* / metabolism


  • Calmodulin
  • Malates
  • Organic Anion Transporters
  • Plant Proteins
  • malic acid
  • Calcium