Mechanism of calmodulin inactivation of the calcium-selective TRP channel TRPV6.
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ABSTRACT:
SUBMITTER: Singh AK
PROVIDER: S-EPMC6093632 | biostudies-literature | 2018 Aug
REPOSITORIES: biostudies-literature
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Project description:Epithelial calcium channel TRPV6 is a member of the vanilloid subfamily of TRP channels that is permeable to cations and highly selective to Ca2+ ; it shows constitutive activity regulated negatively by Ca2+ and positively by phosphoinositol and cholesterol lipids. In this review, we describe the molecular structure of TRPV6 and discuss how its structural elements define its unique functional properties. High Ca2+ selectivity of TRPV6 originates from the narrow selectivity filter, where Ca2+ ions are directly coordinated by a ring of anionic aspartate side chains. Divalent cations Ca2+ and Ba2+ permeate TRPV6 pore according to the knock-off mechanism, while tight binding of Gd3+ to the aspartate ring blocks the channel and prevents Na+ from permeating the pore. The iris-like channel opening is accompanied by an α-to-π helical transition in the pore-lining transmembrane helix S6. As a result of this transition, the intracellular halves of the S6 helices bend and rotate by about 100 deg, exposing different residues to the channel pore in the open and closed states. Channel opening is also associated with changes in occupancy of the transmembrane domain lipid binding sites. The inhibitor 2-aminoethoxydiphenyl borate (2-APB) binds to TRPV6 in a pocket formed by the cytoplasmic half of the S1-S4 transmembrane helical bundle and shifts open-closed channel equilibrium towards the closed state by outcompeting lipids critical for activation. Ca2+ inhibits TRPV6 via binding to calmodulin (CaM), which mediates Ca2+ -dependent inactivation. The TRPV6-CaM complex exhibits 1:1 stoichiometry; one TRPV6 tetramer binds both CaM lobes, which adopt a distinct head-to-tail arrangement. The CaM C-terminal lobe plugs the channel through a unique cation-π interaction by inserting the side chain of lysine K115 into a tetra-tryptophan cage at the ion channel pore intracellular entrance. Recent studies of TRPV6 structure and function described in this review advance our understanding of the role of this channel in physiology and pathophysiology and inform new therapeutic design.
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Project description:Epithelial calcium channel TRPV6 plays vital roles in calcium homeostasis, and its dysregulation is implicated in multifactorial diseases, including cancers. Here, we study the molecular mechanism of selective nanomolar-affinity TRPV6 inhibition by (4-phenylcyclohexyl)piperazine derivatives (PCHPDs). We use x-ray crystallography and cryo-electron microscopy to solve the inhibitor-bound structures of TRPV6 and identify two types of inhibitor binding sites in the transmembrane region: (i) modulatory sites between the S1-S4 and pore domains normally occupied by lipids and (ii) the main site in the ion channel pore. Our structural data combined with mutagenesis, functional and computational approaches suggest that PCHPDs plug the open pore of TRPV6 and convert the channel into a nonconducting state, mimicking the action of calmodulin, which causes inactivation of TRPV6 channels under physiological conditions. This mechanism of inhibition explains the high selectivity and potency of PCHPDs and opens up unexplored avenues for the design of future-generation biomimetic drugs.
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Project description:Transient receptor potential vanilloid 6 (TRPV6) is a calcium channel implicated in multifactorial diseases and overexpressed in numerous cancers. We recently reported the phenyl-cyclohexyl-piperazine cis-22a as the first submicromolar TRPV6 inhibitor. This inhibitor showed a seven-fold selectivity against the closely related calcium channel TRPV5 and no activity on store-operated calcium channels (SOC), but very significant off-target effects and low microsomal stability. Here, we surveyed analogues incorporating structural features of the natural product capsaicin and identified 3OG, a new oxygenated analog with similar potency against TRPV6 (IC50 = 0.082 ± 0.004 ?M) and ion channel selectivity, but with high microsomal stability and very low off-target effects. This natural product-inspired inhibitor does not exhibit any non-specific toxicity effects on various cell lines and is proposed as a new tool compound to test pharmacological inhibition of TRPV6 mediated calcium flux in disease models.
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Project description:Transient receptor potential (TRP) channels are involved in various physiological processes, including sensory transduction. The TRP channel TRPV6 mediates calcium uptake in epithelia and its expression is dramatically increased in numerous types of cancer. TRPV6 inhibitors suppress tumor growth, but the molecular mechanism of inhibition remains unknown. Here, we present crystal and cryo-EM structures of human and rat TRPV6 bound to 2-aminoethoxydiphenyl borate (2-APB), a TRPV6 inhibitor and modulator of numerous TRP channels. 2-APB binds to TRPV6 in a pocket formed by the cytoplasmic half of the S1-S4 transmembrane helix bundle. Comparing human wild-type and high-affinity mutant Y467A structures, we show that 2-APB induces TRPV6 channel closure by modulating protein-lipid interactions. Mutagenesis and functional analyses suggest that the identified 2-APB binding site might be present in other members of vanilloid subfamily TRP channels. Our findings reveal a mechanism of ion channel allosteric modulation that can be exploited for therapeutic design.
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Project description:Herein we report the first photoswitchable inhibitor of Transient Receptor Potential Vanilloid 6 (TRPV6), a selective calcium channel involved in a number of diseases and in cancer progression. By surveying analogs of a previously reported TRPV6 inhibitor appended with a phenyl-diazo group, we identified a compound switching between a weak TRPV6 inhibitor in its dark, E-diazo stereoisomer (Z/E = 3:97, IC50 ≫ 10 μM) and a potent inhibitor as the Z-diazo stereoisomer accessible reversibly by UV irradiation at λ = 365 nm (Z/E = 3:1, IC50 = 1.7 ± 0.4 μM), thereby allowing precise spatiotemporal control of inhibition. This new tool compound should be useful to deepen our understanding of TRPV6.
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Project description:Calcium-selective transient receptor potential vanilloid subfamily member 6 (TRPV6) channels play a critical role in calcium uptake in epithelial tissues. Altered TRPV6 expression is associated with a variety of human diseases, including cancers. TRPV6 channels are constitutively active and their open probability depends on the lipidic composition of the membrane in which they reside; it increases substantially in the presence of phosphatidylinositol 4,5-bisphosphate. Crystal structures of detergent-solubilized rat TRPV6 in the closed state have previously been solved. Corroborating electrophysiological results, these structures demonstrated that the Ca2+ selectivity of TRPV6 arises from a ring of aspartate side chains in the selectivity filter that binds Ca2+ tightly. However, how TRPV6 channels open and close their pores for ion permeation has remained unclear. Here we present cryo-electron microscopy structures of human TRPV6 in the open and closed states. The channel selectivity filter adopts similar conformations in both states, consistent with its explicit role in ion permeation. The iris-like channel opening is accompanied by an ?-to-?-helical transition in the pore-lining transmembrane helix S6 at an alanine hinge just below the selectivity filter. As a result of this transition, the S6 helices bend and rotate, exposing different residues to the ion channel pore in the open and closed states. This gating mechanism, which defines the constitutive activity of TRPV6, is, to our knowledge, unique among tetrameric ion channels and provides structural insights for understanding their diverse roles in physiology and disease.
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Project description:Precise regulation of calcium homeostasis is essential for many physiological functions. The Ca(2+)-selective transient receptor potential (TRP) channels TRPV5 and TRPV6 play vital roles in calcium homeostasis as Ca(2+) uptake channels in epithelial tissues. Detailed structural bases for their assembly and Ca(2+) permeation remain obscure. Here we report the crystal structure of rat TRPV6 at 3.25?Å resolution. The overall architecture of TRPV6 reveals shared and unique features compared with other TRP channels. Intracellular domains engage in extensive interactions to form an intracellular 'skirt' involved in allosteric modulation. In the K(+) channel-like transmembrane domain, Ca(2+) selectivity is determined by direct coordination of Ca(2+) by a ring of aspartate side chains in the selectivity filter. On the basis of crystallographically identified cation-binding sites at the pore axis and extracellular vestibule, we propose a Ca(2+) permeation mechanism. Our results provide a structural foundation for understanding the regulation of epithelial Ca(2+) uptake and its role in pathophysiology.
| S-EPMC4919205 | biostudies-literature
Project description:Transient receptor potential vanilloid subfamily member 6 (TRPV6) is a highly selective calcium channel that has been considered as a part of store-operated calcium entry (SOCE). Despite its first discovery in the early 2000s, the role of this channel in prostate cancer (PCa) remained, until now, obscure. Here we show that TRPV6 mediates calcium entry, which is highly increased in PCa due to the remodeling mechanism involving the translocation of the TRPV6 channel to the plasma membrane via the Orai1/TRPC1-mediated Ca(2+)/Annexin I/S100A11 pathway, partially contributing to SOCE. The TRPV6 calcium channel is expressed de novo by the PCa cell to increase its survival by enhancing proliferation and conferring apoptosis resistance. Xenografts in nude mice and bone metastasis models confirmed the remarkable aggressiveness of TRPV6-overexpressing tumors. Immunohistochemical analysis of these demonstrated the increased expression of clinical markers such as Ki-67, prostate specific antigen, synaptophysin, CD31, and CD56, which are strongly associated with a poor prognosis. Thus, the TRPV6 channel acquires its oncogenic potential in PCa due to the remodeling mechanism via the Orai1-mediated Ca(2+)/Annexin I/S100A11 pathway.
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Project description:Tetrameric ion channels have either swapped or non-swapped arrangements of the S1-S4 and pore domains. Here we show that mutations in the transmembrane domain of TRPV6 can result in conversion from a domain-swapped to non-swapped fold. These results reveal structural determinants of domain swapping and raise the possibility that a single ion channel subtype can fold into either arrangement in vivo, affecting its function in normal or disease states.
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Project description:TRPV6 is a member of the transient receptor potential superfamily of ion channels that facilitates Ca(2+) absorption in the intestines. These channels display high selectivity for Ca(2+), but in the absence of divalent cations they also conduct monovalent ions. TRPV6 channels have been shown to be inactivated by increased cytoplasmic Ca(2+) concentrations. Here we studied the mechanism of this Ca(2+)-induced inactivation. Monovalent currents through TRPV6 substantially decreased after a 40-s application of Ca(2+), but not Ba(2+). We also show that Ca(2+), but not Ba(2+), influx via TRPV6 induces depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2) or PIP(2)) and the formation of inositol 1,4,5-trisphosphate. Dialysis of DiC(8) PI(4,5)P(2) through the patch pipette inhibited Ca(2+)-dependent inactivation of TRPV6 currents in whole-cell patch clamp experiments. PI(4,5)P(2) also activated TRPV6 currents in excised patches. PI(4)P, the precursor of PI(4,5)P(2), neither activated TRPV6 in excised patches nor had any effect on Ca(2+)-induced inactivation in whole-cell experiments. Conversion of PI(4,5)P(2) to PI(4)P by a rapamycin-inducible PI(4,5)P(2) 5-phosphatase inhibited TRPV6 currents in whole-cell experiments. Inhibiting phosphatidylinositol 4 kinases with wortmannin decreased TRPV6 currents and Ca(2+) entry into TRPV6-expressing cells. We propose that Ca(2+) influx through TRPV6 activates phospholipase C and the resulting depletion of PI(4,5)P(2) contributes to the inactivation of TRPV6.
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