Jean-Pierre Kinet

LTRPC7 is a Mg·ATP-regulated divalent cation channel required for cell viability

The molecular mechanisms that regulate basal or background entry of divalent cations into mammalian cells are poorly understood. Here we describe the cloning and functional characterization of a Ca2+- and Mg2+-permeable divalent cation channel, LTRPC7 (nomenclature compatible with that proposed in ref. 1), a new member of the LTRPC family of putative ion channels. Targeted deletion of LTRPC7 in DT-40 B cells was lethal, indicating that LTRPC7 has a fundamental and nonredundant role in cellular physiology. Electrophysiological analysis of HEK-293 cells overexpressing recombinant LTRPC7 showed large currents regulated by millimolar levels of intracellular Mg·ATP and Mg·GTP with the permeation properties of a voltage-independent divalent cation influx pathway. Analysis of several cultured cell types demonstrated small magnesium-nucleotide-regulated metal ion currents (MagNuM) with regulation and permeation properties essentially identical to the large currents observed in cells expressing recombinant LTRPC7. Our data indicate that LTRPC7, by virtue of its sensitivity to physiological Mg·ATP levels, may be involved in a fundamental process that adjusts plasma membrane divalent cation fluxes according to the metabolic state of the cell.

ADP-ribose gating of the calcium-permeable LTRPC2 channel revealed by Nudix motif homology

Free ADP-ribose (ADPR), a product of NAD hydrolysis and a breakdown product of the calcium-release second messenger cyclic ADPR (cADPR), has no defined role as an intracellular signalling molecule in vertebrate systems. Here we show that a 350-amino-acid protein (designated NUDT9) and a homologous domain (NUDT9 homology domain) near the carboxy terminus of the LTRPC2/TrpC7 putative cation channel both function as specific ADPR pyrophosphatases. Whole-cell and single-channel analysis of HEK-293 cells expressing LTRPC2 show that LTRPC2 functions as a calcium-permeable cation channel that is specifically gated by free ADPR. The expression of native LTRPC2 transcripts is detectable in many tissues including the U937 monocyte cell line, in which ADPR induces large cation currents (designated IADPR) that closely match those mediated by recombinant LTRPC2. These results indicate that intracellular ADPR regulates calcium entry into cells that express LTRPC2.

Signalling through the high-affinity IgE receptor FcεRI

The FcεRI complex forms a high-affinity cell-surface receptor for the Fc region of antigen-specific immunoglobulin E (IgE) molecules. FcεRI is multimeric and is a member of a family of related antigen/Fc receptors which have conserved structural features and similar roles in initiating intracellular signalling cascades. In humans, FcεRI controls the activation of mast cells and basophils, and participates in IgE-mediated antigen presentation. Multivalent antigens bind and crosslink IgE molecules held at the cell surface by FcεRI. Receptor aggregation induces multiple signalling pathways that control diverse effector responses. These include the secretion of allergic mediators and induction of cytokine gene transcription, resulting in secretion of molecules such as interleukin-4, interleukin-6, tumour-necrosis factor-α and granulocyte-macrophage colony-stimulating factor. FcεRI is therefore central to the induction and maintenance of an allergic response and may confer physiological protection in parasitic infections.

Recruitment of Tyrosine Phosphatase HCP by the Killer Cell Inhibitory Receptor

Cytolysis of target cells by natural killer (NK) cells and by some cytotoxic T cells occurs unless prevented by inhibitory receptors that recognize MHC class I on target cells. Human NK cells express a p58 inhibitory receptor specific for HLA-C. We report association of the tyrosine phosphatase HCP with the p58 receptor in NK cells. HCP association was dependent on tyrosine phosphorylation of p58. Phosphotyrosyl peptides corresponding to the p58 tail bound and activated HCP in vitro. Furthermore, introduction of an inactive mutant HCP into an NK cell line prevented the p58-mediated inhibition of target cell lysis. These data imply that the inhibitory function of p58 is dependent on its tyrosine phosphorylation and on recruitment and activation of HCP.

Amplification of CRAC current by STIM1 and CRACM1 (Orai1)

Depletion of intracellular calcium stores activates store-operated calcium entry across the plasma membrane in many cells. STIM1, the putative calcium sensor in the endoplasmic reticulum, and the calcium release-activated calcium (CRAC) modulator CRACM1 (also known as Orai1) in the plasma membrane have recently been shown to be essential for controlling the store-operated CRAC current (ICRAC). However, individual overexpression of either protein fails to significantly amplify ICRAC. Here, we show that STIM1 and CRACM1 interact functionally. Overexpression of both proteins greatly potentiates ICRAC, suggesting that STIM1 and CRACM1 mutually limit store-operated currents and that CRACM1 may be the long-sought CRAC channel.

TRPM4 Is a Ca2+-Activated Nonselective Cation Channel Mediating Cell Membrane Depolarization

Calcium-activated nonselective (CAN) cation channels are expressed in various excitable and nonexcitable cells supporting important cellular responses such as neuronal bursting activity, fluid secretion, and cardiac rhythmicity. We have cloned and characterized a second form of TRPM4, TRPM4b, a member of the TRP channel family, as a molecular candidate of a CAN channel. TRPM4b encodes a cation channel of 25 pS unitary conductance that is directly activated by [Ca2+]i with an apparent K(D) of approximately 400 nM. It conducts monovalent cations such as Na+ and K+ without significant permeation of Ca2+. TRPM4b is activated following receptor-mediated Ca2+ mobilization, representing a regulatory mechanism that controls the magnitude of Ca2+ influx by modulating the membrane potential and, with it, the driving force for Ca2+ entry through other Ca2+-permeable pathways.

CRACM1 Multimers Form the Ion-Selective Pore of the CRAC Channel

Receptor-mediated Ca(2+) release from the endoplasmic reticulum (ER) is often followed by Ca(2+) entry through Ca(2+)-release-activated Ca(2+) (CRAC) channels in the plasma membrane . RNAi screens have identified STIM1 as the putative ER Ca(2+) sensor and CRACM1 (Orai1; ) as the putative store-operated Ca(2+) channel. Overexpression of both proteins is required to reconstitute CRAC currents (I(CRAC); ). We show here that CRACM1 forms multimeric assemblies that bind STIM1 and that acidic residues in the transmembrane (TM) and extracellular domains of CRACM1 contribute to the ionic selectivity of the CRAC-channel pore. Replacement of the conserved glutamate in position 106 of the first TM domain of CRACM1 with glutamine (E106Q) acts as a dominant-negative protein, and substitution with aspartate (E106D) enhances Na(+), Ba(2+), and Sr(2+) permeation relative to Ca(2+). Mutating E190Q in TM3 also affects channel selectivity, suggesting that glutamate residues in both TM1 and TM3 face the lumen of the pore. Furthermore, mutating a putative Ca(2+) binding site in the first extracellular loop of CRACM1 (D110/112A) enhances monovalent cation permeation, suggesting that these residues too contribute to the coordination of Ca(2+) ions to the pore. Our data provide unequivocal evidence that CRACM1 multimers form the Ca(2+)-selective CRAC-channel pore.

Phosphatidylinositol‐3,4,5‐trisphosphate (PtdIns‐3,4,5‐P3)/Tec kinase‐dependent calcium signaling pathway: a target for SHIP‐mediated inhibitory signals

Tec family non‐receptor tyrosine kinases have been implicated in signal transduction events initiated by cell surface receptors from a broad range of cell types, including an essential role in B‐cell development. A unique feature of several Tec members among known tyrosine kinases is the presence of an N‐terminal pleckstrin homology (PH) domain. We directly demonstrate that phosphatidylinositol‐3,4,5‐trisphosphate (PtdIns‐3,4,5‐P3) interacting with the PH domain acts as an upstream activation signal for Tec kinases, resulting in Tec kinase‐dependent phospholipase Cγ (PLCγ) tyrosine phosphorylation and inositol trisphosphate production. In addition, we show that this pathway is blocked when an SH2‐containing inositol phosphatase (SHIP)‐dependent inhibitory receptor is engaged. Together, our results suggest a general mechanism whereby PtdIns‐3,4,5‐P3 regulates receptor‐dependent calcium signals through the function of Tec kinases.

Btk/Tec kinases regulate sustained increases in intracellular Ca2+ following B‐cell receptor activation

Brutons tyrosine kinase (Btk) is essential for B‐lineage development and represents an emerging family of non‐receptor tyrosine kinases implicated in signal transduction events initiated by a range of cell surface receptors. Increased dosage of Btk in normal B cells resulted in a striking enhancement of extracellular calcium influx following B‐cell antigen receptor (BCR) cross‐linking. Ectopic expression of Btk, or related Btk/Tec family kinases, restored deficient extracellular Ca2+ influx in a series of novel Btk‐deficient human B‐cell lines. Btk and phospholipase Cγ (PLCγ) co‐expression resulted in tyrosine phosphorylation of PLCγ and required the same Btk domains as those for Btk‐dependent calcium influx. Receptor‐dependent Btk activation led to enhanced peak inositol trisphosphate (IP3) generation and depletion of thapsigargin (Tg)‐sensitive intracellular calcium stores. These results suggest that Btk maintains increased intracellular calcium levels by controlling a Tg‐sensitive, IP3‐gated calcium store(s) that regulates store‐operated calcium entry. Overexpression of dominant‐negative Syk dramatically reduced the initial phase calcium response, demonstrating that Btk/Tec and Syk family kinases may exert distinct effects on calcium signaling. Finally, co‐cross‐linking of the BCR and the inhibitory receptor, FcγRIIb1, completely abrogated Btk‐dependent IP3 production and calcium store depletion. Together, these data demonstrate that Btk functions at a critical crossroads in the events controlling calcium signaling by regulating peak IP3 levels and calcium store depletion.

New developments in FcεRI regulation, function and inhibition

The high-affinity Fc receptor for IgE (FcεRI), a multimeric immune receptor, is a crucial structure for IgE-mediated allergic reactions. In recent years, advances have been made in several important areas of the study of FcεRI. The first area relates to FcεRI-mediated biological responses that are antigen independent. The second area encompasses the biological relevance of the distinct signalling pathways that are activated by FcεRI; and the third area relates to the accumulated evidence for the tight control of FcεRI signalling through a broad array of inhibitory mechanisms, which are being developed into promising therapeutic approaches.