1. Structure and Function of TPC1 Vacuole SV Channel Gains Shape
Rainer Hedrich, Thomas D. Mueller, Dirk Becker, Irene Marten 
Molecular Plant 
Volume 11, Issue 6, Pages (June 2018)
DOI: /j.molp
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2. Figure 1
Impact of TPC1 Channel Variants with Different Susceptibilities to Inhibitory Luminal Ca2+ on Vacuolar K+ Transport.
The current–voltage curves depicted in (A) demonstrate that in contrast to fou2 and ouf8, the wild-type TPC1 outward currents, reflecting K+ uptake into the vacuole, are suppressed at high vacuolar Ca2+ loads. Contrary to fou2 (D454N), the ability to release luminal K+ into the cytosol via TPC1 channels (B) is impaired in the TPC1 pore-channel mutant ouf8 (D454N M629I) at increased vacuolar Ca2+ concentration. Note that according to the sign convention for plant endomembranes from 1992 (Bertl et al., 1992), the membrane voltages refer to the electrical potential at the cytosolic side of the vacuolar membrane, with the electrical potential at the luminal side set to zero. Before 1992, the designated tonoplast membrane voltage reflected the electrical potential for the luminal side relative to the cytosolic side and an opposite direction of the membrane ion currents was monitored (cf. Hedrich and Neher, 1987). Note also that the plant vacuole sign convention is not consistently applied for animal endosomes.
Molecular Plant  , DOI: ( /j.molp )
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3. Figure 2
Voltage-Dependent Gating of TPC1 Is Controlled by Cytosolic Calcium.
(A) Schematic view of the voltage-dependent gating process of TPC1 illustrating the synergetic effects of voltage (depolarization) and cytosolic Ca2+ on the voltage-sensing domain (VSD).
(B) At resting vacuolar membrane potentials of about −30 mV, high vacuolar Ca2+ concentration stabilizes the VSD (i.e., S10 helix, see text) in its resting state. An initial depolarization of the vacuolar membrane potential primes TPC1 channels for Ca2+-dependent gating: positively charged VSD domains in the TPC1 dimer are unlocked and slide upward. The moving VSDs pull on S4–S5 linker flanking the pore region, thereby driving TPC1 into a pre-activated but still closed conformation. When cytosolic [Ca2+] transients exceed a critical threshold, determined by the Ca2+ affinity of the TPC1 EF hands, a proposed conformational change results in dilatation of the pore and opening of the permeation pathway.
Molecular Plant  , DOI: ( /j.molp )
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4. Figure 3
Stimulatory Effect of Cytosolic Ca2+ on Voltage-Dependent TPC1 Channel Transport Function.
(A) Illustration of the stimulatory effect of increasing cytosolic Ca2+ levels on the single channel activity of wild-type (WT) and fou2 at a positive membrane voltage. “C” denotes the current level with all channels being in the closed state while “O1” to “O3” define the current levels at which 1–3 TPC1 channels are simultaneously open.
(B) The depolarization-induced activation of macroscopic TPC1 currents is reproduced at low (green) and high (red) cytosolic Ca2+ levels for wild-type and fou2. Arrows indicate the instantaneous jump from a hyperpolarizing to a depolarizing membrane voltage.
(C) The voltage-dependent relative open-channel probability (rel. Po) demonstrates that in comparison with wild-type, the higher fou2 channel activity shown in (A) and (B) is linked to the shift in the voltage threshold for TPC1 channel activation to more negative membrane voltages at an elevated cytosolic Ca2+ level. Interestingly, fou2 and the wild-type-like ouf8 mutant show a similar voltage-dependent TPC1 activation behavior at stimulatory high cytosolic Ca2+ levels.
Molecular Plant  , DOI: ( /j.molp )
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5. Figure 4 Evolution of TPC Voltage-Sensing Domains.
Bacterial NaV channels (1×6TM) alike Shaker-like K+ channels exhibit single VSD (S4), which carries four Arg residues participating in voltage sensing. TPC channels from plants and animals (2×6TM) display two VSDs (S4 and S10) that differ significantly with respect to charge clustering. In contrast to vertebrate TPC1 channels harboring three Arg residues in S4 (VSD1), only two positively charged residues are found in S4 of “green” TPC1 channels according to structural alignments. This probably explains why VSD1 in the Arabidopsis TPC1 does not contribute to voltage sensing (see text). Sequence comparisons reveal that for S10 (VSD2) the situation is the other way around: plant TPC1 channels harbor three arginines while human TPC1 channels show only two. Nevertheless, both VSD1 and VSD2 seem to contribute to voltage-dependent gating in human TPC1. Sequence alignments were performed using MUSCLE, and sequence logos were obtained employing WebLogo 3. Arginines (R) shown in red letters are supposed to participate in voltage sensing according to structural modeling. Red dots indicate those that have been experimentally verified by structure–function analyses. Black arrows refer to Arg residues that upon mutation result in non-functional TPC1 channels but locate outside the VSD (S5–S5 linker; S10–S11 linker) according to published crystal structures.
Molecular Plant  , DOI: ( /j.molp )
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6. Figure 5 Evolution of TPC Cytosolic Ca2+-Sensing Domains (EF Hands).
Phylogenetic analysis of cytosolic Ca2+-binding EF hands of plant TPC1-like channels reveals clade-specific signatures. The typical consensus motif for calcium binding is a characteristic DxDxxG motif. A canonical EF-hand often consists of a Ca2+-binding helix-loop-helix domain, where two antiparallel DxDxxG calcium-binding loops are flanked by interacting incoming and exiting α helices. Seven key equivalent structural positions are present within all EF-hand domains. Six residues involved in Ca2+ binding are in positions 1, 3, 5, 7, 9, and 12 and are designated as “X,” “Y,” “Z,” “-X,” “-Y,” “-Z.” The seventh residue is at position “X-4” and is represented by an invariant Phe residue in TPC EF1 and EF2. Sequence alignments were performed with MUSCLE, and phylogenetic trees were calculated using RAxML. Trees were visualized using the Interactive tree of life (iTOL) v3 online tool. Species abbreviations are: (Charopyceae) Ccu, Cylindrocystis cushleckae; Cir, Coleochaete irregularis; Efi, Entransia fimbriat; Kfl, Klebsormidium flaccidum; Kni, Klebsormidium nitens; Msp, Mougeotia sp. (Lycopods) Hlu, Huperzia lucidula; Hse, Huperzia selago. (Bryophytes) Cco, Conocephalum conicum; Mpo, Marchantia polymorpha; Mvi, Megaceros vincentianus; Nae, Nothoceros aenigmaticus; Ppa, Physcomitrella patens; Ppi, Porella pinnata; Ppu, Ptilidium pulcherrimum; Rbe, Riccia beyrichiana; Rli, Radula lindenbergiana. (Polypodiales) Ate, Adiantum tenerum; Ehy, Equisetum hyemale; Lyja, Lygodium japonicum; Ovu, Ophioglossum vulgatum; Polic, Polypodium glycyrrhiza. (Gymnosperms) Ala, Abies lasiocarpa; Pipa, Pinus parviflora; Pra, Pinus_radiata. (Basal angiosperms) Nnu, Nelumbo nucifera; Atr, Amborella trichopoda; (Solanales) Can, Capsicum annuum; Nsi, Nicotiana sylvestris; Nta, Nicotiana tabacum; Nta, Nicotiana tabacum; Stu, Solanum tuberosum. (Caryophyllales) Bvu, Beta vulgaris; Cqu, Chenopodium quinoa; Dmu, Dionaea muscipula; Mcr, Mesembryanthemum crystallinum. (Brassicales) Aal, Arabis alpina; Aly, Arabidopsis lyrata; Ath, Arabidopsis thaliana; Bol, Brassica oleracea; Bra, Brassica rapa FPsc; Cgr, Capsella grandiflora; Csa, Camelina sativa; Esa, Eutrema salsugineum; Nca, Noccaea caerulescens; Tha, Tarenaya hassleriana. (Poales) Bdi, Brachypodium distachyon; Hvu, Hordeum vulgare; Osit, Oryza sativa Indica group; Osj, Oryza sativa Japonica group; Pvi, Panicum virgatum; Sbi, Sorghum bicolor; Sit, Setaria italica; Tae, Triticum aestivum; Zea, Zea mays. (Fabales) Car, Cicer arietinum; Gma, Glycine max; Gso, Glycine soja; Lja, Lotus japonicus; Lsa, Lathyrus_sativus; Luan, Lupinus angustifolius; Mtr, Medicago truncatula; Pan, Phaseolus angularis; Pvu, Phaseolus vulgaris; Tpr, Trifolium pratense; Van, Vigna angularis; Vicfa, Vicia faba.
Molecular Plant  , DOI: ( /j.molp )
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