The L530R variation associated with recurrent kidney stones impairs\nthe structure and function of TRPV5
TRPV5 is a Ca2+-selective channel that plays a key role in the reabsorption of Ca2+ ions in the kidney. Recently, a rare L530R variation (rs757494578) of TRPV5 was found to be associated with recurrent kidney stones in a founder population. However, it was unclear to what extent this variation alters the structure and function of TRPV5. To evaluate the function and expression of the TRPV5 variant, Ca2+ uptake in Xenopus oocytes and western blot analysis were performed. The L530R variation abolished the Ca2+ uptake activity of TRPV5 in Xenopus oocytes. The variant protein was expressed with drastic reduction in complex glycosylation. To assess the structural effects of this L530R variation, TRPV5 was modeled based on the crystal structure of TRPV6 and molecular dynamics simulations were carried out. Simulation results showed that the L530R variation disrupts the hydrophobic interaction between L530 and L502, damaging the secondary structure of transmembrane domain 5. The variation also alters its interaction with membrane lipid molecules. Compared to the electroneutral L530, the positively charged R530 residue shifts the surface electrostatic potential towards positive. R530 is attracted to the negatively charged phosphate group rather than the hydrophobic carbon atoms of membrane lipids. This shifts the pore helix where R530 is located and the D542 residue in the Ca2+-selective filter towards the surface of the membrane. These alterations may lead to misfolding of TRPV5, reduction in translocation of the channel to the plasma membrane and/or impaired Ca2+ transport function of the channel, and ultimately disrupt TRPV5-mediated Ca2+ reabsorption.
1. Introduction<!>2.1 Ca2+ uptake assay<!>2.2 Western blot analyses<!>2.3 System modelling<!>2.4 Molecular dynamic simulations<!>3. Results and discussion<!>3.1 The L530R variation abolishes the Ca2+ transport\nactivity of TRPV5<!>3.2 The L530R variation damages the secondary structure of TM5<!>3.3 The L530R variation causes a shift of pore helix and residue D542 towards\nmembrane surface
<p>TRPV5, one of the two epithelial Ca2+ channels with a high
Ca2+ selectivity, is mainly expressed in the distal
convoluted tubule (DCT) and connecting tubule (CNT) in the kidney [1; 2]. It regulates the first step in transcellular
Ca2+ transport and is considered as a gatekeeper for active
Ca2+ reabsorption [3]. The knockout of TRPV5 in mice resulted in severe
hypercalciuria and bone abnormalities [2]. Furthermore, altered expression of TRPV5 in mouse models of
human diseases is associated with vitamin D-deficiency rickets, altered estrogen
levels and postmepausal osteoporosis, and parathyroid hormone-related disorders
[4].</p><p>Several nonsynonymous single nucleotide polymorphisms (SNPs) in TRPV5,
including A8V (rs4252372), R154H (rs4236480), A563T (rs4252499) and L712F
(rs4252509), have been found in African Americans with high allele frequency
[5; 6]. Our previous works indicate that the A563T variation
increases TRPV5-mediated Ca2+ transport [6] and affects the structure and dynamics of the
pore regions of TRPV5 [7].
This variation may contribute to an increased renal Ca2+
reabsorption in African Americans compared to Caucasians. The R154H variation, which
is present in both African and non-African populations at a relatively high allele
frequency, usually causes a lower Ca2+ uptake activity in TRPV5
when combined with other variations [6]. However, in most cases, the decrease in Ca2+
uptake does not reach statistical significance [6]. Interestingly, this SNP has been found to be
associated with the occurrence of multiple stones in a cohort of 365 kidney stone
patients in Taiwan [8].
Recently, a L530R variation (rs757494578) in TRPV5 was found to be associated with
recurrent kidney stones in 2636 individuals from a founder population in Iceland
[9]. This variation
introduces a positive charged residue into a hydrophobic region of the pore helix
and likely affects the function of TRPV5; however, the effects of this mutation have
not been examined. To understand the role of TRPV5 in kidney stone disease, we
assessed the Ca2+ transport function of the L530R variant by
monitoring radiotracer Ca2+ uptake in Xenopus
oocytes. To gain new insights into the structural changes produced in TRPV5 with the
L530R variation, we performed molecular dynamic simulations of TRPV5 using the
recently published crystal structure of TRPV6 [10], a close homologue of TRPV5 [11; 12]. Our results are expected to provide further evidence for a
relationship between genetic variations in TRPV5 and the risk of kidney stone
disease.</p><!><p>Ca2+ uptake by Xenopus oocytes was
performed as described previously [6; 13]. The animal
protocol used in this study was approved by the Institutional Animal Care and
Use Committee (IACUC) of the University of Alabama at Birmingham. The L530R
variation of TRPV5 was generated using the QuikChange site-directed mutagenesis
kit (Stratagene, La Jolla, CA) in the FLAG-tagged human TRPV5 and the mutation
was confirmed by sequencing. The capped complementary RNAs (cRNAs) of FLAG
tagged TRPV5 with L530 (reference) and R530 (variant) were prepared using
mMESSAGE mMACHINE™ SP6 Transcription Kit (ThermoFisher). Oocytes were
microinjected with either the reference or the variant cRNA at 12.5 ng/oocyte or
water. Oocytes were cultured in 0.5×L-15 solution and assayed 2 days
after injection by radiotracer 45Ca2+ uptake in
standard Ca2+ uptake solution. After uptake, oocytes were
washed six times and then lysed in 10% SDS solution. Radioactivity of
each oocyte was determined using a scintillation counter. Statistical
significance is defined as P < 0.05 by a
Student's t-test.</p><!><p>Lysates were extracted from ten oocytes/group injected with water, L530
or R530 TRPV5 cRNA. Monoclonal anti-FLAG antibody (F7425, 1:1000 dilution) was
purchased from Sigma–Aldrich (St. Louis, MO). Appropriate horseradish
peroxidase (HRP)-conjugated secondary antibodies (1:10000 dilution) were
purchased from Santa Cruz Biotechnology. Chemiluminescence signals were detected
using SuperSignal West Pico Chemiluminescent Substrate kit (Pierce
Biotechnology, Rockford, IL).</p><!><p>The modeling of human TRPV5 was performed based on the structure of rat
TRPV6 using MODELLER 9.13 software [14]. The template of TRPV6 with Ca2+ ions
was obtained from the Protein Data Bank with an ID of 5IWP [10]. The sequence of TRPV5 used in
modeling contains the linker helix (LH) 1–2, pre-transmembrane (TM)
helix (pre-S1), TM helices 1–6 (S1–S6), and the TRP domain. The
alignment of the human TRPV5 sequence used in the modeling and the corresponding
rat TRPV6 sequence is presented in Fig. S1. Since we are only using human TRPV5 in this study,
the species will not be specified hereafter. The identity between the sequences
is 83.1%, which is much higher than the threshold of 30% that is
considered to be reliable for modeling membrane proteins [15]. The modeled TRPV5 tetramer is
shown in Fig. 1A. The L530R variation was
introduced into all four subunits of TRPV5 using the mutagenesis function of
PyMOL [16]. The two TRPV5
systems containing leucine or arginine residues at amino-acid position 530 are
denoted as L530 and R530, respectively.</p><p>To mimic the membrane environment, the modeled TRPV5 structures were
embedded in a lipid bilayer composed of 299
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids using CHARMM-GUI
membrane builder [17].
Each system (L530 or R530) was then hydrated with a
total of 32,854 TIP3P water molecules on both sides of the bilayer. The
approximate dimensions of the resultant simulation box were 126 Å
× 126 Å × 110 Å along the x, y, and z axes,
respectively. Na+ and Cl− ions were added
to the system to neutralize it and maintain a 150 mM NaCl concentration. The
parameters of a FF14SB force field [18] were assigned to the protein, ions, and water molecules,
and a FFlipid14 force field [19] was used for POPC lipids.</p><!><p>To investigate the effect of the L530R variation on the structural change
in TRPV5, two 400 ns molecular dynamic (MD) simulations were performed using the
AMBER14 simulation package [20]. The simulation protocol is similar to our previous studies
[21; 22; 23; 24; 25], and it can be found in the Supplementary Material.
Before data analysis, the root mean square deviation (RMSD) for the CÎ±
atoms of TRPV5 was calculated to assess the equilibration of the simulation.
Simulation reached an equilibration state after 200 ns in both L530
and R530 systems (Fig. S2). Thus, the last 200 ns simulations were used for analyses
by using the CPPTRAJ program of AMBER14. The DSSP method was applied to
determine whether an amino acid residue belonged to an α helix
[26]. The
electrostatic potential for TRPV5 was calculated by APBS [27]. The VMD software
[28] was used for
structure visualization.</p><!><p>In order to understand the effect of the L530R variation on TRPV5, we
evaluated the function and expression of TRPV5 using radiotracer
45Ca2+ uptake in Xenopus oocytes
and western blot analysis. We modeled the structure of the TRPV5 containing the
L530R variant based on the crystal structure of rat TRPV6. The major findings are
described below.</p><!><p>The functional effect of L530R variation on TRPV5 was evaluated by the
Ca2+ uptake ability when TRPV5 variants were expressed in
Xenopus oocytes. Compared to the Ca2+
uptake observed in oocytes expressing TRPV5 with L530, Ca2+
uptake of TRPV5 with R530 was decreased to a level not statistically different
from that of water injected oocytes (negative control) (Fig. 1B). This indicates that Ca2+
transport activity was totally abolished by the L530R variation. This could
result from a lack of expression of TRPV5 R530 protein, a rapid degradation of
the protein, a lack of expression of the protein in the plasma membrane, or a
defect in transport function. TRPV5 proteins expressed in
Xenopus oocytes generally exhibit two major bands when
assess in western blot: a lower band that is core-glycosylated, and a higher
band that is complexly glycosylated. We have shown previously that the
complex-glycosylated band is that expressed in the plasma membrane
[6]. The western blot
analysis showed that the complex-glycosylated band of TRPV5 (band B in Fig. 1C) was significantly reduced by the
L530R variation. This suggested that the L530R variation reduced the level of
TRPV5 at the plasma membrane. However, the total loss of Ca2+
uptake function suggests that the structural change induced by L530R variation
may also impair the Ca2+ transport function as well.</p><!><p>In order to understand the structural changes introduced by L530R
variation, the structure of TRPV5 was first constructed by homology modeling
using the crystal structure of TRPV6. In the modeled TRPV5 (Fig. 1A), the four aspartate residues at 542 (D542)
form a ring in the middle of the pore as a Ca2+-selective
filter [7]. Residue L530
is located in the pore helix and points away from residue D542. Since residue
L530 is distant from D542, it is not likely that the L530R variation impacts the
structure and function of TRPV5 through a direct interaction between residue 530
and residue D542. We therefore investigated whether the L530R variation affects
its nearby residues.</p><p>In the modeled TRPV5, residue L530 is on the protein surface and is
surrounded by hydrophobic residues leucine 502 (L502), tyrosine 526 (Y526) and
alanine 505 (A505) (Fig.
S3). The L530R variation introduces a positive charge which would
alter hydrophobic interactions. To test this proposition, the electrostatic
potential maps for L530 and R530 systems were
calculated (Fig. 2A). The positive
potential is shown in blue, while the negative potential is shown in red. The
surface electrostatic potential for residue 530 is labeled in a dotted circle in
Fig. 2A. As expected, the electrostatic
potential for residue 530 was shifted from neutral (white color for residue
L530) to positive (blue color for residue R530). The changed electrostatic
potential may directly alter the interaction between residue 530 and the
hydrophobic residue L502. Fig. 2B shows
that the hydrophobic side chain of L530 interacts with L502; however, the
charged side chain of R530 is oriented away and loses contact with L502.</p><p>Since residue 530 and L502 are both located in α helix
structures (residue 530 in pore helix and L502 in TM helix 5), secondary
structure analysis was performed to assess whether the altered interaction can
cause structural changes in TRPV5. To this end, the helix occupancy for each
residue was calculated from the equilibrated simulation data (Fig. S4). Comparison
between L530 and R530 systems showed that the helix
occupancy did not change for the total helices (54.7% vs.
55.4%), for the pore helix (99.2% vs. 96.7%) and for TM
helix 5 (S5) (81.0% vs. 79.2%). However, due to the altered
interaction between residue 530 and L502, the helix occupancy for L502 was
significantly decreased in monomers 1 and 4 of the L530R tetramer (100%
and 99.9% in L530 vs. 4.3% and 16.2% in
R530). This altered the helical structure of S5 and divided S5
into two parts (Fig 2B).</p><!><p>In the membrane environment, residue 530 is exposed to lipid molecules.
Thus, the L530R variation would also alter the interaction between residue 530
and lipid molecules. To examine this proposition, radial distribution functions
(RDFs) of residue 530 with the phosphorus (P) atoms and the carbon (C) atoms of
POPC lipids were calculated (Fig. 3A). RDF
calculates the probability of finding specific atoms away from the given atoms
in a distance function [29]. It can describe the strength of the interaction between two
groups of atoms, as a large peak indicates a strong interaction while a small
peak indicates a weak interaction. The RDF results show that P atoms of POPC
have a small peak with residue L530 (around 13.8Å), whereas they have
two large peaks with residue R530 (around 5.3 Å and 8.9Å). In
addition, C atoms of POPC have a slightly larger peak with residue L530 (around
7.8Å) than that with residue R530 (around 9.1Å). To give a clear
view of the interaction between residue 530 and lipid molecules, simulation
snapshots are represented in Fig. 3B. The
hydrophobic side chain of L530 is surrounded by C atoms of POPC, while the
charged side chain of R530 is coordinated with the P atoms of POPC (Fig. 3B).</p><p>To further study the interaction between residue 530 and membrane
lipids, the mass density describing the location of residues along the bilayer
normal (z axis) was calculated for residue 530, pore helix, and residue D542
(Fig. 4A). To give a clear view of the
positions of residue 530 and pore helix relative to the membrane surface, final
simulation structures are represented in Fig.
4B. The cyan dashed lines in Fig.
4A and cyan lines in Fig. 4B are
the density peaks of the P atoms of POPC lipids, which indicate the surfaces of
the membrane. Results indicate that when the leucine residue at 530 of TRPV5 was
replaced by arginine, the residue moved closer to the membrane surface (Fig. 4B) due to the strong interaction
between the arginine residue and P atoms of POPC (Fig. 3A). The movement of residue R530 further raised the pore helix
where it is located towards the membrane surface. Furthermore, the residue D542
in the Ca2+-selective filter also shifted towards the
membrane surface in the R530 system.</p><p>This study integrated biochemical and computational approaches to
investigate the effects of L530R variation on the structure and function of
TRPV5. The Ca2+ uptake experiments indicated that the L530R
variation results in a total loss-of-function in TRPV5. Based on the western
blot results, the mutant TRPV5 protein was synthesized, however, the complexly
glycosylated form of TRPV5 was dramatically reduced. As we have previously shown
that the complexly glycosylated form is the one detected at the oocyte plasma
membrane [6], the plasma
membrane expression of the variant was likely significantly reduced. While the
unglycosylated TRPV5 can still go to the plasma membrane [13], the variant channel was likely
defective in Ca2+ transport despite reaching the plasma
membrane.</p><p>The molecular dynamic simulations provided some insights into the
aberrant changes that occurred in the structure of TRPV5 with the L530R
variation. The most significant change was that the positively charged arginine
produced a positive shift in the electrostatic potential in the lipid membrane
environment. This not only disrupted the interaction between residue 530 and the
hydrophobic residue L502, but also altered the interaction between the
transmembrane domains of TRPV5 and the surrounding lipid molecules. L530 in the
pore helix was surrounded by Y526 in the same helix and L502 and A505 in TM5.
The hydrophobic interactions between these residues may stabilize the overall
hydrophobic core helix structures of TRPV5. In the R530 variant of TRPV5,
however, the charged residue R530 disrupted the hydrophobic interaction with
L502, leading to a damaged secondary structure of TM5 in two of the four
monomers. This might impair the core hydrophobic helix structure and result in
the misfolding of TRPV5. In addition, the shift of pore helix towards the
membrane surface due to the L530R variation may alter the folding of TRPV5 as
well. The starting model in the simulation was the properly folded TRPV5,
however, it is possible that the majority of R530 variants could not reach this
conformation in actuality. If misfolding occurs, the misfolded proteins will not
pass the "quality control" in the endoplasmic reticulum and they
will not move forward through the trans-Golgi network where
complex-glycosylation takes place. Instead, the misfolded proteins will undergo
endoplasmic reticulum-associated protein degradation (ERAD) [30]. This could explain the
significant reduction in glycosylation complexity in TRPV5 containing R530 as
shown in Figure 1C. Given that the L530R
variation resulted a total loss of Ca2+ uptake function in
TRPV5, even if some of the variant TRPV5 proteins reached the plasma membrane,
they were likely dysfunctional. The negatively charged phosphate group in lipid
molecules attracted R530 residue and brought the pore helix and the
Ca2+ filter closer to the plasma membrane. The altered
interaction between R530 residue and membrane lipids may also contribute to the
disruption of the Ca2+ transport function of the channel.
Together with the potentially altered secondary structure of TM5, the
Ca2+ transport function of TRPV5 is likely impaired.</p><p>The L530R variation is associated with a significantly increased risk of
recurrent kidneys stones in a founder population [9]. Knockout [2] or mutation [31] of the TRPV5 gene in mice results in
drastic urinary Ca2+ wasting. It is likely the
loss-of-function mutation will cause significant hypercalciuria in the L530R
carriers in an autosomal dominant fashion similar to what has been observed in
mice [31]. Because the
mutation is very rare with the minor allele frequency of 0.13% in this
founder population, homozygous L530R is unlikely present in this population, and
thus the L530R carriers would have one normal TRPV5 allele. Since TRPV5 tetramer
is formed as a functional unit, it is expected that the carriers of L530R will
have functional homotetramers of "wild-type" TRPV5,
heterotetramers of "wild-type" and variant TRPV5, as well as
nonfunctional homotetramers of variant TRPV5. This would result in a significant
reduction of TRPV5-mediated Ca2+ reabsorption in L530R
carries. Mice lacking TRPV5 alone do not form kidney stones under normal
conditions [32]. However,
when the H+- ATPase is also disrupted in the collecting duct,
tubular calcium phosphate crystals are formed in the renal medulla
[32]. Taken together,
it is likely that the reduction of the Ca2+ transport
function of TRPV5 in L530R variation carriers will result in hypercalciuria.
When other environmental or genetic factors are also present, calcium oxalate
crystallization and kidney stone formation may occur. This could explain the
recently reported association between this loss-of-function variation of TRPV5
and recurrent kidney stones [9].</p>