Protein-coding gene in the species Homo sapiens
TRPV6 is a membrane calcium (Ca2+) channel protein which is particularly involved in the first step in Ca2+absorption in the intestine.
Classification.
Transient Receptor Potential Vanilloid subfamily member 6 (TRPV6) is an epithelial Ca2+ channel that belongs to the transient receptor potential family (TRP) 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 Ca2+-selectivity of TRPV5 and TRPV6 makes these channels distinct from the other four TRPV channels (TRPV1-TRPV4). TRPV5 and TRPV6 are involved in Ca2+ 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-onset 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.
Identification.
Peng "et al" identified TRPV6 in 1999 from rat duodenum in an effort to search for Ca2+ transporting proteins involved in Ca2+absorption. TRPV6 was also called calcium transport protein 1 (CaT1) initially although the names epithelial calcium channel 2 (ECaC2) and CaT1-like (CaT-L) were also used in early studies to describe the channel. The human and mouse orthologs of TRPV6 were cloned by Peng et al and Weber et al, respectively. The name TRPV6 was confirmed in 2005.
Gene location, chromosomal location, and phylogeny.
The human "TRPV6" gene is located on chromosomal locus 7q33-34 close 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-a.a-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.
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 Ca2+-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 (originally noted as CaT-La and CaT-Lb). 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(157, in parentheses are annotated amino acid numbering), M418(378), and M721(681) whereas the derived allele codes for R197(157), V418 (378) and T721(681). 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, Ca2+ and vitamin D3 levels in food, hormonal status, location within the tissue, cellular location, reproductive status, and weaning status (see Section Regulation).
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, cecum, 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.
Primary and 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 (ARD) 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 (S1 through S6), a pore-loop (also called P-loop), amphipathic TRP helix, C-terminal hook, and a six-residue β-strand (β3) (Figure 1).
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 (Figure 2). 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 inter-subunit interactions to form a central ion pore (Figure 1). 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 (formed by the β-hairpin structure in the linker domain) 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 (538TIID541) containing four Aspartate 541 (D541) side chains (one from each protomer) is critical for Ca2+ 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 Ca2+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 symmetrical 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. Intra-cellular 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 Ca2+ ion binds in the selectivity filter formed by D541 and permits Na+ permeation. At high Ca2+ concentration, Ca2+ permeation occurs by a knock-off mechanism that involves the formation of short-lived conformations involving binding of three Ca2+ ions to residue D541.
Channel gating.
The conformational changes involved in channel opening are hinged around the residue Alanine 566 (A566) and occur in the pore-lining helix S6 (Figure 3). 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 by phosphatidylinositol 4,5-bisphosphate (PIP2) and calmodulin (CaM).
The influx of Ca2+ inside the cell triggers negative feedback mechanisms to suppress TRPV6 activity and prevent Ca2+ overload. TRPV6 channel activity is regulated by the intracellular level of phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) and interactions with Ca2+-Calmodulin (CaM) complex. The depletion of PIP2 or CaM-binding inactivates TRPV6. The influx of Ca2+ in TRPV6 expressing cells activates phospholipase C (PLC) 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 Ca2+ and PIP2 concentration.
Interacting proteins.
Among 20+ TRPV6 interactors identified so far, the functional consequences of Ca2+-binding protein Calmodulin (CaM) and Glucuronidase Klotho have been most extensively characterized [36, 37, 41, 42]. Functional consequences of TRPV6 channel activation are summarized in the table below).
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.
Physiological functions.
The Ca2+-selective channel proteins TRPV6 and TRPV5 cooperate to maintain calcium concentration in specific organs. TRPV6 functions as apical Ca2+ entry channels mediating transcellular transport of this ion in the intestine, placenta, and possibly some other exocrine organs. TRPV6 also plays important roles in maternal-fetal calcium transport, keratinocyte differentiation, and Ca2+ homeostasis in the endolymphatic system of the vestibular system, and maintenance of male fertility.
"C"a2+ absorption in intestine.
Two routes of Ca2+ absorption are recognized: paracellular transport and transcellular transport (see Figure 4). A high-Ca2+-diet favors paracellular transport of the ion across the length of the intestine allowing them to pass between the intercellular tight junctions that connect epithelial cells. In contrast under conditions when [Ca2+] in the lumen of the intestine is lower in