The function of the two-pore channel TPC1 depends on dimerization of its carboxy-terminal helix

Abstract

Two-pore channels (TPCs) constitute a family of intracellular cation channels with diverse permeation properties and functions in animals and plants. In the model plant Arabidopsis, the vacuolar cation channel TPC1 is involved in propagation of calcium waves and in cation homeostasis. Here, we discovered that the dimerization of a predicted helix within the carboxyl-terminus (CTH) is essential for the activity of TPC1. Bimolecular fluorescence complementation and co-immunoprecipitation demonstrated the interaction of the two C-termini and pointed towards the involvement of the CTH in this process. Synthetic CTH peptides dimerized with a dissociation constant of 3.9 µM. Disruption of this domain in TPC1 either by deletion or point mutations impeded the dimerization and cation transport. The homo-dimerization of the CTH was analyzed in silico using coarse-grained molecular dynamics (MD) simulations for the study of aggregation, followed by atomistic MD simulations. The simulations revealed that the helical region of the wild type, but not a mutated CTH forms a highly stable, antiparallel dimer with characteristics of a coiled-coil. We propose that the voltage- and Ca²⁺-sensitive conformation of TPC1 depends on C-terminal dimerization, adding an additional layer to the complex regulation of two-pore cation channels.

Electronic supplementary material
The online version of this article (doi:10.1007/s00018-016-2131-3) contains supplementary material, which is available to authorized users.

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ORIGINAL ARTICLE
The function of the two-pore channel TPC1 depends
on dimerization of its carboxy-terminal helix
Nina Larisch
1
•Sonja A. Kirsch
2
•Alexandra Schambony
3
•Tanja Studtrucker
1
•
Rainer A. Bo
¨ckmann
2
•Petra Dietrich
1
Received: 12 August 2015 / Revised: 7 December 2015 / Accepted: 4 January 2016 / Published online: 18 January 2016
The Author(s) 2016. This article is published with open access at Springerlink.com
Abstract Two-pore channels (TPCs) constitute a family
of intracellular cation channels with diverse permeation
properties and functions in animals and plants. In the
model plant Arabidopsis, the vacuolar cation channel TPC1
is involved in propagation of calcium waves and in cation
homeostasis. Here, we discovered that the dimerization of a
predicted helix within the carboxyl-terminus (CTH) is
essential for the activity of TPC1. Bimolecular fluores-
cence complementation and co-immunoprecipitation
demonstrated the interaction of the two C-termini and
pointed towards the involvement of the CTH in this pro-
cess. Synthetic CTH peptides dimerized with a dissociation
constant of 3.9 lM. Disruption of this domain in TPC1
either by deletion or point mutations impeded the dimer-
ization and cation transport. The homo-dimerization of the
CTH was analyzed in silico using coarse-grained molecular
dynamics (MD) simulations for the study of aggregation,
followed by atomistic MD simulations. The simulations
revealed that the helical region of the wild type, but not a
mutated CTH forms a highly stable, antiparallel dimer with
characteristics of a coiled-coil. We propose that the volt-
age- and Ca
2?
-sensitive conformation of TPC1 depends on
C-terminal dimerization, adding an additional layer to the
complex regulation of two-pore cation channels.
Keywords Vacuole Calcium signaling 
MD simulation Patch-clamp 
Microscale thermophoresis Mutation
Introduction
Two-pore cation channels (TPCs) are intracellular ion
channels residing in the vacuolar membrane of plant cells,
and in lysosomes and endosomes of mammalian cells [1,
2]. Depending on their biophysical properties and host
species, they play diverse roles, ranging from cation and
pH homeostasis, sensing of the metabolic state, cell-to-cell
signaling, control of the membrane potential and mem-
brane trafficking to pigmentation, and even to the control
of Ebola virus invasion in animal cells [1–8]. The under-
lying molecular mechanisms are in most cases fairly
unknown.
TPCs belong to the superfamily of voltage-gated ion
channels that are built from Shaker-like domains of 6
transmembrane segments (S1-S6) and a pore-forming helix
between S5 and S6. One TPC monomer contains two
Shaker-like domains connected by a cytosolic linker, and
the channel functions as a dimer [1,9,10]. The cytosolic
N-terminus contains a dileucine motif, which is responsible
for targeting of the channel to the tonoplast in plant cells or
lysosomes in animal cells [11,12]. Two EF-hand motifs in
the linker domain of plant, but not animal TPCs mediate
binding and channel activation by cytosolic calcium ions
[13,14]. In contrast, animal TPC1 and TPC2 are directly
Electronic supplementary material The online version of this
article (doi:10.1007/s00018-016-2131-3) contains supplementary
material, which is available to authorized users.
&Petra Dietrich
petra.dietrich@fau.de
1
Molecular Plant Physiology, Department of Biology,
University of Erlangen-Nu
¨rnberg, Staudtstrasse 5, 91058
Erlangen, Germany
2
Computational Biology, Department of Biology, University
of Erlangen-Nu
¨rnberg, Staudtstrasse 5, 91058 Erlangen,
Germany
3
Developmental Biology, Department of Biology, University
of Erlangen-Nu
¨rnberg, Staudtstrasse 5, 91058 Erlangen,
Germany
Cell. Mol. Life Sci. (2016) 73:2565–2581
DOI 10.1007/s00018-016-2131-3 Cellular and Molecular Life Sciences
123
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activated by phosphatidyl-inositol-3,5-bisphosphate
PI(3,5)P
2
[6,7,15] and have been identified as mediators
of nicotinic acid adenine dinucleotide phosphate
(NAADP)-induced Ca
2?
-release from endolysosomes [9,
12,16–18].
While animal cells express two or more different
channels, in most plant species there is only one TPC
isoform, TPC1 [1,2]. TPC1 encodes the slow vacuolar
(SV) channel [19] and mediates the passage of K
?
and
Na
?
, and other monovalent as well as divalent cations,
although a direct involvement in calcium release from the
vacuole in vivo is strongly debated [20–25].
Recently, a role for TPC1 in a rapid, long-distance
signaling system based on Ca
2?
waves has been uncovered
[3]. In response to a localized salt stimulus at the Ara-
bidopsis root, the calcium wave mainly travels through the
cortex and endodermis at speeds of up to 420 lm/s, and is
required for activation of stress responsive genes in the
shoot. Lack of TPC1 largely reduces the travel speed of
this trigger wave, while TPC1 overexpression accelerates
it. TPC1 thus plays an important role in long-distance
signaling in response to salt stress [3]. How the density or
distribution of TPC1 in the vacuolar membrane affects the
speed of the long-distance calcium wave remains largely
unknown.
Deregulation of the voltage-dependent activity of TPC1
leads to imbalanced cation homeostasis in the vacuole of
fou2, which has a threefold higher Ca/K ratio as compared
to the wild type [26]. The expression profile of fou2
resembles that of wild type plants under K starvation, and
fou2 plants produce more oxylipins in response to
wounding [27,28]. The fou2/TPC1D454 N mutation
introduces an amino acid exchange in the binding site for
luminal Ca
2?
, which abolishes the inhibition of TPC1 by
luminal calcium ions and shifts the activity range towards
more negative potentials [26,29]. These results demon-
strate the important role of TPC1 for cation homeostasis
and vacuolar storage function.
A tight regulation of SV channels prevents loss of
potassium and other cations from the vacuole, and many
factors down-regulating or blocking TPC1 have been
identified, including luminal calcium ions [30], protons
[31], sodium ions [32], and polyamines [33]. A further
negative regulation is mediated by polyunsaturated fatty
acids, which like luminal Ca
2?
ions shift the voltage
dependence towards more negative potentials [34]. In
comparison to these ionic and metabolic factors, less is
known about interactions of TPC1 with regulatory proteins
and their sites of interaction. 14-3-3 proteins rapidly reduce
SV currents [35,36], and regulation by kinases and phos-
phatases is most likely [37,38].
Except for a few cases like calcium binding [13,29]or
block by polyamines [33], little is known about the struc-
ture function relation in TPC1. Here, we report that the
predicted carboxy-terminal helical regions of two TPC1
monomers are prone to dimerization. These dimers are
shown to be essential for the function of the (dimerized)
TPC1 channel. We employed wild type coarse-grained and
atomistic simulations, as well as coarse-grained mutant
simulations, which revealed that the wild type C-terminal
domain forms a stable antiparallel coiled-coil. In contrast,
the mutant showed a highly promiscuous dimerization
pattern, pointing to a significantly decreased affinity. We
suggest that the dimerization of the wild type TPC1 car-
boxyl-termini stabilizes the channel in a conformation,
which is sensitive to Ca
2?
-binding and depolarization,
adding an additional layer to the complex regulation of
two-pore cation channels.
Materials and methods
Plant material and growth conditions
Arabidopsis thaliana Col-0 wild type and tpc1-2mutant
plants [20] were used. After seed stratification at 4 C for
3 days, plants were grown on soil in a growth chamber
under 8 h light/16 h dark conditions at 22 C. Nicotiana
benthamiana wild type plants were cultivated on soil in the
greenhouse at 22 C under a 16 h light/8 h dark cycle and
used after 6 weeks for infiltration.
Cloning procedures
Cloning procedures for C-terminal GFP-fusions are
described elsewhere [11], and eGFP was used in all cases.
PCR-primers were designed accordingly with the respec-
tive cloning sites and with or without stop-codon and
purchased from Sigma. For construct details and primer
sequences see Supplemental Table 1 and 2.
For BiFC analyses the gateway entry vector pENTR-D-
TOPO and the destination vectors pDEST–
GW
VYCE and
pDEST–
GW
VYNE were used for C-terminal fusion of the
Venus C- and N-terminus, respectively, and pDEST–
VYCE(R)
GW
and pDEST–VYNE(R)
GW
for N-terminal
tagging [39].
For the Co-IP tests the C-terminally tagged GFP-fusions
were used as templates to amplify the soluble C-terminus
(amino acids 673–733) of TPC1 or TPC1-3LP fused to
GFP with PCR. The constructs were subcloned into the
pENTR-D-TOPO vector and afterwards brought into the
expression vectors with EcoRI and XhoI (New England
2566 N. Larisch et al.
123
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Biolabs), so that they were N-terminally tagged either with
6x Myc (pCS2?MT) or with the FLAG-tag (pCS2 ?Flag)
[40].
Protoplast isolation and transformation
Expression of GFP-fusion proteins for electrophysiological
measurements or confocal microscopy was performed in A.
thaliana tpc1-2mesophyll protoplasts as described [11,
13]. Briefly, leaves were enzymatically digested to release
mesophyll protoplasts, which were then transformed by the
polyethylene glycol method. Protoplasts were used for
electrophysiological measurements and subcellular local-
ization of GFP fluorescence 2–4 days after transformation.
Confocal microscopy
Fluorescence signals were detected and documented with a
TCS SP2 confocal laser scanning microscope and the Leica
Confocal Software (Leica Microsystems). A 488 nm Argon
laser was used for excitation of GFP and of the auto-flu-
orescence of chlorophyll. GFP-signals were detected from
500 to 556 nm and chlorophyll signals from 675 to
767 nm. Venus was excited with a 543 nm helium–neon
laser and detected in the range from 520 to 556 nm, the
corresponding chlorophyll signals were detected from 637
to 736 nm. Images were processed with Photoshop (Adobe
Systems).
Electrophysiological recordings
Patch-clamp experiments were performed on transformed
vacuoles harboring two-pore channels as identified by GFP
fluorescence. The pipette (luminal) solution consisted of
100 mM K-gluconate, 2 mM MgCl
2
, 10 mM EGTA,
10 mM MES, pH 5.5/Tris. The bath (cytosolic) solution
contained 50 mM K-gluconate, 1 mM CaCl
2
,1mM
MgCl
2
, 2 mM DTT, 10 mM HEPES, pH 7.5/Tris. Solu-
tions were adjusted to 430 mosmol/kg by adding
D-sorbitol. For analysis of the TPC1-S706 mutants two
further calcium concentrations of the bath solution were
used in which 1 mM CaCl
2
was substituted by 0.2 mM or
0.05 mM CaCl
2
, respectively. Competition assays using
the CTH peptide were performed on endogenous TPC1
channels in vacuoles isolated from Col-0 plants. Peptides
were dissolved at 10 mM in bath solution supplemented
with 1 % DMSO and 0.1 % pluronic, and diluted [600-
fold for application in patch-clamp recordings. Currents
were recorded either in the whole-vacuolar or cytosolic
side-out configuration of the patch-clamp technique, using
an EPC10 amplifier and the program PULSE (HEKA
electronics, Lambrecht, Germany). Recordings and data
analysis were performed as described earlier [11]. Relative
open probabilities (P
o
) were determined from tail currents
at -53 mV, following pulses to the different test voltages.
Tail currents were normalized to the maximum values at
1mM Ca
2?
, plotted as a function of the applied test
voltage, and fitted according to the Boltzmann equation:
PO¼A
1þexpzF
RTðVoVÞ
where V
o
and zare parameters for the voltage at half-
maximum open probability and the apparent number of
gating charges, respectively, and Adescribes the maximum
conductance that is reached under the experimental con-
ditions. For comparison of the voltage dependence in the
presence of different calcium concentrations, the gating
charge as determined for TPC1 in the presence of 1 mM
Ca
2?
was held constant for fits at lower Ca
2?
, as this
parameter does not dependent on the cytosolic Ca
2?
con-
centration [23].
BiFC, tobacco infiltration, and fluorescence
quantification
BiFC studies were performed as described [41] using a
Split Venus system [39]. The Agrobacterium strain C58C1
[42] harboring the plasmid of interest and the helper strain
p19 [43] were grown in LB medium supplemented with
100 lg/ml Kanamycin at 29 C overnight. The cultures
were brought to an optical density (OD
600
) of 1.0 in infil-
tration buffer (10 mM MES, 10 mM MgCl
2
, 100 lM
acetosyringone, pH 5.7/KOH) and the combinations to test
for co-expression mixed at equal amounts. The p19 helper
strain (OD
600
=1.0) was added to these mixtures in a 1:1
ratio. Suspensions were incubated for 2 h before infiltration
into the abaxial side of 6-week-old N. benthamiana leaves.
Each leaf was infiltrated with several plasmid combinations
and a positive control (TPC1/pDEST-
GW
VYCE and TPC1/
pDEST-
GW
VYNE) as well as a negative control (p19
alone) at separated areas to monitor comparability of the
transient expression and to define background fluorescence.
For fluorescence quantification leaf disks (Ø 6 mm)
were cut out of the infiltrated areas with a cork borer 2.5
day after infiltration. Leaf disks were placed upside-down
in a black 96-well plate prefilled with 70 llH
2
O per well
to minimize dehydration effects. Fluorescence was mea-
sured with a plate-reader (Infinite F200, Tecan) with an
excitation wavelength of 485 ±20 nm and an emission
wavelength of 525 ±25 nm [41].
Cell culture, transfection, Co-IP and western blot
analysis
HEK293T cells were propagated in DMEM GIBCO Glu-
tamax (Life Technologies) supplemented with 10 % fetal
The function of the two-pore channel TPC1 depends on dimerization of its carboxy-terminal helix 2567
123
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calf serum. Cells were seeded in 6–well plates (20–30 %
confluency) and transfected the next day using 10 ll Roti-
Fect (Carl Roth) per 2 lg total DNA (1 lg DNA per
plasmid). Two days after transfection cells were washed
with 2 ml PBS buffer (Life Technologies) and harvested
for immunoprecipitation in 150 ll cold lysis buffer
(10 mM HEPES, 150 mM NaCl, 1 % Nonidet P40, 5 %
glycerol, pH 7,4/KOH) supplemented with protease inhi-
bitors and phosphatase inhibitors (Complete mini EDTA
free and PhosStop, Roche). Cells were disrupted mechan-
ically with a syringe (cannula diameter 0.55 mm). The
lysate was cleared by centrifugation at 13.500 rpm and
4C for 10 min. Total protein concentrations were mea-
sured with a bicinchoninic acid protein assay (Applichem),
typically the total protein amount was 500–600 lg. Load-
ing controls of 50 lg of the lysate were taken accordingly
and filled up to 20 ll with lysis buffer before adding 5 ll
of 5x sample buffer (250 mM Tris, 5 % SDS, 25 % glyc-
erol, 0.25 % bromophenol blue, 250 mM b–
mercaptoethanol, pH 6.8/HCl) and boiling. For immuno-
precipitation, 3 lg of mouse anti-c-Myc Epitope antibody
9E10 (sc-40, Santa Cruz) was used per sample. Samples
were incubated with the antibody for 45 min at 4 C with a
rotator, then 10 ll of protein G Dynabeads (Life Tech-
nologies) equilibrated in the lysis buffer was added to each
sample and incubation continued for another 45 min.
Samples were washed three times with cold lysis buffer
before eluting three times with 20 ll of 100 mM glycine
(pH 2.5/HCl). After neutralizing the pH 15 ll of 5x sample
buffer was added and samples were boiled.
Proteins of the loading controls and samples were sep-
arated with SDS-PAGE (12 % gel) and transferred to a
PVDF membrane in duplicate. Membranes were blocked
for 1 h with blocking reagent (Roche) and incubated over
night at 4 C with rabbit anti-Myc or anti-Flag antibodies.
After washing three times with TBST buffer, the mem-
branes were incubated for 1 h at RT with anti-rabbit
antibody coupled to alkaline phosphatase. Membranes
were washed three times with TBST, followed by two
times with alkaline phosphatase buffer (100 mM NaCl,
100 mM Tris, pH 9.5/HCl). Colorimetric detection of the
proteins was performed with NBT/BCIP substrate (Roche)
in alkaline phosphatase buffer until a sufficient staining
was achieved. All antibodies used for the western blots
were purchased from Cell Signaling Technology.
Molecular dynamics simulations
All simulations were carried out using GROMACS 4.6.x
[44]. The secondary structure of the TPC1 C-terminal part
(679–733) was addressed applying different prediction
tools (Table 1). The consensus helical sequence
RSQRVDTLLHHMLGDEL, i.e., those residues that were
predicted by at least three out of four prediction tools, was
converted into an a-helical structure using PyMOL [45],
and the atomistic structure was changed to coarse-grained
representation with the aid of martinize [46]. Aggregation
of two TPC helices was studied from 100 simulations with
randomized starting structures, using the previously
developed DAFT algorithm [47], in conjunction with the
polarizable Martini [48] force field version 2.2; the two a-
helices were put in a dodecahedron simulation box at a
specified distance to each other. Finally, the solvent was
added using insane [49,50]. In this manner, each generated
system contained two 17 amino acids long helices and
approximately 5300 polarizable water beads. The usage of
a polarizable water model instead of a standard water
model was shown to be more appropriate for adsorption
studies of small peptides to a bilayer since the standard
water resulted in overestimation of binding, probably due
to a difference in electrostatic screening [51]. In addition to
the wild type TPC1 systems, coarse-grained systems of the
TPC1-3LA mutant were set up in the same manner as
described above and simulations were performed under the
same simulation conditions.
Five atomistic [force field Amber 14SB, 52] starting
configurations were generated from selected coarse-grained
frames of the wild type simulations using the backward
method [53]. The K
?
-ion concentration of 100 mM was
chosen similar to the cytoplasmic salt concentrations in
plants [54]. Additionally, the unit cells were altered to
smaller ones, containing approximately 5700 TIP3P [55]
water molecules as well as counterions (Cl
-
).
The modeled sequence is a small part of a larger protein
sequence. To describe the helices accurately, the termini
were kept neutral in all coarse-grained simulations or
acetylated and amidated, respectively, in the atomistic
simulations.
Simulation details
After automatically generating each 100 coarse-grained
(CG) starting structures using DAFT, the wild type and
mutant systems were subjected to a relaxation and equili-
bration process using martinate [56]. The energy was
minimized using steepest-descent (500 steps) and a 10 ps
position-restrained simulation with a time step of 2 fs.
Subsequently, the systems were relaxed in a 100 ps NpT
simulation with an integration time step of 20 fs under
constant pressure pand temperature T. In all simulations,
the isotropic pressure of 1 bar was kept constant using a
weak coupling scheme [57] with a 3 ps time constant. The
temperature was maintained at 310 K with the v-rescale
thermostat and a time constant of 1 ps [58]. The relative
dielectric constant was globally set to 2.5 and long-range
electrostatic interactions were treated using particle-mesh
2568 N. Larisch et al.
123
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Ewald [59] summation with a real-space cutoff of 1.2 nm.
Dispersion interactions were described by a Lennard-Jones
12–6 potential that was shifted to zero between 0.9 nm and
1.2 nm. The production simulations were run for 250 ns
each, using a time step of 20 fs.
Selected backmapped coarse-grained (CG) structures of
the wild type were studied at atomistic resolution at 300 K
and 1 bar using the Amber14SB force field. The simulation
length was 200 ns (4 simulations) and 270 ns (1 simulation),
respectively, with an integration time step of 2 fs. The
temperature was kept constant using the Nose
´-Hoover [60,
61] thermostat with a time constant of 0.5 ps. The pressure
was modulated isotropically with the Parinello-Rahman [62]
barostat and a time constant of 10 ps. The relative permit-
tivity was set to 1. Long-range Coulomb interactions were
calculated with the particle-mesh Ewald summation and a
real-space cutoff of 1 nm. Van der Waals interactions were
treated with a cutoff of 1 nm as typical for Amber force fields
[63]. Furthermore, long-range dispersion corrections were
applied for the energy and pressure.
Data analysis
Relative orientations of the C-terminal helices of AtTPC1
and mutated AtTPC1-3LA were described using Euler
angles (Fig. 7b), which characterize the relative orientation
of two peptides [47,64]. The matrix with Euler angles was
obtained by least-square fitting a structure on a reference
structure. To investigate the stability of formed dimers, the
tilt angle between the dimers was determined over the last
50 ns of each simulation and its distribution plotted in a
histogram. The tilt, ranging from 0to 180, was then
divided into three subintervals (0–50,50–130, and
130–180) in the case of wild type dimers and four
subintervals (0–35,35–100, 100–150, and 150–
180) in the case of mutant dimers. These intervals were
chosen according to the minima in the histogram
(Fig. S6a). Subsequently, the number of transitions
between these intervals was counted for each simulation
and further evaluated.
MicroScale thermophoresis (MST) binding assay
For MST experiments, peptides corresponding to the con-
sensus helical sequence (RSQRVDTLLHHMLGDEL) and
the 3LA mutant (RSQRVDTAAHHMAGDEL) were syn-
thesized (Peptide Speciality Laboratories GmbH,
Heidelberg, Germany). To allow for label-free MST
binding experiments, additional wild type and mutant
peptides were synthesized, which were C-terminally
extended by addition of two tryptophanes and a short linker
(AAWW).
Labelfree MicroScale Thermophoresis binding experi-
ments were performed in cooperation with the 2bind
GmbH (Regensburg, Germany), using 750 nM tryptophane
containing target peptide in PBS pH 7.5, 1 % DMSO,
0.1 % Pluronic with varied concentrations of the ligand
peptide at 80 % MST power, 100 % LED power in
hydrophilic zero background capillaries on a Monolith
NT.labelfree device at 25 C (NanoTemper Technologies,
Munich, Germany). Normalized fluorescence data sets
(WT peptide and MT peptide) were analyzed in the ther-
mophoresis and temperature jump. For determination of the
binding affinity (K
D
) of the wild type peptide, the recorded
fluorescence was normalized to the fraction bound
(0 =unbound, 1 =bound), and fitted using the K
D
fit
formula derived from the law of mass action. Technical
duplicates were performed for each experimental setup.
Sequence information
Sequence data from this article can be found in the EMBL/
GenBank data libraries under the following accession
numbers: Aly-Arabidopsis lyrata (D7M2M4); Ath-
Table 1 Secondary structure prediction of the TPC1 carboxyl-terminus
CCoil, Hhelical, Esheet, Tbeta-turn
a
Yang et al. [85], Roy et al. [86], Zhang [87]
b
Xu, Zhang [88]
c
Remmert et al. [89], So
¨ding [90], So
¨ding et al. [91]
d
Kelley, Sternberg [92]
The function of the two-pore channel TPC1 depends on dimerization of its carboxy-terminal helix 2569
123
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Arabidopsis thaliana (B9DFD5); Bna-Brassica napus
(A0A078G686); Bol-Brassica oleracea (A0A0D3E0B2);
Cru-Capsella rubella (R0GT49); Csa-Cucumis sativus
(A0A0A0K5Q7); Egr-Eucalyptus grandis (A0A059A094);
Gma-Glycine max (1M3S8); Gso-Glycine
soja(A0A0B2R1M3); Hvu-Hordeum vulgare (Q6S5H8);
Jcu-Jatropha curcas (A0A067JJP4); Mtr-Medicago trun-
catula (A0A072VBZ7); Nta-Nicotiana tabacum
(Q75VR1); Osa-Oryza sativa (Q5QM84); Ptr-Populus
trichocarpa (U5FYB3); Sit-Setaria italica (K3XEV7); Sly-
Solanum lycopersicum (K4CFU2); Tae-Triticum aestivum
(Q6YLX9); Tca-Theobroma cacao (A0A061E309); Zma-
Zea mays (B6SP34).
Results
AC-terminal region is essential for TPC1 function
We previously reported that deletion of the last 55 amino
acids of AtTPC1, corresponding to the cytosolic carboxyl-
terminus, resulted in a mutant (TPCDC) which is correctly
targeted to the tonoplast, but lacks channel function [11].
This indicates an essential role of this region for the activity
of TPC1, but not for the targeting or trafficking process.
These previous results were obtained from electrophysi-
ological recordings of excised patches and thus, due to the
small membrane area, a little residual activity of TPC1DC
may have escaped observation. We now compared slow
vacuolar (SV) currents in the whole-vacuolar configuration
of the patch-clamp technique after expression of wild type
TPC1 and the TPC1DC mutant, respectively. Both channel
variants were expressed as GFP-fusions in the tpc1-2
knockout background, allowing localization and electro-
physiological analysis of the introduced TPC1 versions as
homo-dimers [11,13]. In the presence of 1 mM CaCl
2
in the
bath solution, which fully activates the TPC1 wild type
(Fig. 1a), no currents were obtained from vacuoles
expressing the truncated channel (Fig. 1d). This result sup-
ports the conclusion that the C-terminus is necessary for the
voltage- and Ca
2?
-dependent activity of TPC1 [11].
To investigate whether a specific sub-region of the
carboxyl-terminus is involved in this regulation, two
additional truncated channel versions were created:
TPC1DC8 contained the residues 1–725, and TPC1DC29
the residues 1–704. Both mutants were localized in the
tonoplast, indicating their expression, efficient ER export,
and correct targeting (Fig. 1b, c). The lack of the last 8
amino acids did not interfere with the activity of TPC1DC8
(Fig. 1b), and the amplitudes and current–voltage behavior
of the mutant were wild type like (Fig. 1b, e). In contrast,
although TPC1DC29 was expressed normally (Fig. 1c,
Fig. S1), this mutant stayed silent like the TPC1DC mutant
lacking the whole carboxyl-terminus (Fig. 1c–e). These
results identified amino acids 705–725 to include a region
indispensable for channel function.
Mutation in a predicted helical domain abolishes
TPC1 activity
So far, no structural information is available about TPCs of
plant or animal origin. To address the secondary structure
of the C-terminus, different prediction tools were applied,
TPC1
TPC1 C8
TPC1 C29
TPC1 C
2 nA
200 ms
2 nA
200 ms
2 nA
200 ms
2 nA
200 ms
a
b
c
d
e
300
200
100
Iss (pA/pF)
150100500
-50
V (mV)
Fig. 1 The C-terminus is essential for the function of TPC1. a–
dConfocal fluorescence overlay images of the GFP (green) and
chlorophyll (red) signals (scales represent 5 lm), and representative
whole-vacuolar current responses of TPC1-GFP variants as indicated
on the left. Applied voltages ranged from -73 to ?147 mV in 20 mV
intervals, starting from a holding potential of -53 mV. eCurrent–
voltage relations of whole-vacuolar steady-state currents determined
from traces as shown in (a–d) for TPC1-GFP (closed circles,n=4),
TPC1DC8-GFP (open circles,n=5), TPC1DC29-GFP (open
rhombi,n=6), and TPC1DC-GFP (closed rhombi,n=4) normal-
ized to the vacuolar membrane capacitance. Data represent mean
values ±SE
2570 N. Larisch et al.
123
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which revealed a consensus helical sequence to span amino
acids 707–723 (Table 1).
To assess the role of this a-helix we investigated a
mutant with an altered secondary structure. Substitution of
three leucines (L714, 715, and 719) by prolines as a potent
helix-breaker [65] resulted in the mutant TPC1-3LP,
which, according to the secondary structure prediction
tools listed in Table 1, lacks the a-helix. In a comple-
mentary experiment, a second point mutant, TPC1-3LA,
was chosen to replace bulky hydrophobic leucines by the
comparably small alanine favoring an a-helical confor-
mation [66]. The prediction tools as listed in Table 1
supported the assumption of a helical secondary structure
in the C-terminus of TPC1-3LA. The fluorescence emerg-
ing from the GFP-tagged mutant was detected in the
tonoplast of transformed cells, but whole-vacuolar currents
of TPC1-3LP produced only about 7 % of the wild type
amplitude (Fig. 2a, b). Interestingly, TPC1-3LA resulted in
a loss of SV currents to a similar extent as TPC1-3LP
(Fig. 2c). Comparable expression levels between wild type
and mutants were indicated by similar GFP intensities, both
on the single cell level and for the protoplast suspensions
(Fig. 2b–c, Fig. S1). Residual currents produced by both
mutants displayed the typical voltage- and time-dependent
characteristics of the SV channel. In excised patches, no
macroscopic currents were resolved, but the presence of
single-channels with a conductance of 58 pS, similar to
that of the wild type [14,20,67], showed that the single
channel amplitudes were not affected by the mutation
(Fig. S2).
A current reduction was also obtained, when a point
mutation was introduced at Ser706, which is located at the
N-terminal end of the helix (Table 1). Since Ser706 rep-
resents a putative phosphorylation site, it was replaced
either by the phosphorylation-mimicking Asp, introducing
a negative charge, or by Ala. In both cases, the whole-
vacuolar current amplitudes were largely reduced, by 58 %
and 53 % for TPC1-S706D and TPC1-S706A, respectively
(Fig. 2d, Fig. S3). The current reductions were also
observed at non-saturating Ca
2?
-concentrations of 0.2 mM
and 0.05 mM (Fig. 2d). Similar open probabilities of wild
type and mutants at saturating Ca
2?
concentrations (1 mM)
and 0.2 mM, corresponding to the half-maximal activation
concentration [13], showed that the current reductions did
not result from a largely impaired Ca
2?
-dependent shift of
the voltage dependence (Fig. S3). Besides shifting the
voltage dependence to less negative potentials, an elevation
of the cytosolic Ca
2?
concentration also increases the
maximum conductance [13]. While TPC1-S706 wild type
currents were reduced to 53 ±10 % at 107 mV following
the reduction of Ca
2?
from 1 mM to 200 lM, this value
was 28 ±3 % for TPC1-S706A and 24 ±4 % for TPC1-
S706D (Fig. 2d). The S706 mutation therefore appears to
modestly affect the Ca
2?
-sensitivity of channels via the
link between the Ca
2?
-binding and change in the number
of voltage-sensitive channels.
Together, these results show that Ser706 appears to have
a structural role instead of being involved in channel reg-
ulation by phosphorylation. Furthermore we conclude that
mutations in or near the C-terminal helix reduce the
number of open channels rather than affecting the voltage
dependence itself.
An alignment of the TPC1 carboxyl-termini of different
species shows that the C-terminal helix (CTH), including
S706 as well as L714, L715, and L719, is highly conserved
among plants (Fig. 2e), implicating an important function
of this domain. Our results strongly suggest that vacuoles
harboring mutations in the C-terminal helix formed less
functional two-pore channels. As helical structures are
often involved in mediating protein–protein interactions,
the CTH of TPC1 may be involved in protein–protein
interactions required as a prerequisite for channel gating or
stabilization of the open state.
TPC1 dimerizes via its C-termini
One possibility for a CTH-mediated interaction would be a
dimerization of the cytosolic C-termini of two TPC1 sub-
units. To test this possibility, interaction studies were
performed using bimolecular fluorescence complementa-
tion (BiFC) and co-immunoprecipitation (Co-IP).
Split Venus BiFC was tested for the full-length chan-
nels, either the wild type TPC1 or the TPC1-3LP mutant.
BiFC signals from wild type channels could be observed
when two C-terminally tagged TPC1 subunits were co-
infiltrated in Nicotiana benthamiana (Fig. 3a). The fluo-
rescence signals emerged from the vacuolar membrane and
in few cells additionally from the preceding endomem-
branes of the secretory pathway, such as endoplasmic
reticulum or Golgi apparatus. This shows that the C-ter-
minal parts of TPC1 come into close contact to one
another, allowing for fluorophore formation. Patch-clamp
analysis of tpc1-2 Arabidopsis cells co-transformed with
the C-terminally tagged TPC1 BiFC-constructs resolved
typical SV currents, supporting the hypothesis that a
dimerization via the C-termini results in voltage-sensitive
channels (Fig. 3b).
TPC1-3LP C-terminally tagged with the Venus halves
produced BiFC signals that were reduced compared to the
wild type (Fig. 3d). Confocal fluorescence microscopy
revealed that compared to the wild type, TPC1-3LP tagged
with the Venus halves was less efficiently transported to
the vacuole (Fig. 3c), since the fluorescence emerged not
only from the vacuolar membrane, but also to significant
amounts from other endomembranes, mostly the ER. This
localization pattern of TPC1-3LP with BiFC was not seen
The function of the two-pore channel TPC1 depends on dimerization of its carboxy-terminal helix 2571
123
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with GFP-tagged TPC1-3LP in Arabidopsis tpc1-2cells
(Fig. 2b, Fig. S1) and might hint to a slowed trafficking
associated with the tobacco expression system compared to
the Arabidopsis mesophyll protoplasts. Nevertheless, the
reduced BiFC signals of the helix-breaking point mutant
TPC1-3LP (Fig. 3c) in comparison to the wild type may
indicate that the dimer formation via the C-terminus was
hampered. However, the flexibility of the C-termini and
Venus halves would still allow the formation of the
fluorophore.
200 ms
2 nA
200 ms
2 nA
200 ms
2 nA
TPC1 TPC1-3LATPC1-3LP
abc
d
200
150
100
50
Iss (pA/pF)
150100500
-50
V (mV)
200
150
100
50
Iss (pA/pF)
150100500
-50
V (mV)
200
150
100
50
Iss (pA/pF)
150100
50
0-50
V (mV)
Sly NLIVAFVLEAFQAEMDLEAAANCADGDD--KESRSERRRNVGTKTRSQRVDFLLHHMLSSELTECSHDDP---- 739
Nta NLIVAFVLEAFQAEVDLEASARCVDGDD--KEAKSERRRNVGTKTRSQRVDFLLHHMLRSELTECSNENP---- 735
Cru NLVVAFVLEAFFTELDLEEEEKCQGQD---SQERRNRRRSAGSKSRSQRVDTLLHHMLGDELSKPECSTSDT-- 732
Ath NLVVAFVLEAFFTELDLEEEEKCQGQD---SQEKRNRRRSAGSKSRSQRVDTLLHHMLGDELSKPECSTSDT-- 733
Aly NLVVAFVLEAFFAELDLEEEENCQGED---SQERRNRRRSAGTKSRSQRVDTLLHHMLGDELSKPECSTSDT-- 732
Bol NLIVAFVLEAFFTELDLEEEEKCEGQD---SQERRNRRRSAGSKSRSQRVDTLLHHMLGDELSKPECSTTATDT 736
Bna NLIVAFVLEAFFTELDLEEEEKCEGQD---SQERRNRRRSAGSKSRSQRVDTLLHHMLGDELSKPECSTTATDT 738
Gso NLIIAFVLEAFFAEMELESSETCEGNDKEVEGDKY-RKRSIGTKTRSQRVDALLHHMLSAELCQNESSSTQTS- 739
Gma NLIIAFVLEAFFAEMELESSETCEGNGKEVEGDKY-RKRSIGTKTRSQRVDALLHHMLSAELCQNEPSSTQTS- 738
Mtr NLIIAFVLEAFFAEIELEEAETGDGNDKEVAGERYPRRRALGTKSRSQRVDALLHHMLSAELGQNQTSST---- 737
Ptr NLVMAFVLEGFFAEMELETAEKCEAEDKE-GSNSKSRRRSVGTKTRSQRVDNLLHHMLSAELEKPECSNA---- 726
Jcu NLVVAFVLEAFFAEMDLEKPEECEDED---ARTSKPRR--IGTKSRSQRVDILLHHMLSAELHDNQSSNA---- 735
Egr NLVVAFVLEAFFAEMDLESSENCEGQDEE-IRGR--RSRSVGTKTRSQRVDVLLHHMLSAELDKAKCTCPQP-- 737
Tca NLVVAFVLEAFFTEMDLETSGNCEEDDKD-AGSGKYRRRLVGTKTRSQRIDILLHHMLSAELDKGQSSASSTP- 737
Csa NLVVAFVLEAFFAELDIESSENGEEQDQD-KDSRKDRPRFVGTKTRSRKVDILLHHMLSAELDDKDSD------ 738
Zma NLIVAFVLEAFFAEMELEKAGEADTQDS--TPQGRNKRRSMRARTKGTMVDILLHHMLSNELDGSQNSD----- 749
Sit NLIVAFVLEAFFAEMELEKAGESDMQDS--TPQGRNKRRSMRVKTKGTMVDILLHHMLSNELDGSQNTDQ---- 745
Osa NLIVAFVLEAFFAEMELEKDGEADIQDP--TLEGRNRRRSVRVRTKGTMVDILLHHMLSNELDGSQNRDQ---- 757
Tae NLIVAFVLEAFFAEMELEKGEEVDIQNP--TSGGIKKRRSMRVRSKGTMVDILLHHMLSNELDGSQNS------ 742
Hvu NLIVAFVLEAFFAEMELEKGEEVDIQSP--TSGGIKKRRSMRVRSKGTMVDILLHHMLSNELDGSQNS------ 742
**::*****.* :*:::* . : :::. :******* ** .
e
2572 N. Larisch et al.
123
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In contrast, combinations with N-terminally tagged
TPC1 subunits did not result in Venus-fluorescence making
intramolecular interactions of the N-terminus seem unli-
kely (Fig. 3d). Functional expression and tonoplast
localization of an N-terminally tagged TPC1 were verified
using a GFP tag (Fig. S4).
In an independent approach, the soluble C-termini
(residues 673–732) of wild type TPC1 and of TPC1-3LP
were assayed for co-immunoprecipitation in a heterologous
system. The proteins were N-terminally tagged with a
6xMyc-tag for precipitation or a FLAG-tag for detection of
co-precipitated proteins, and in addition C-terminally
enlarged by a GFP-fusion to provide better handling, and
co-expressed in HEK293T cells. These experiments con-
firmed that the TPC1 C-terminus bound to itself and could
be detected with both antibodies in the immuno-precipitate
(Fig. 4). As a control, the empty vector pCS2 or with
6xMyc-GFP was co-transfected with the Flag- or Myc-
tagged C-terminus, respectively, since GFP is known to
have a tendency to dimerize itself. Compared to the wild
type, the C-terminus of the TPC1-3LP-mutant showed a
reduced ability to dimerize, reflected by a weaker signal
bFig. 2 Point mutations within a conserved C-terminal region reduce
TPC1 activity. a–cRepresentative whole-vacuolar current responses
(top) and current–voltage relations (bottom) of corresponding steady-
state currents of TPC1-GFP variants as indicated at the top. Applied
voltages ranged from -73 to ?147 mV in 20 mV intervals, starting
from a holding potential of -53 mV; current–voltage curves are
shown starting from -43 mV. Additionally shown in band care
confocal fluorescence overlay images of the GFP (green) and
chlorophyll (red) signals of corresponding mesophyll protoplasts
(scales represent 5 lm). aTPC1-GFP as in Fig. 1(closed circles,
n=4), bTPC1-3LP-GFP (open squares,n=7), cTPC1-3LA-GFP
(open circles,n=8). dCurrent–voltage relations of excised cytosolic
side-out vacuolar membrane patches of TPC1-GFP (closed circles),
TPC1-S706A-GFP (upward triangle), and TPC1-S706D-GFP (down-
ward triangle) expressing cells as well as tpc1-2mutants (open
circles), with cytosolic [Ca
2?
] of 1 mM (left), 0.2 mM (middle), or
0.05 mM (right), respectively (n=3–7). eC-terminal alignment of
TPC1 from different plant species. Gray bar indicates the end of
transmembrane segment S12, as predicted for AtTPC1 [84]. Green
bar denotes the predicted C-terminal helix for AtTPC1. Aly-
Arabidopsis lyrata;Ath-Arabidopsis thaliana;Bna-Brassica napus;
Bol-Brassica oleracea;Cru-Capsella rubella;Csa-Cucumis sativus;
Egr-Eucalyptus grandis;Gma-Glycine max;Gso-Glycine soja;Hvu-
Hordeum vulgare;Jcu-Jatropha curcas;Mtr-Medicago truncatula;
Nta-Nicotiana tabacum;Osa-Oryza sativa;Ptr-Populus trichocarpa;
Sit-Setaria italica;Sly-Solanum lycopersicum;Tae-Triticum aes-
tivum;Tca-Theobroma cacao;Zma-Zea mays
ab
cd
Fig. 3 The C-termini of two TPC1 monomers interact with each
other. aConfocal fluorescence overlay images of the Venus (yellow)
and chlorophyll (red) signals of a tobacco cell co-expressing TPC1
fused to Venus-Ct (TPC1-VC) and TPC1 fused to Venus-Nt (TPC1-
VN), shown at two different magnifications (scales represent 5 lm).
The BiFC signals emerge from the tonoplast. bWhole-vacuolar
current response of a tpc1-2cell co-expressing the same constructs as
in (a). cConfocal fluorescence overlay image of the YFP (yellow) and
chlorophyll (red) signals of a tobacco cell co-expressing TPC1-3LP-
VC and TPC1-3LP-VN (scales represent 5 lm). The BiFC signals
emerge from endomembranes and tonoplast. dFluorescence signal
intensities of BiFC experiments in relative units, determined from leaf
disks with a fluorescence reader. VC-TPC1 and VN-TPC1 (n=23),
TPC1-VC and VN-TPC1 (n=23), VC-TPC1 and TPC1-VN
(n=23), TPC1-VC and TPC1-VN (n=38), TPC1-3LP-VC and
TPC1-3LP-VN (n=11). As negative controls leaf disks only
expressing the helper strain p19 (n=28) and not infiltrated leaves
(n=39) were used
The function of the two-pore channel TPC1 depends on dimerization of its carboxy-terminal helix 2573
123
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from the Co-IP, while the expression level was comparable
to the wild type (Fig. 4).
The two independent protein–protein interaction assays
demonstrated the dimerization of the TPC1 C-terminus and
suggest that this interaction is mediated by the C-terminal a-
helix (CTH). The ability of the CTH to homo-dimerize was
further directly measured in microscale thermophoresis
(MST) experiments. Titration of the wild type peptide cor-
responding to the CTH (Table 1) against the wild type
peptide labeled by tryptophane induced a dose-dependent
change in mobility (Fig. 5a), indicative of the dimer for-
mation. From the change in fluorescence a binding affinity of
3.85 ±1.1 lM was determined (Fig. 5c). In contrast, titra-
tion curves of the 3-LA peptide revealed no interactions of
this mutant peptide in a concentration range from 3 nM to
100 lM (Fig. 5b,c).
The CTH mediates formation of an antiparallel
coiled-coil dimer
The CTH-mediated dimerization was further analyzed in
silico, both for the wild type and for the CTH-3LA mutant.
To this end, the dimerization was first analyzed from 100
aggregation simulations (250 ns each) for each system.
Each dimerization simulation started from two C-terminal
TPC a-helices at an initial peptide center of mass (COM)
distance between 5.5 and 6.5 nm solvated in a box of water
at coarse-grained resolution. Dimers were formed for both
systems in all simulations within tens of nanoseconds,
reflecting the observed enhanced stickiness of the coarse-
grained MARTINI force field [47].
Fig. 4 The C-terminus of TPC1 interacts with itself. Co-immuno-
precipitation of TPC1 C-terminal proteins co-expressed in HEK 293T
cells (combinations as indicated) and analyzed by western blotting.
Precipitation was achieved via the Myc-tag (IP anti-Myc), and
proteins detected via antibodies against the Myc- (WB anti-Myc) or
the Flag-tag (WB anti-Flag). The Flag-tagged C-terminus of TPC1
(Flag-Ct) was co-precipitated with the Myc-tagged C-terminus of
TPC1 (Myc-Ct, lane 4), whereas only background interaction was
observed with Myc-tagged GFP (lanes 2 and 5). Introduction of the
3LP mutation resulted in visibly weaker interaction of the two C-
termini (lane 8). Similar results for the interaction of the TPC1 C-
termini were obtained in 3 independent experiments. The presence of
the proteins was verified in the cell lysate. Calculated protein weights
were Myc-Ct (46 kDa), Flag-Ct (37 kDa), 3LP-Myc-Ct (47 kDa),
3LP-Flag-Ct (38 kDa), Myc-eGFP (37 kDa), Myc (10 kDa), Flag
(1 kDa)
a
c
b
Fig. 5 Synthetic CTH peptides dimerize. a,bMST time traces
(normalized fluorescence) of 16 capillaries containing 750 nM
tryptophane-labeled wild type (a) or 3LA mutant (b) CTH peptide
and unlabeled wild type (a) or 3LA mutant (b) CTH peptide at
concentrations between 3 nM and 100 lM. Thermodiffusion is
reduced with increasing peptide concentrations. cThe normalized
fluorescence of the MST traces was converted to the fraction bound
and plotted against the concentration of the ligand. A K
D
of
3.85 ±1.1 lM was determined for the interaction of the wild type
CTH peptides (open circles), while no interaction was measured for
the 3LA mutant (closed circles)
2574 N. Larisch et al.
123
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However, the dimerized CTH-3LA adopted significantly
different relative orientations as compared to wild type
dimers: Overall, the mutant exhibited more flexibility in
the bound state (Fig. 6, Fig. S5). The angle between the
helical axes, called tilt, describes the parallel or antiparallel
orientation of the bound peptides (Fig. 7b). The distribu-
tion of this angle (Fig. 6), calculated over the last 50 ns of
the trajectories, is in the case of the CTH-3LA dimers very
diffuse, pointing to a drastically enhanced flexibility and
thus less stable dimers as compared to the wild type
dimers. The number of transitions between the orientations
(see ‘‘Data Analysis’’) strengthens this conclusion: While
the majority of wild type dimers were stable in their ori-
entation (median of orientation transitions is 0), the number
of transitions in mutant dimers was significantly increased
(median is 4.5, Fig. S6). This flexibility in the relative
orientation of mutated helices most likely results from a
reduction in steric hindrance. Leucines consist of two
coarse-grained beads (four residual methyl groups), while
alanine exhibits only one backbone bead (one residual
methyl group). Since the majority of wild type dimers
ended up in a conformation, where leucines are buried in
the interface (see below), this exchange to the smaller
alanines caused an increased flexibility in the tilt angle.
Furthermore, alanines are not as hydrophobic as leucines
and thus, they are more prone to face the solvent.
The relative instability and promiscuity of the CTH-3LA
dimers observed in MD simulations corresponds to the lack
of dimer formation in MST experiments. In the following,
we focused on the analysis of dimer formation for the CTH
wild type peptide.
Within approximately 50 ns, the wild type peptides
approached each other in all simulations to a distance
below 1 nm, reflecting dimerization (81 % of the mono-
mers dimerized within 25 ns). Figure 7a shows the center
of mass distances for each simulation. With increasing
simulation time, cluster formation is seen, with one cluster
at a COM distance of &1 nm (c1) and a second cluster at
&1.45 nm (c2). Peptides in dimers of cluster c2 adopted a
shifted configuration (see below).
The orientation of the C-terminal dimers of TPC1 was
analyzed using Euler angles [47,64]. A tilt angle below 50
indicates that the helices are in a parallel conformation,
while an angle above 130reflects an antiparallel orienta-
tion (Fig. 7b). The latter configuration was adopted in
86 % of the coarse-grained simulations, characterized by
an average tilt angle of 160. In 7 % of the simulations, the
dimers ended up in a parallel orientation (average tilt of
18), the remaining simulations ended up in diverse inter-
mediate configurations. To reach the antiparallel
conformation most of the dimers followed a specific
dimerization pathway (Fig. 7c): At larger intermolecular
distances (3–7 nm), i.e., at the beginning of the simulation,
the distribution of tilt angles was random (Fig. 7c). Upon
approach, the tilt was constrained to values of 10–50, the
peptides thus oriented in a parallel head-to-tail configura-
tion. Subsequently, the peptides reoriented and ended up in
the antiparallel configuration (130–180, Fig. 7c).
To evaluate the frequency of the different dimer con-
formations at the end of the simulations, combinations of
the binding position band rotation angle /(compare
Fig. 7b) obtained from the trajectories were plotted as a
two-dimensional kernel density map (Fig. 7d). While the
position of one monomer to a reference monomer is
defined by the position b, the exact binding site that faces
the reference structure is given by the phase /, the rotation
around its helical axis (Fig. 7b).
In the bound state, i.e., at the end of the simulations,
positively and negatively charged amino acids come into
close proximity, in particular in the antiparallel configu-
ration (Fig. 7d). These antiparallel dimers (cluster A in
Fig. 7d) are electrostatically favored by proximity of the N-
terminal arginines of one monomer and the C-terminal
glutamic and aspartic acids of the second monomer.
Additionally, the hydrophobic residues leucine and valine
are positioned at the dimer interface.
Besides the main cluster A, four additional clusters (B–
E) were identified. The second-largest cluster B consists of
antiparallel dimers that interact as well through electro-
static interactions. Due to a decreased number of
hydrophobic residues at the dimer interface, this confor-
mation is expected to be metastable. Parallel dimers were
found in clusters C and D. In these arrangements, the
helices are shifted with respect to each other, allowing for a
0.000 0.025
0 20 40 60 80 100 120 140 160 180
0.000 0.010
0 20 40 60 80 100 120 140 160 180
Wild type
Mutant
DensityDensity
Tilt
Tilt
Fig. 6 CTH dimer tilt distribution reflects an increased flexibility of
mutant dimers. Tilt angles between coarse-grained dimers were
determined over the last 50 ns from each simulation and plotted in a
histogram (x-axis: tilt, y-axis: density). The density distribution of tilt
angles between mutant dimers (bottom) is compared to wild type
dimers (top) very diffuse, pointing to an increased flexibility of
mutant dimers
The function of the two-pore channel TPC1 depends on dimerization of its carboxy-terminal helix 2575
123
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better packing of hydrophobic residues (parallel dimer,
Fig. 7d). Therefore, the center of mass distances between
the corresponding monomers is increased as compared to
the antiparallel dimers (compare c2 in Fig. 7a). For cluster
E (tilt from 50–130) the inner leucines are in close
proximity to each other. Furthermore, in this configuration
the central aspartic acids are able to interact with the
arginines.
To evaluate the binding strength, the non-bonded
interaction energy was calculated for the different confor-
mations. In agreement with the high frequency of
antiparallel dimers (cluster A and B), their mean interac-
tion energy was lowest among all dimers with a value of
&-600 kJ/mol, while the parallel dimers interact with
&-420 kJ/mol, and the rest found in cluster E with
&-500 kJ/mol.
For further analysis of the stability of the dimers formed
at coarse-grained (CG) resolution, five CG conformations
corresponding to the main clusters A–E as indicated in
Fig. 7d were backmapped to atomistic resolution and
simulated for 200 ns (cluster A, C–E) or 270 ns (cluster B).
The two initial parallel dimer configurations (C and D)
changed in the atomistic simulations to an antiparallel
orientation within less than 10 ns, indicating a relative
instability of the parallel configurations. The dimers of
clusters D and E adopted an antiparallel configuration
(position d,ein Fig. 7d) during the atomistic simulations,
with the three inner leucines and valine at the helical
interface stabilized by two salt bridges at the termini
(Fig. 8a, iv and v). This orientation is indicative of coiled-
coil packing, burying hydrophobic residues within the core
flanked by stabilizing ionic interactions at the helical sides.
Less stable conformations were observed for the other
dimers (clusters A, B, C). Here, leucines and valines were
tilt
tilt
0 50 100 150 200 250
0
1
2
3
4
5
6
7
Distance (nm)
Time (ns)
a
b
c
c2
c1
d
Parallel
Antiparallel
COM Distance (nm)
012345 67
180
20
40
60
80
100
120
140
160
-150 -100 150100500-50
β
Φ
50 3002502001501000 350
bFig. 7 In silico dimerization study of the carboxy-terminus of TPC1.
aCenter of mass (COM) distance between the two helices (residues
707–723), with initial values between 5.5 and 6.5 nm, as a function of
simulation time. Different colored lines represent 100 independent
coarse-grained (CG) simulations. c2 and c1 indicate two populations
of dimers distinguished by their COM distance. bSchematic repre-
sentation of angles used to analyze the orientation of bound helices. b
describes the position of the binding partner to the reference structure
(colored in gray), /is the rotation around the helical axis, and the tilt
angle depicts the tilt between the peptides. Lower tilt values (B50)
point to parallel binding and larger (C130) to antiparallel binding.
Helices were colored in rainbow scheme from N-terminus (blue)toC-
terminus (red). cCenter of mass distances of two monomers forming
homo-dimers in antiparallel arrangement, shown for all simulations
resulting in antiparallel configurations. dLeft: Kernel density for b
and /angle combinations in the last 50 ns of the CG simulations,
describing the relative orientation of peptides. The yellow area
indicates a high density, white color a low density. Antiparallel
dimers were found in clusters A and B, parallel dimers in C and D,
others in E. Two selected conformations from clusters D and E were
finally observed at positions in phase space d and e, respectively, after
backmapping to atomistic resolution followed by atomic-scale
molecular dynamics simulations. Right Orientation for the antiparallel
and parallel homo-dimers. Glutamic and aspartic acids are colored
red, arginine green, and hydrophobic residues light brown. For
clarity, only interacting residues are colored
2576 N. Larisch et al.
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partially exposed to the solvent (Fig. 8a, ii and iii) or
hydrogen bonds within the helix were broken and conse-
quently the helix disrupted (Fig. 8a, i and ii). Additionally,
hydrophilic histidines were partially found at the interface.
The combined coarse-grained and atomistic study thus
strongly suggests that the wild type, but not the mutant
CTHs of two TPC1 monomers form a stable helical dimer
in an antiparallel coiled-coil conformation (Fig. 8b).
Together with the results of the mutagenesis study, we
propose the formation of an antiparallel CTH dimer as a
prerequisite for TPC1 activity. This conclusion is further
supported by the ability of the synthetic CTH peptide to
rapidly inhibit TPC1 currents, when applied to the active
channels in electrophysiological recordings (Fig. S7).
a
b
i ii iii
iv v
C
C
N
N
Fig. 8 CTH dimer configurations observed in combined coarse-
grained/atomistic simulations. aMetastable (i,ii,iii) and stable (iv,
v) dimer configurations. For the stable dimer arrangement, the
hydrophobic residues are found mainly at the interfacial region, and
stabilizing salt bridges at the termini. For clarity, interfacial
methionines are hidden. The less stable configurations show partially
exposed hydrophobic (light brown) amino acids and a disrupted
helical conformation. Glutamic and aspartic acids are colored red and
arginine green.bModel for the functional TPC1 with the antiparallel
CTH dimer enlarged (Shaker domains I and II are differently colored
in each monomer). A head-to-tail conformation is assumed for the
TPC1 dimer, as suggested by Rietdorf et al. [10]
The function of the two-pore channel TPC1 depends on dimerization of its carboxy-terminal helix 2577
123
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Discussion
Here, we identified the presence of a carboxy-terminal
helix (CTH) in TPC1 and documented its role for channel
dimerization and function. Loss of the CTH rendered the
channel inactive while point mutations within the CTH
resulted in severely reduced channel activity. Corre-
spondingly, BiFC analysis and Co-IP experiments reported
on the interaction of the C-terminus, which was reduced by
mutations within the CTH. A residual current of 10 % in
the TPC1-3LA and -3LP mutant may indicate that in
contrast to the CTH peptide the complete C-terminus may
dimerize to a small extent even with three leucines
replaced. Although we cannot exclude the possibility that
in addition to the CTH other parts of the C-terminus are
involved in the dimerization process, the lack of the CTH
was sufficient to render the channel silent.
Dimerization of the CTH was directly shown in MST
assays and MD simulations. The sequential multiscale
simulations revealed that the wild type CTH dimer
preferably adopts an antiparallel coiled-coil conformation.
This antiparallel orientation is in accordance with an
assumed head-to-tail configuration of the channels [10].
The fact that leucine to alanine mutations in the inner helix
drastically impaired channel function is in accordance with
the observation that although alanine is a residue of high
helix propensity, it can decrease the stability of the helix
(dimer) [66,68]. Indeed, coarse-grained MD simulations
discovered mutated dimers that exhibit a significant
increase of flexibility, pointing to less stable dimers. These
results were corroborated by MST analyses, which showed
that the wild type CTH dimerized with a K
d
of 3.9 lM
while the mutants did not interact. Apparently, the CTH is
not required for correct assembly of the channel in the
membrane. However, our results suggest that the CTH is
crucial for stabilization of the TPC1 dimer and coupled
with dimerization also for channel gating.
Gating of ion channels by their soluble termini is a
common feature of ion channels [69–73], and often mod-
ification of the C-terminus induces alterations of the
voltage dependence [74–77]. In comparison, mutations
within and near the CTH did not feedback onto the voltage
dependence of TPC1. In this respect, TPC1 shares struc-
tural and functional properties with voltage-dependent
sodium (Na
V
) channels. Deletion of the complete or distal
part of the C-terminus from a prokaryotic Na
V
resulted in a
complete or almost complete loss of sodium currents,
respectively, while the trafficking of the channel and the
voltage dependence of activation were unaffected [70].
Combined MD simulations and electron paramagnetic
resonance spectroscopy showed that a C-terminal helix of
this channel forms a coiled-coil bundle involving four
subunits, and that this tetramerization is essential for cou-
pling to channel opening via a proximal C-terminal linker
following S6. Most notably, the linker contains a nega-
tively charged cluster, and several glutamate residues are
also conserved in the plant TPC C-termini following S6
(Fig. 2). A role of the coiled-coil in stabilizing the sodium
channel tetramer or dimer in case of TPC1, and in enabling
the opening and closing of the pore during gating without
disrupting the quaternary structure may thus represent a
mechanism also valid for two-pore channels, which has to
be further evaluated in future studies.
Besides voltage changes, TPC1 is activated by binding
of Ca
2?
ions to the EF-hands in the central linker domain
between transmembrane S6 and S7, which apparently sta-
bilizes the open state [13,78,79]. The rabbit skeletal
muscle type 1 ryanodine receptor RyR1 is an intracellular
Ca
2?
release channel, which also belongs to the six-
transmembrane superfamily. Similar to TPC1, activation of
RyR1 by Ca
2?
involves cytosolic EF-hands, here present in
the N-terminus, while the C-terminus homodimerizes. An
essential function of the RyR1 C-terminus requires the last
15 amino acids, deletion of which abolishes channel
function [80]. Recently, the crystal structure of the RyR1
revealed a putative mechanism for Ca
2?
-mediated gating
involving the C-terminus [73,81]. In RyR1 Ca
2?
-depen-
dent changes in the conformation of the N-terminal EF-
hand containing domain are transmitted to the pore via
contacts with the C-terminal domain, inducing a change of
the cytosolic aperture of the channel and stabilizing the
open state [73,81]. A RyR1-like allosteric mechanism may
therefore also account for the role of the C-terminus in the
Ca
2?
- and voltage-dependent activity of TPC1.
TPC1 is a large-conductance channel and is therefore
tightly regulated to prevent ion leakage from the vacuole
[reviewed in 2]. The many ionic cytosolic regulators, such as
H
?
,Ca
2?
,Mg
2?
,K
?
, and Na
?
, exert their effects via
alteration of the voltage dependence of the channel. In
contrast, 14-3-3 proteins inhibit TPC1 activity by about
90 % within 10 s without any changes in the voltage
dependence [36]. A similar reduction in channel activity
without affecting the voltage dependence was observed for
the CTH mutants described here. Likewise, application of
the synthetic CTH peptide to the vacuolar membrane rapidly
inhibited TPC1 currents (Fig. S7). It is therefore tempting to
speculate that the 14-3-3 protein GRF6, which regulates
TPC1 in Arabidopsis [35], interferes with the dimerization of
the carboxyl-termini. A region partly overlapping with the
CTH contains two serines (S706, S708) and constitutes a
putative 14-3-3 binding site [705-KSRSQR, 82]. Accumu-
lation of GRF6 dimers may thus push the CTHs apart, which
will destabilize the TPC1 dimer and induce a rapid closure of
the channel pore. Alternatively, 14-3-3 proteins may act
2578 N. Larisch et al.
123
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

more indirectly, because a second putative 14-3-3 binding
site is predicted to be located in the linker domain between
the two EF-hands at T359 [83]. Given a RyR1-like allosteric
mechanism, 14-3-3 binding in the linker may interfere with
the coordinated mechanism, which links Ca
2?
binding to the
activation of the channel via the C-terminal a-helical region.
In any case, the coiled-coil mediated dimerization of wild
type CTH’s as a prerequisite for channel opening adds a very
rapid regulatory mechanism to shut down this large vacuolar
conductance, either by destabilizing the TPC1 dimer, or by
preventing the allosteric coupling to the channel pore.
Acknowledgments This work was supported by the German Sci-
ence Foundation (DFG): Research Training Group 1962-Dynamic
Interactions at Biological Membranes. Computer time was provided
by the Computing Center of the University Erlangen-Nu
¨rnberg
(RRZE). We would like to thank Joanna Bogdanska-Urbaniak and
Gudrun Steingra
¨ber for technical assistance, and Norbert Sauer (FAU
Erlangen-Nu
¨rnberg) for sharing the confocal microscope.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
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