Abstract

Two Pore Channels (TPCs) are cation-selective voltage- and ligand-gated ion channels in membranes of intracellular organelles of eukaryotic cells. In plants, the TPC1 subtype forms the slowly activating vacuolar (SV) channel, the most dominant ion channel in the vacuolar membrane. Controversial reports about the permeability properties of plant SV channels fueled speculations about the physiological roles of this channel type. TPC1 is thought to have high Ca2+ permeability, a conclusion derived from relative permeability analyses using the Goldman-Hodgkin-Katz (GHK) equation. Here, we investigated in computational analyses the properties of the permeation pathway of TPC1 from Arabidopsis thaliana. Using the crystal structure of AtTPC1, protein modeling, molecular dynamics (MD) simulations, and free energy calculations, we identified a free energy minimum for Ca2+, but not for K+, at the luminal side next to the selectivity filter. Residues D269 and E637 coordinate in particular Ca2+ as demonstrated in in silico mutagenesis experiments. Such a Ca2+-specific coordination site in the pore explains contradicting data for the relative Ca2+/K+ permeability and strongly suggests that the Ca2+ permeability of SV channels is largely overestimated from relative permeability analyses. This conclusion was further supported by in silico electrophysiological studies showing a remarkable permeation of K+ but not Ca2+ through the open channel.