Bidirectional Allosteric Coupling between PIP2 Binding and the Pore of the Oncochannel TRPV6.

The epithelial ion channel TRPV6 plays a pivotal role in calcium homeostasis. Channel function is intricately regulated at different stages, involving the lipid phosphatidylinositol-4,5-bisphosphate (PIP2). Given that dysregulation of TRPV6 is associated with various diseases, including different types of cancer, there is a compelling need for its pharmacological targeting. Structural studies provide insights on how TRPV6 is affected by different inhibitors, with some binding to sites else occupied by lipids. These include the small molecule cis-22a, which, however, also binds to and thereby blocks the pore. By combining calcium imaging, electrophysiology and optogenetics, we identified residues within the pore and the lipid binding site that are relevant for regulation by cis-22a and PIP2 in a bidirectional manner. Yet, mutation of the cytosolic pore exit reduced inhibition by cis-22a but preserved sensitivity to PIP2 depletion. Our data underscore allosteric communication between the lipid binding site and the pore and vice versa for most sites along the pore.

Photocrosslinking-induced CRAC channel-like Orai1 activation independent of STIM1.

Ca2+ release-activated Ca2+ (CRAC) channels, indispensable for the immune system and various other human body functions, consist of two transmembrane (TM) proteins, the Ca2+-sensor STIM1 in the ER membrane and the Ca2+ ion channel Orai1 in the plasma membrane. Here we employ genetic code expansion in mammalian cell lines to incorporate the photocrosslinking unnatural amino acids (UAA), p-benzoyl-L-phenylalanine (Bpa) and p-azido-L-phenylalanine (Azi), into the Orai1 TM domains at different sites. Characterization of the respective UAA-containing Orai1 mutants using Ca2+ imaging and electrophysiology reveal that exposure to UV light triggers a range of effects depending on the UAA and its site of incorporation. In particular, photoactivation at A137 using Bpa in Orai1 activates Ca2+ currents that best match the biophysical properties of CRAC channels and are capable of triggering downstream signaling pathways such as nuclear factor of activated T-cells (NFAT) translocation into the nucleus without the need for the physiological activator STIM1.

TRPV6 Regulation by Cis-22a and Cholesterol.

The highly calcium-selective transient receptor potential vanilloid-type channel TRPV6 is important for epithelial Ca2+ transport. Proper regulation of the inherently constitutively active TRPV6 channels is intricate in preserving Ca2+ homeostasis, whereby structural and functional data suggest that lipids hold an essential role. Altered expression levels or specific TRPV6 mutations may lead to diseases, hence, TRPV6 represents an interesting target for pharmacological modulation. Recent cryo-EM data identified that the specific TRPV6 blocker cis-22a binds, apart from the pore, to a site within the tetrameric channel that largely matches a lipid binding pocket, LBS-2. Therein, cis-22a may replace a lipid such as cholesterol that is bound in the open state. Based on site-directed mutagenesis and functional recordings, we identified and characterized a series of residues within LBS-2 that are essential for TRPV6 inhibition by cis-22a. Additionally, we investigated the modulatory potential of diverse cholesterol depletion efforts on TRPV6 activity. While LBS-2 mutants exhibited altered maximum currents, slow Ca2+-dependent inactivation (SCDI) as well as less inhibition by cis-22a, TRPV6 activity was resistant to cholesterol depletion. Hence, lipids other than cholesterol may predominate TRPV6 regulation when the channel is expressed in HEK293 cells.

CRAC channel opening is determined by a series of Orai1 gating checkpoints in the transmembrane and cytosolic regions.

The initial activation step in the gating of ubiquitously expressed Orai1 calcium (Ca2+) ion channels represents the activation of the Ca2+-sensor protein STIM1 upon Ca2+ store depletion of the endoplasmic reticulum. Previous studies using constitutively active Orai1 mutants gave rise to, but did not directly test, the hypothesis that STIM1-mediated Orai1 pore opening is accompanied by a global conformational change of all Orai transmembrane domain (TM) helices within the channel complex. We prove that a local conformational change spreads omnidirectionally within the Orai1 complex. Our results demonstrate that these locally induced global, opening-permissive TM motions are indispensable for pore opening and require clearance of a series of Orai1 gating checkpoints. We discovered these gating checkpoints in the middle and cytosolic extended TM domain regions. Our findings are based on a library of double point mutants that contain each one loss-of-function with one gain-of-function point mutation in a series of possible combinations. We demonstrated that an array of loss-of-function mutations are dominant over most gain-of-function mutations within the same as well as of an adjacent Orai subunit. We further identified inter- and intramolecular salt-bridge interactions of Orai subunits as a core element of an opening-permissive Orai channel architecture. Collectively, clearance and synergistic action of all these gating checkpoints are required to allow STIM1 coupling and Orai1 pore opening. Our results unravel novel insights in the preconditions of the unique fingerprint of CRAC channel activation, provide a valuable source for future structural resolutions, and help to understand the molecular basis of disease-causing mutations.

Inactivation-mimicking block of the epithelial calcium channel TRPV6.

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.

Natural product inspired optimization of a selective TRPV6 calcium channel inhibitor.

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 µM) 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.

Photoswitchable Inhibitor of the Calcium Channel TRPV6.

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.

Interferon inducible X-linked gene CXorf21 may contribute to sexual dimorphism in Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease, characterised by increased expression of type I interferon (IFN)-regulated genes and a striking sex imbalance towards females. Through combined genetic, in silico, in vitro, and ex vivo approaches, we define CXorf21, a gene of hitherto unknown function, which escapes X-chromosome inactivation, as a candidate underlying the Xp21.2 SLE association. We demonstrate that CXorf21 is an IFN-response gene and that the sexual dimorphism in expression is magnified by immunological challenge. Fine-mapping reveals a single haplotype as a potential causal cis-eQTL for CXorf21. We propose that expression is amplified through modification of promoter and 3'-UTR chromatin interactions. Finally, we show that the CXORF21 protein colocalises with TLR7, a pathway implicated in SLE pathogenesis. Our study reveals modulation in gene expression affected by the combination of two hallmarks of SLE: CXorf21 expression increases in a both an IFN-inducible and sex-specific manner.

Lipid-independent control of endothelial and neuronal TRPC3 channels by light.

Lipid-gated TRPC channels are highly expressed in cardiovascular and neuronal tissues. Exerting precise pharmacological control over their activity in native cells is expected to serve as a basis for the development of novel therapies. Here we report on a new photopharmacological tool that enables manipulation of TRPC3 channels by light, in a manner independent of lipid metabolism and with higher temporal precision than lipid photopharmacology. Using the azobenzene photoswitch moiety, we modified GSK1702934A to generate light-controlled TRPC agonists. We obtained one light-sensitive molecule (OptoBI-1) that allows us to exert efficient, light-mediated control over TRPC3 activity and the associated cellular Ca2+ signaling. OptoBI-1 enabled high-precision, temporal control of TRPC3-linked cell functions such as neuronal firing and endothelial Ca2+ transients. With these findings, we introduce a novel photopharmacological strategy to control native TRPC conductances.

A novel STIM1-Orai1 gating interface essential for CRAC channel activation.

Calcium signalling through store-operated calcium (SOC) entry is of crucial importance for T-cell activation and the adaptive immune response. This entry occurs via the prototypic Ca2+ release-activated Ca2+ (CRAC) channel. STIM1, a key molecular component of this process, is located in the membrane of the endoplasmic reticulum (ER) and is initially activated upon Ca2+ store depletion. This activation signal is transmitted to the plasma membrane via a direct physical interaction that takes place between STIM1 and the highly Ca2+-selective ion channel Orai1. The activation of STIM1 induces an extended cytosolic conformation. This, in turn, exposes the CAD/SOAR domain and leads to the formation of STIM1 oligomers. In this study, we focused on a small helical segment (STIM1 α3, aa 400-403), which is located within the CAD/SOAR domain. We determined this segment's specific functional role in terms of STIM1 activation and Orai1 gating. The STIM1 α3 domain appears not essential for STIM1 to interact with Orai1. Instead, it represents a key domain that conveys STIM1 interaction into Orai1 channel gating. The results of cysteine crosslinking experiments revealed the close proximity of STIM1 α3 to a region within Orai1, which was located at the cytosolic extension of transmembrane helix 3, forming a STIM1-Orai1 gating interface (SOGI). We suggest that the interplay between STIM1 α3 and Orai1 TM3 allows STIM1 coupling to be transmitted into physiological CRAC channel activation.