TRPV6


TRPV6 is a membrane calcium channel protein which is particularly involved in the first step in calcium absorption in the intestine.

Classification

Transient Receptor Potential Vanilloid subfamily member 6 is an epithelial calcium channel that belongs to the transient receptor potential family of proteins. The TRP family is a group of channel proteins critical for ionic homeostasis and the perception of various physical and chemical stimuli. TRP channels can detect temperature, osmotic pressure, olfaction, taste, and mechanical forces. The human genome encodes for 28 TRP channels, which include six TRPV channels. The high calcium-selectivity of TRPV5 and TRPV6 makes these channels distinct from the other four TRPV channels. TRPV5 and TRPV6 are involved in calcium transport, whereas TRPV1 through TRPV3 are heat sensors with different temperature threshold for activation, and TRPV4 is involved in sensing osmolarity. Genetic defects in TRPV6 gene are linked to transient neonatal hyperparathyroidism and early onsite chronic pancreatitis. Dysregulation of TRPV6 is also involved in hypercalciuria, kidney stone formation, bone disorders, defects in keratinocyte differentiation, skeletal deformities, osteoarthritis, male sterility, Pendred syndrome, and certain sub-types of Cancer.

Discovery

Peng et al identified TRPV6 in 1999 from rat duodenum in an effort to search for calcium transporting proteins involved in calcium absorption. TRPV6 was also called calcium transport protein 1 initially although the names epithelial calcium channel 2 and CaT1-like were also used in early studies to describe the channel. The human and mouse orthologues of TRPV6 were cloned by Weber et al and Peng et al, respectively. The name TRPV6 was confirmed in 2005.

Gene location and phylogeny

The human TRPV6 gene is located on chromosomal locus 7q33-34 in close proximity to its homolog TRPV5 on 7q35. The TRPV6 gene in human encodes for 2906 bp-long mRNA. In contrast to most other proteins, which initiate translation with an AUG codon, TRPV6 translation is initiated by non-AUG-codon-mediated reading. TRPV6 protein bears a 40-aa-long N-terminal extension in placenta and in some physiological settings in comparison to the annotated version of the protein used in biological studies. However, it is still to be determined whether the long version of the TRPV6 protein is the dominant form in different tissues.
SpeciesHumanRatMouse
Chromosomal location7q33-q344q226B2
Annotated aa length725727727
In vivo aa lengtha765767767
RefSeq nucleotideNM_018646NM_053686NM_022413
RefSeq proteinNP_061116NP_446138NP_071858

aTo be verified in different tissues.
It has been hypothesized that Trpv5 and Trpv6 genes were generated from a single ancestral gene by gene duplication events. Phylogenetic analysis has shown that TRPV6 paralogs in mammals, sauropsids, amphibians, and chondrichthyes arose out of independent duplication events in the ancestor of each group. It is speculated that two specialized calcium-selective Trpv homologs arose as an adaptation to achieve a greater degree of functional specialization for navigating distinct renal challenges of terrestrial animals.
Two alleles of the TRPV6 gene have been identified in humans. These alleles exhibit coupled polymorphisms generating two versions of the same gene.  The polymorphisms give rise to an “ancestral variant” and a “derived variant” that differ in five bases and three amino acids. The ancestral allele codes for C197, M418, and M721 whereas the derived allele codes for R197, V418 and T721. The frequency of the ancestral TRPV6 allele varies across different population groups. It is hypothesized that selection pressures that could have changed TRPV6 allele distribution include changes in patterns of milk consumption, domestication of animals, change in ultraviolet light exposure due to trans-equatorial migration, genomic adaptations providing immune advantages to populations encountering new pathogens.

Tissue distribution

The TRPV6 protein is expressed in epithelial tissues such as the intestine, kidney, placenta, epididymis, and exocrine glands such as pancreas, prostate and salivary, sweat, and mammary glands. TRPV6 protein expression in humans has been demonstrated in the esophagus, stomach, small intestine, colon, pancreas, mammary glands, ovary, thyroid, and prostate by immunohistochemistry approaches. TRPV6 expression mainly confines on the apical membrane of epithelial cells. In the intestine, the protein is expressed on the brush-border membrane of enterocyte.
Differences in the TRPV6 expression profile have been reported possibly due to variation in assay-dependent such primer design, hybridization probes, PCR vs. northern blotting, semi-quantitative PCR vs. RT-PCR, and antibodies used for immunodetection. TRPV6 expression profile is also influenced by age, gender, calcium and vitamin D3 levels in food, hormonal status, location within the tissue, cellular location, reproductive status, and weaning status.
In humans, TRPV6 transcripts have been detected in the placenta, pancreas, prostate cancer, and duodenum and the prostate by northern blotting; and in duodenum, jejunum, placenta, pancreas, testis, kidney, brain, and colon by semi-quantitative PCR.  In rodents, TRPV6 expression has been validated in the duodenum, caecum, small intestine, colon, placenta, pancreas, prostate, and epididymis by Northern Blotting. In mouse, TRPV6 transcript abundance measured by RT PCR is as follows: prostate > stomach, brain > lung > duodenum, cecum, heart, kidney, bone > colon > skeletal muscle > pancreas.  
Data from Human Protein Atlas and RNA-Seq based suggest TRPV6 mRNA is low in most tissues except for the placenta, salivary gland, pancreas, and prostate. TRPV6 mRNA is expressed in the apical domain of murine osteoclasts of cortical bone. Cortical and trabecular osteocytes do not express TRPV6 mRNA whereas osteoblasts show weak expression.

Structure and biophysical properties

Secondary structure

Overall, four subunits of TRPV6 arrange to form a tetrameric channel displaying a four-fold symmetry. Beginning from N-terminus and moving towards the C-terminus of the protein, each TRPV6 polypeptide contains: an N-terminal helix, an ankyrin repeat domain containing six ankyrin repeats, a β-hairpin structure linker domain made up two β-strands, a helix-turn-helix motif, a pre-SI helix, TM domain made up of six TM helices, a pore-loop, amphipathic TRP helix, C-terminal hook, and a six-residue β-strand.

Tertiary and quaternary structure

The TRPV6 channel protein displays four-fold symmetry and contains two main compartments: a 30 Å-tall transmembrane domain with a central ion channel pore and a ~70 Å-tall and a ~110 Å-wide intracellular skirt enclosing a 50 Å × 50 Å cavity wide cavity underneath the ion channel . The clustering of four TRPV6 subunits forms an aqueous pore exhibiting a fourfold symmetry. A pre-SI helix links the intracellular portion of the protein to the TM domain through a linker domain made up of β-hairpin structure and a helix-turn-helix motif. Helices S1 through S4 form a transmembrane helical bundle or TM domain that is inserted almost perpendicularly to the plane of the plasma membrane .
The pore module elements are made up of S5, S6, and the P-loop in TM domains. The pore module from each TRPV6 polypeptide participates in intersubunit interactions to form a central ion pore. The pore-forming elements of each TRPV6 subunit also interact with S1-S4 domains of the adjacent polypeptide in a domain-swapped arrangement. Intersubunit interactions also occur between S1-S2 extracellular loops and S5-P and S6-P loops of the neighboring TRPV6 subunits. The conserved N-linked glycosylation site on the S1-S2 loop is required for by the Klotho-mediated activation. The intracellular skirt portion of the TRPV6 protein is mainly made up of the ankyrin repeats. The TRP domain is oriented parallel to the membrane and participates in hydrophobic interactions with the TM domain and the hydrophilic interactions in the intracellular skirt. The N-terminal helix, C-terminal hook, and β-sheets in the channel participates in intersubunit interactions with the ARDs to provides a framework for holding the elements of the intracellular skirt together.

Pore architecture and cation binding sites

The TRPV6 pore has four main elements, namely, the extracellular vestibule, a selectivity filter, a hydrophobic cavity, and a lower gate. Facing the central lumen of the channel, a four-residue selectivity filter containing four D541 side chains is critical for calcium selectivity and other biophysical properties of the channel. This filter forms a negatively charged ring that discriminates between ions based on their size and charge. Mutations in the critical pore-forming residue of TRPV6 blocks calcium uptake, a strategy has been used to generate TRPV6 loss-of-function models to examine the role of the channel in animal physiology. Four different types of cation binding sites are thought to exist in the TRPV6 channel. Site 1 is located in the central pore and shares the same plane that is occupied by the key selective residues D541. Site 2 is thought to be present about 6-8 Å below Site 1 followed by Site 3 which is located in the central pore axis about 6.8 Å below Site 2. Site 2 and 3 are thought to interact with partially-hydrated to equatorially-hydrated Ca2+ ions. Finally, four symmetric cation binding sites in the extracellular vestibule mediate the recruitment of cations towards the extracellular vestibule of TRPV6 and are referred to as recruitment sites.

Ion permeation

The conductance of TRPV6 for divalent cations follows the preference: Ca2+ > Ba2+ > Sr2+ > Mn2. Intracellular Mg2+ inhibits TRPV6 and contributes to the strong inward rectification exhibited by the channel . TRPV6 uptake activity is inhibited by divalent Pb2, Cu2+, Cd2+, Zn2, Co2+, Fe2+, and trivalent cations La3+, Fe3+, Gd3+. The concentration of ions to achieve the inhibition ranges from 1 to 10 μM. . The TRPV6 protein is constitutive with a single-channel conductance of 42-58 ps . At low Ca2+ concentrations, a single calcium ion binds in the selectivity filter formed by aspartates D541 and permits Na+ permeation. At high Ca2+ concentration, calcium permeation occurs by a knock-off mechanism that involves the formation of short-lived conformations involving binding of three calcium to residue D541.

Channel gating

The conformational changes involved in channel opening are hinged around the residue A566 and occur in the pore-lining helix S6 . The upper portion of S6 helix undergoes an α-to-π helical transition which forces the lower portion of the helix to turn by 100 degrees and tilt away from the pore axis by 11 degrees .  This conformational change moves the lower portion of the helix gating the pore and thereby widens the pore size. The conformational change alters the residues facing the pore axis and triggers the formation of new electrostatic bonds subunit and salt bridges that offset the high energetic cost of unfavorable α-to-π helical transition that occurs during channel opening .

Regulation

The influx of Ca2+ inside the cell triggers negative feedback mechanisms to suppress TRPV6 activity and prevent calcium overload  . TRPV6 channel activity is regulated by phospholipid PIP2 levels and interactions with Ca2+-Calmodulin complex . The depletion of PIP2 or calmodulin-binding inactivates TRPV6 . The influx of Ca2+ in TRPV6 expressing cells activates phospholipase C which in turn hydrolyzes PIP2. Depletion in PIP2 levels results in a decline in channel activity since most TRP channels require this lipid for activation . The lipid PIP2 can override Ca2+-CaM-mediated inhibition of TRPV6. Overall, TRPV6 inactivation by calmodulin is orchestrated by a balance of intracellular calcium and PIP2 concentration.

Interacting proteins

Among 20+ TRPV6 interactors identified so far, the functional consequences of Ca2+-binding protein Calmodulin
InteractorConsequence
BSPRYN/A
Calbindin-D28kN/A
CalmodulinInhibition
Cyclophilin BActivation
FYNPO4lyation
I-MFAN/A
KlothoActivation, Glycosylation
NHERF4Activation
NIPSNAP1Inhibition
NUMBInhibition
PTENN/A
PTP1BDePO4lyation
RAB11AActivation,
Increase in Plasma membrane level
RGS2N/A
RYR1N/A
S100A10Activation,
Increase in Plasma membrane level
SRCPO4lyation
TRPC1Retains in ER, Inhibition
TRPML3N/A
TRPV5Tetramer formation,
New Channel creation

Abbreviations
Protein Interactor
BSPRY: B-Box and Spry Domain Containing Protein; FYN: Fyn Kinase Belonging Src Family of Kinases; I-MFA: Myo D Family Inhibitor; NHERF: Na Exchanger Regulatory Factor; NIPSNAP14-Nitrophenylphosphatase Domain and Non-Neuronal SNAP25-Like Protein Homolog 1; Numb: Drosophila mutation that removes most of the sensory neurons in the developing peripheral nervous system; PTP: Protein Tyrosine Phosphatase; Rab11a: Member RAS Oncogene Family; RGS2: Regulator Of G-Protein Signaling 2; RyR1: Ryanodine Receptor 1; TRPC1: Transient receptor potential canonical 1; TRPML3: Transient receptor potential Mucolipin-3.

Regulation of expression

Vitamin D

Expression of TRPV6 is vitamin D dependent in mice and humans. Its expression was greatly reduced in animals that do not express the vitamin D receptor.
Vitamin D treatment of human colon cancer cells, Caco-2, increased expression of TRPV6 transcripts, and also stimulated the transport of calcium, probably through increased TRPV6 expression. In human duodenal explants, TRPV6 transcript expression was increased 3-fold after 6h incubation with the active form of vitamin D, 1,25-dihydroxycholecalciferol.