Reinhold Penner

CRACM1 Is a Plasma Membrane Protein Essential for Store-Operated Ca2+ Entry

Store-operated Ca2+ entry is mediated by Ca2+ release–activated Ca2+ (CRAC) channels following Ca2+ release from intracellular stores. We performed a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins that inhibit store-operated Ca2+ influx. A secondary patch-clamp screen identified CRACM1 and CRACM2 (CRAC modulators 1 and 2) as modulators of Drosophila CRAC currents. We characterized the human ortholog of CRACM1, a plasma membrane–resident protein encoded by gene FLJ14466. Although overexpression of CRACM1 did not affect CRAC currents, RNAi-mediated knockdown disrupted its activation. CRACM1 could be the CRAC channel itself, a subunit of it, or a component of the CRAC signaling machinery.

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.

A Unified Nomenclature for the Superfamily of TRP Cation Channels

The TRP superfamily includes a diversity of non-voltage-gated cation channels that vary significantly in their selectivity and mode of activation. Nevertheless, members of the TRP superfamily share significant sequence homology and predicted structural similarities. Currently, most of the genes and proteins that comprise the TRP superfamily have multiple names and, in at least one instance, two distinct genes belonging to separate subfamilies have the same name. Moreover, there are many cases in which highly related proteins that belong to the same subfamily have unrelated names. Therefore, to minimize confusion, we propose a unified nomenclature for the TRP superfamily.The current effort to unify the TRP nomenclature focuses on three subfamilies (TRPC, TRPV, and TRPM) that bear significant similarities to the founding member of this superfamily, Drosophila TRP, and which include highly related members in worms, flies, mice, and humans (Table 1)(Table 1). Members of the three subfamilies contain six transmembrane segments, a pore loop separating the final two transmembrane segments, and similarity in the lengths of the cytoplasmic and extracellular loops. In addition, the charged residues in the S4 segment that appear to contribute to the voltage sensor in voltage-gated ion channels are not conserved. The TRP-Canonical (TRPC) subfamily (formerly short-TRPs or STRPs) is comprised of those proteins that are the most highly related to Drosophila TRP. The TRPV subfamily (formerly OTRPC), is so named based on the original designation, Vanilloid Receptor 1 (VR1), for the first mammalian member of this subfamily (now TRPV1). The name for the TRPM subfamily (formerly long-TRPs or LTRPs) is derived from the first letter of Melastatin, the former name (now TRPM1) of the founding member of this third subfamily of TRP-related proteins. Based on amino acid homologies, the mammalian members of these three subfamilies can be subdivided into several groups each (Table 2Table 2 and Figure 1Figure 1) .Table 1Number of TRP Genes in Worms (C. elegans), Flies (Drosophila melanogaster), Mice, and HumansSubfamilyWormsFliesMiceHumansTRPC3376aaTRPV5255TRPM4188aTRPC2 is a pseudogene and is not counted.Table 2Nomenclature of the Mammalian TRP SuperfamilyNameGroupFormer NamesAccession NumbersTRPC11TRP1CAA61447, AAA93252TRPC1TRPC22TRP2X89067, AAD17195, AAD17196, AAG29950, AAG29951, AAD31453,TRPC2CAA06964TRPC33TRP3AAC51653TRPC3TRPC44TRP4CAA68125, BAA23599TRPC4TRPC54TRP5AAC13550, CAA06911, CAA06912TRPC5TRPC63TRP6NP_038866TRPC6TRPC73TRP7AAD42069, NP_065122TRPC7TRPV11VR1AAC53398OTRPC1TRPV21VRL-1AAD26363, AAD26364, BAA78478OTRPC2GRCTRPV3 (not assigned)TRPV42OTRPC4AAG17543, AAG16127, AAG28027, AAG28028, AAG28029,VR-OACCAC20703TRP12VRL-2TRPV53ECaC1CAB40138CaT2TRPV63CaT1AAD47636ECaC2CAC20416CaT-LCAC20417TRPM11MelastatinAAC13683, AAC80000TRPM22TRPC7BAA34700LTRPC2TRPM31KIAA1616AA038185LTRPC3TRPM43TRPM4H18835LTRPC4TRPM53MTR1AAF26288LTRPC5TRPM64Chak2AF350881TRPM74TRP-PLIKAAF73131Chak1LTRPC7TRPM82TRP-p8AC005538Indicated are the suggested gene and protein names, the groups within each subfamily, the former names, and accession numbers.Figure 1Phylogenetic Tree of the TRP SuperfamilyThe tree, which was adapted from Clapham et al., 2001 (Nat. Rev. Neurosci. 2, 387–396), was calculated using the neighbor-joining method and human, rat, and mouse sequences.View Large Image | View Hi-Res Image | Download PowerPoint SlideThe numbering system for the mammalian TRPC, TRPV, and TRPM proteins takes into account the order of their discovery and, in as many cases as possible, the number that has already been assigned to the genes and proteins (Table 2)(Table 2). In the case of the TRPV proteins, the numbering system is also based in part on the groupings of the TRPV proteins. New members of each subfamily will maintain the same root name and, with the exception of TRPV3, will be assigned the next number in the sequence. Currently, TRPV3 is unassigned to maintain the TRPV1/ TRPV2 and TRPV5/TRPV6 groupings and so that the former OTRPC4 could be renamed TRPV4. The next TRPV protein will be designated TRPV3.We hope this new nomenclature will add clarity to the field and simplify the naming of new members of the TRP superfamily. We recommend that accession numbers be used whenever it is necessary to unambiguously specify a given variant resulting from alternative mRNA splicing. Finally, this nomenclature has been approved by the HUGO Gene Nomenclature Committee and we recommend that this system be used in all future publications concerning TRPC, TRPV, and TRPM subfamily members.

Regulation of Vertebrate Cellular Mg2+ Homeostasis by TRPM7

TRPM7 is a polypeptide with intrinsic ion channel and protein kinase domains whose targeted deletion causes cells to experience growth arrest within 24 hr and eventually die. Here, we show that while TRPM7s kinase domain is not essential for activation of its channel, a functional coupling exists such that structural alterations of the kinase domain alter the sensitivity of channel activation to Mg(2+). Investigation of the relationship between Mg(2+) and the cell biological role of TRPM7 revealed that TRPM7-deficient cells become Mg(2+) deficient, that both the viability and proliferation of TRPM7-deficient cells are rescued by supplementation of extracellular Mg(2+), and that the capacity of heterologously expressed TRPM7 mutants to complement TRPM7 deficiency correlates with their sensitivity to Mg(2+). Overall, our results indicate that TRPM7 has a central role in Mg(2+) homeostasis as a Mg(2+) uptake pathway regulated through a functional coupling between its channel and kinase domains.

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.

Regulation of calcium influx by second messengers in rat mast cells

Biphasic increases in the free intracellular calcium concentration, consisting of a large initial transient followed by a sustained elevation, are frequently observed in non-excitable cells following stimulation. In rat peritoneal mast cells a cAMP- and Ca-activated chloride current can interact with IP3-dependent calcium influx to provide the sustained elevation of intracellular Ca concentration following transient IP3-induced release of calcium from intracellular stores. This novel combination of second messenger systems provides a flexible means to modulate calcium-dependent processes such as exocytosis.

TRPM7 Provides an Ion Channel Mechanism for Cellular Entry of Trace Metal Ions

Trace metal ions such as Zn2+, Fe2+, Cu2+, Mn2+, and Co2+ are required cofactors for many essential cellular enzymes, yet little is known about the mechanisms through which they enter into cells. We have shown previously that the widely expressed ion channel TRPM7 (LTRPC7, ChaK1, TRP-PLIK) functions as a Ca2+- and Mg2+-permeable cation channel, whose activity is regulated by intracellular Mg2+ and Mg2+·ATP and have designated native TRPM7-mediated currents as magnesium-nucleotide–regulated metal ion currents (MagNuM). Here we report that heterologously overexpressed TRPM7 in HEK-293 cells conducts a range of essential and toxic divalent metal ions with strong preference for Zn2+ and Ni2+, which both permeate TRPM7 up to four times better than Ca2+. Similarly, native MagNuM currents are also able to support Zn2+ entry. Furthermore, TRPM7 allows other essential metals such as Mn2+ and Co2+ to permeate, and permits significant entry of nonphysiologic or toxic metals such as Cd2+, Ba2+, and Sr2+. Equimolar replacement studies substituting 10 mM Ca2+ with the respective divalent ions reveal a unique permeation profile for TRPM7 with a permeability sequence of Zn2+ ≈ Ni2+ >> Ba2+ > Co2+ > Mg2+ ≥ Mn2+ ≥ Sr2+ ≥ Cd2+ ≥ Ca2+, while trivalent ions such as La3+ and Gd3+ are not measurably permeable. With the exception of Mg2+, which exerts strong negative feedback from the intracellular side of the pore, this sequence is faithfully maintained when isotonic solutions of these divalent cations are used. Fura-2 quenching experiments with Mn2+, Co2+, or Ni2+ suggest that these can be transported by TRPM7 in the presence of physiological levels of Ca2+ and Mg2+, suggesting that TRPM7 represents a novel ion-channel mechanism for cellular metal ion entry into vertebrate cells.