Christian Harteneck

OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarity.

Ca2+-permeable channels that are involved in the responses of mammalian cells to changes in extracellular osmolarity have not been characterized at the molecular level. Here we identify a new TRP (transient receptor potential)-like channel protein, OTRPC4, that is expressed at high levels in the kidney, liver and heart. OTRPC4 forms Ca2+-permeable, nonselective cation channels that exhibit spontaneous activity in isotonic media and are rapidly activated by decreases in, and are inhibited by increases in, extracellular osmolarity. Changes in osmolarity of as little as 10% result in significant changes in intracellular Ca2+ concentration. We propose that OTRPC4 is a candidate for a molecular sensor that confers osmosensitivity on mammalian cells.

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.

From worm to man: three subfamilies of TRP channels

A steadily increasing number of cDNAs for proteins that are structurally related to the TRP ion channels have been cloned in recent years. All these proteins display a topology of six transmembrane segments that is shared with some voltage-gated channels and the cyclic-nucleotide-gated channels. The TRP channels can be divided, on the basis of their homology, into three TRP channel (TRPC) subfamilies: short (S), long (L) and osm (O). From the evidence available to date, this subdivision can also be made according to channel function. Thus, the STRPC family, which includes Drosophila TRP and TRPL and the mammalian homologues, TRPC1-7, is a family of Ca2+-permeable cation channels that are activated subsequent to receptor-mediated stimulation of different isoforms of phospholipase C. Members of the OTRPC family are Ca2+-permeable channels involved in pain transduction (vanilloid and vanilloid-like receptors), epithelial Ca2+ transport and, at least in Caenorhabditis elegans, in chemo-, mechano- and osmoregulation. The LTRPC family is less well characterized.

Cloning and Functional Expression of a Human Ca2+-Permeable Cation Channel Activated by Calcium Store Depletion

Depletion of intracellular calcium stores generates a signal that activates Ca2+-permeable channels in the plasma membrane. We have identified a human cDNA, TRPC1A, from a human fetal brain cDNA library. TRPC1A is homologous to the cation channels trp and trpl in Drosophila and is a splice variant of the recently identified cDNA Htrp-1. Expression of TRPC1A in CHO cells induced nonselective cation currents with similar permeabilities for Na+, Ca2+, and Cs+. The currents were activated by intracellular infusion of myo inositol 1,4,5-trisphosphate or thapsigargin. Expression of TRPC1A significantly enhanced increases in the intracellular free calcium concentration induced by Ca2+ restitution after prolonged depletion. Similar results were obtained in Sf9 cells. We conclude that TRPC1A encodes a Ca2+-permeable cation channel activated by depletion of intracellular calcium stores.

Expression of soluble guanylyl cyclase: Catalytic activity requires two enzyme subunits

Purified soluble guanylyl cyclase consists of two subunits (70 and 73 kDa) whose primary structures were recently determined. The availability of cDNA clones coding for either subunit allowed to study the question of the functional roles of the two subunits in expression experiments. Enzyme subunits were expressed in COS‐7 cells by transfection with expression vectors containing the coding region for the 70 of 73 kDa subunit of the enzyme. No significant elevation in the activity of soluble guanylyl cyclase was observed in cells transfected with cDNA coding for one of the subunits. In contrast, transfection of cells with cDNAs coding for both subunits resulted in a marked increase in activity of soluble guanylyl cyclase. Enzyme activity was stimulated about 50‐fold by sodium nitroprusside. The results indicate that formation of cyclic GMP by soluble guanylyl cyclase requiresboth 70 and 73 kDa subunits.

Molecular cloning and expression of a new α-subunit of soluble guanylyl cyclase Interchangeability of the α-subunits of the enzyme

A cDNA coding for a new subunit of soluble guanylyl cyclase with a calculated molecular mass of 81.7 kDa was cloned and sequenced. On the basis of sequence homology, the new subunit appears to be an isoform of the α1‐subunit and was designated α2 as the new subunit is very similar to the α1‐subunit in the middle and C‐terminal part: it is quite diverse in the N‐terminal part. Preceding experiments had shown that coexpression of the α1‐ and β1‐subunits is necessary to obtain a catalytically active guanylyl cyclase in COS cells [(1990) FEBS Lett. 272, 221–223]. The finding that the α2‐subunit was able to replace the α1‐ but not the β1‐subunit in expression experiments demonstrates the interchangeability of the α‐subunit isoforms of soluble guanylyl cyclase.

The primary structure of the larger subunit of soluble guanylyl cyclase from bovine lung Homology between the two subunits of the enzyme

The primary structure of the larger subunit of the soluble guanylyl cyclase from bovine lung, which catalyzes the formation of cyclic GMP from GTP, has been determined. Two clones, isolated from two bovine libraries yielded a total of 3261 bp with a coding region of 2073 bp. The open reading frame encodes a protein of 691 amino acids and a molecular mass of 77 500. The deduced amino acid sequence reveals regions which are, to a large extent, homologous to the sequence of the smaller subunit of the enzyme as well as to the sequences of other gyanylyl and adenylyl cyclases.

G PROTEINS ENDOGENOUSLY EXPRESSED IN SF 9 CELLS : INTERACTIONS WITH MAMMALIAN HISTAMINE RECEPTORS

Expression of functionally active mammalian histamine H1- and H2-receptors was recently demonstrated in Sf9 cells. Either receptor elicited phosphoinositide degradation leading to an increased cytoplasmic calcium concentration. In the present study we focussed on identifying the Sf9 guanine nucleotide-binding proteins (G proteins) involved. Immunodetection of Sf9 membranes showed expression of Gα isoforms belonging to all four G protein subfamilies. During prolonged baculovirus infection of Sf9 cells, binding of guanosine 5’-o-(3-thiotriphosphate) as well as the intensities of G protein immunoreactivity, pertussis toxin-mediated ADP-ribosylation, GTP azidoanilide labelling of Gα, and phosphate-labelling of Gβ declined in cell membranes. Some 48h after infection with mammalian histamine receptor-encoding viruses virtually no functional coupling of ligand-activated receptors to insect G proteins was observed despite a high level of expressed receptors. In contrast, Sf9 cells infected only for 28h allowed studies on histamine-induced G protein coupling. In membranes obtained from H1-receptor-expressing cells, histamine increased incorporation of GTP azidoanilide into G q/11-like proteins whereas in membranes containing H2-receptors histamine enhanced GTP azidoanilide-labelling of G q/11-like and Gs-like proteins. In fura-loaded H1- and H2-receptor-expressing cells histamine induced the release of calcium from intracellular stores. This study shows firstly that Sf9 G proteins couple to mammalian histamine receptors and secondly that H1-receptors activate only Gq/11, whereas H2-receptors activate Gq/11 and Gs, but neither receptor couples to Gi/o or G12. Finally, the time following baculovirus infection is critical for studying the functional coupling between recombinantly expressed and endogenous signal transduction components.

THE DROSOPHILA CATION CHANNEL TRPL EXPRESSED IN INSECT SF9 CELLS IS STIMULATED BY AGONISTS OF G-PROTEIN-COUPLED RECEPTORS

Structures and regulations of vertebrate channels responsible for sustained calcium elevations after hormone stimulation are largely unknown. Therefore, the Drosophila photoreceptor channels, trp and trpl, which are assumed to be involved in calcium influx, serve as model system. trpl expressed in Sf9 cells showed spontanous activity. Hormonal stimulations of calcium influx (detected by fura‐2) and of an outwardly rectifying current were observed in Sf9 cells coinfected with baculoviruses encoding trpl and various heptahelical receptors for histamine, thrombin, and thromboxane A2, all known to cause phospholipase C‐β activation in mammalian cells. Although the identity of the G‐proteins and of possible second messengers involved need to be clarified, it is clear that trpl represents a receptor/G‐protein regulated cation channel.

A Variant of the α2 Subunit of Soluble Guanylyl Cyclase Contains an Insert Homologous to a Region within Adenylyl Cyclases and Functions as a Dominant Negative Protein

A variant of the α2 subunit of soluble guanylyl cyclase (α2i) containing 31 additional amino acids was identified in a number of cell lines and tissues. The in-frame sequence of the insert was within the proposed catalytic domain of guanylyl cyclases and was homologous to a region within the putative catalytic domain of adenylyl cyclases. Messenger RNA for the new variant was detected in some but not all cell lines and tissues expressing the α2 subunit. The novel form, as well as the α2 subunit lacking the insert, were coexpressed with the β1 subunit in Sf9 and COS-7 cells; α2/β1 coexpression yielded a NO-sensitive recombinant protein, whereas the coexpressed α2i/β1 subunits exhibited no guanylyl or adenylyl cyclase activities. Because both subunits (α2i/β1) copurified, the novel variant retains its ability to heterodimerize. In coexpression experiments, the α2i subunit competed with the α2 subunit for dimerization with the β1 subunit, thereby reducing α2/β1-catalyzed guanylyl cyclase activity. These data show that the novel variant functions as a dominant negative protein and that post-transcriptional mRNA processing represents a potential mechanism for regulation of NO-sensitive guanylyl cyclase acitivity.