TRPV3 is a temperature-sensitive vanilloid receptor-like protein

Vanilloid receptor-1 (VR1, also known as TRPV1) is a thermosensitive, nonselective cation channel that is expressed by capsaicin-sensitive sensory afferents and is activated by noxious heat, acidic pH and the alkaloid irritant capsaicin. Although VR1 gene disruption results in a loss of capsaicin responses, it has minimal effects on thermal nociception. This and other experiments--such as those showing the existence of capsaicin-insensitive heat sensors in sensory neurons--suggest the existence of thermosensitive receptors distinct from VR1. Here we identify a member of the vanilloid receptor/TRP gene family, vanilloid receptor-like protein 3 (VRL3, also known as TRPV3), which is heat-sensitive but capsaicin-insensitive. VRL3 is coded for by a 2,370-base-pair open reading frame, transcribed from a gene adjacent to VR1, and is structurally homologous to VR1. VRL3 responds to noxious heat with a threshold of about 39 degrees C and is co-expressed in dorsal root ganglion neurons with VR1. Furthermore, when heterologously expressed, VRL3 is able to associate with VR1 and may modulate its responses. Hence, not only is VRL3 a thermosensitive ion channel but it may represent an additional vanilloid receptor subunit involved in the formation of heteromeric vanilloid receptor channels.     

Polycystin-L is a calcium-regulated cation channel permeable to calcium ions.

Polycystic kidney diseases are genetic disorders in which the renal parenchyma is progressively replaced by fluid-filled cysts. Two members of the polycystin family (polycystin-1 and -2) are mutated in autosomal dominant polycystic kidney disease (ADPKD), and polycystin-L is deleted in mice with renal and retinal defects. Polycystins are membrane proteins that share significant sequence homology, especially polycystin-2 and -L (50% identity and 71% similarity). The functions of the polycystins remain unknown. Here we show that polycystin-L is a calcium-modulated nonselective cation channel that is permeable to sodium, potassium and calcium ions. Patch-clamp experiments revealed single-channel activity with a unitary conductance of 137 pS. Channel activity was substantially increased when either the extracellular or intracellular calcium-ion concentration was raised, indicating that polycystin-L may act as a transducer of calcium-mediated signalling in vivo. Its large single-channel conductance and regulation by calcium ions distinguish it from other structurally related cation channels.     

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.     

A TRPV family ion channel required for hearing in Drosophila

The many types of insect ear share a common sensory element, the chordotonal organ, in which sound-induced antennal or tympanal vibrations are transmitted to ciliated sensory neurons and transduced to receptor potentials. However, the molecular identity of the transducing ion channels in chordotonal neurons, or in any auditory system, is still unknown. Drosophila that are mutant for NOMPC, a transient receptor potential (TRP) superfamily ion channel, lack receptor potentials and currents in tactile bristles but retain most of the antennal sound-evoked response, suggesting that a different channel is the primary transducer in chordotonal organs. Here we describe the Drosophila Nanchung (Nan) protein, an ion channel subunit similar to vanilloid-receptor-related (TRPV) channels of the TRP superfamily. Nan mediates hypo-osmotically activated calcium influx and cation currents in cultured cells. It is expressed in vivo exclusively in chordotonal neurons and is localized to their sensory cilia. Antennal sound-evoked potentials are completely absent in mutants lacking Nan, showing that it is an essential component of the chordotonal mechanotransducer.     

cGMP-dependent cation channel of retinal rod outer segments

Light-modulated cytoplasmic cGMP simultaneously controls plasma membrane Na+ conductance in visual excitation and Ca2+ entry into rods by direct interaction with the cation channel. Cytoplasmic Ca2+ in turn may set operating points and contribute to the dynamics of several enzymes that regulate cGMP levels in the dark, recovery from excitation and receptor adaptation or down regulation. Similar channels may couple electrical activity to internal nucleotide metabolism in other tissues. We here report the identification, partial purification and behaviour after reconstitution of a protein of relative molecular mass 39,000 (Mr 39K) present in both disk and plasma membranes from bovine rod outer segments that mediates these cGMP-dependent cation fluxes. Its cGMP agonist specificity, kinetic cooperativity, ionic selectivity, membrane density and other features closely match the properties of the visual cGMP-dependent conductance inferred from electrophysiological measurements.     

Crystal structure of the channelrhodopsin light-gated cation channel

Channelrhodopsins (ChRs) are light-gated cation channels derived from algae that have shown experimental utility in optogenetics; for example, neurons expressing ChRs can be optically controlled with high temporal precision within systems as complex as freely moving mammals. Although ChRs have been broadly applied to neuroscience research, little is known about the molecular mechanisms by which these unusual and powerful proteins operate. Here we present the crystal structure of a ChR (a C1C2 chimaera between ChR1 and ChR2 from Chlamydomonas reinhardtii) at 2.3?? resolution. The structure reveals the essential molecular architecture of ChRs, including the retinal-binding pocket and cation conduction pathway. This integration of structural and electrophysiological analyses provides insight into the molecular basis for the remarkable function of ChRs, and paves the way for the precise and principled design of ChR variants with novel properties.     

Inhibition of nociceptors by TRPV1-mediated entry of impermeant sodium channel blockers

Most local anaesthetics used clinically are relatively hydrophobic molecules that gain access to their blocking site on the sodium channel by diffusing into or through the cell membrane. These anaesthetics block sodium channels and thereby the excitability of all neurons, not just sensory neurons. We tested the possibility of selectively blocking the excitability of primary sensory nociceptor (pain-sensing) neurons by introducing the charged, membrane-impermeant lidocaine derivative QX-314 through the pore of the noxious-heat-sensitive TRPV1 channel. Here we show that charged sodium-channel blockers can be targeted into nociceptors by the application of TRPV1 agonists to produce a pain-specific local anaesthesia. QX-314 applied externally had no effect on the activity of sodium channels in small sensory neurons when applied alone, but when applied in the presence of the TRPV1 agonist capsaicin, QX-314 blocked sodium channels and inhibited excitability. Inhibition by co-applied QX-314 and capsaicin was restricted to neurons expressing TRPV1. Injection of QX-314 together with capsaicin into rat hindpaws produced a long-lasting (more than 2 h) increase in mechanical and thermal nociceptive thresholds. Long-lasting decreases in pain sensitivity were also seen with regional injection of QX-314 and capsaicin near the sciatic nerve; however, in contrast to the effect of lidocaine, the application of QX-314 and capsaicin together was not accompanied by motor or tactile deficits.     

Synaptic activity at calcium-permeable AMPA receptors induces a switch in receptor subtype

Activity-dependent change in the efficacy of transmission is a basic feature of many excitatory synapses in the central nervous system. The best understood postsynaptic modification involves a change in responsiveness of AMPAR (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor)-mediated currents following activation of NMDA (N-methyl-D-aspartate) receptors or Ca2+-permeable AMPARs. This process is thought to involve alteration in the number and phosphorylation state of postsynaptic AMPARs. Here we describe a new form of synaptic plasticity--a rapid and lasting change in the subunit composition and Ca2+ permeability of AMPARs at cerebellar stellate cell synapses following synaptic activity. AMPARs lacking the edited GluR2 subunit not only exhibit high Ca2+ permeability but also are blocked by intracellular polyamines. These properties have allowed us to follow directly the involvement of GluR2 subunits in synaptic transmission. Repetitive synaptic activation of Ca2+-permeable AMPARs causes a rapid reduction in Ca2+ permeability and a change in the amplitude of excitatory postsynaptic currents, owing to the incorporation of GluR2-containing AMPARs. Our experiments show that activity-induced Ca2+ influx through GluR2-lacking AMPARs controls the targeting of GluR2-containing AMPARs, implying the presence of a self-regulating mechanism.     

Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma membrane Cl- channel regulated by cyclic AMP-dependent phosphorylation and by intracellular ATP. Mutations in CFTR cause cystic fibrosis partly through loss of cAMP-regulated Cl- permeability from the plasma membrane of affected epithelia. The most common mutation in cystic fibrosis is deletion of phenylalanine at residue 508 (CFTR delta F508) (ref. 10). Studies on the biosynthesis and localization of CFTR delta F508 indicate that the mutant protein is not processed correctly and, as a result, is not delivered to the plasma membrane. These conclusions are consistent with earlier functional studies which failed to detect cAMP-stimulated Cl- channels in cells expressing CFTR delta F508 (refs 16, 17). Chloride channel activity was detected, however, when CFTR delta F508 was expressed in Xenopus oocytes, Vero cells and Sf9 insect cells. Because oocytes and Sf9 cells are typically maintained at lower temperatures than mammalian cells, and because processing of nascent proteins can be sensitive to temperature, we tested the effect of temperature on the processing of CFTR delta F508. Here we show that the processing of CFTR delta F508 reverts towards that of wild-type as the incubation temperature is reduced. When the processing defect is corrected, cAMP-regulated Cl- channels appear in the plasma membrane. These results reconcile previous contradictory observations and suggest that the mutant most commonly associated with cystic fibrosis is temperature-sensitive.     

Protein synthesis required to anchor a mutant p53 protein which is temperature-sensitive for nuclear transport

The p53 protein is rendered temperature-sensitive by a point mutation. Rat cells transformed by this mutant p53 and an activated ras oncogene grow well at 37 degrees C but cease DNA synthesis and cell division when shifted to 32 degrees C. Immunostaining demonstrates that the mutant p53 protein is in the nucleus of the arrested cells at 32 degrees C but in the cytoplasm of the growing cells at 37 degrees C. This is the first example of a protein which is temperature-sensitive for nuclear transport. The translocation from cytoplasm to nucleus and vice versa occurs 6 h after temperature shift and is coincident with the inhibition of DNA synthesis; transport from cytoplasm to nucleus does not require protein synthesis. Remarkably, inhibition of protein synthesis at 37 degrees C also results in the rapid appearance of mutant p53 in the cell nucleus. These results suggest the presence of a short-lived protein responsible for holding p53 in the cytoplasm at 37 degrees C but not at 32 degrees C. Analysis of a non-temperature-sensitive mutant p53 protein shows that its cytoplasmic location is sensitive to protein synthesis inhibitors but not to temperature.     

The mitochondrial calcium uniporter is a highly selective ion channel

During intracellular Ca2+ signalling mitochondria accumulate significant amounts of Ca2+ from the cytosol. Mitochondrial Ca2+ uptake controls the rate of energy production, shapes the amplitude and spatio-temporal patterns of intracellular Ca2+ signals, and is instrumental to cell death. This Ca2+ uptake is undertaken by the mitochondrial Ca2+ uniporter (MCU) located in the organelle's inner membrane. The uniporter passes Ca2+ down the electrochemical gradient maintained across this membrane without direct coupling to ATP hydrolysis or transport of other ions. Carriers are characterized by turnover numbers that are typically 1,000-fold lower than ion channels, and until now it has been unclear whether the MCU is a carrier or a channel. By patch-clamping the inner mitochondrial membrane, we identified a previously unknown Ca2+-selective ion channel sensitive to inhibitors of mitochondrial Ca2+ uptake. Our data indicate that this unique channel binds Ca2+ with extremely high affinity (dissociation constant < or =2 nM), enabling high Ca2+ selectivity despite relatively low cytoplasmic Ca2+ concentrations. The channel is inwardly rectifying, making it especially effective for Ca2+ uptake into energized mitochondria. Thus, we conclude that the properties of the current mediated by this novel channel are those of the MCU.     

Activation of the TRPC1 cation channel by metabotropic glutamate receptor mGluR1

Group I metabotropic glutamate receptors (consisting of mGluR1 and mGluR5) are G-protein-coupled neurotransmitter receptors that are found in the perisynaptic region of the postsynaptic membrane. These receptors are not activated by single synaptic volleys but rather require bursts of activity. They are implicated in many forms of neural plasticity including hippocampal long-term potentiation and depression, cerebellar long-term depression, associative learning, and cocaine addiction. When activated, group I mGluRs engage two G-protein-dependent signalling mechanisms: stimulation of phospholipase C and activation of an unidentified, mixed-cation excitatory postsynaptic conductance (EPSC), displaying slow activation, in the plasma membrane. Here we report that the mGluR1-evoked slow EPSC is mediated by the TRPC1 cation channel. TRPC1 is expressed in perisynaptic regions of the cerebellar parallel fibre-Purkinje cell synapse and is physically associated with mGluR1. Manipulations that interfere with TRPC1 block the mGluR1-evoked slow EPSC in Purkinje cells; however, fast transmission mediated by AMPA-type glutamate receptors remains unaffected. Furthermore, co-expression of mGluR1 and TRPC1 in a heterologous system reconstituted a mGluR1-evoked conductance that closely resembles the slow EPSC in Purkinje cells.     

The voltage-sensitive sodium channel is a bell-shaped molecule with several cavities

Voltage-sensitive membrane channels, the sodium channel, the potassium channel and the calcium channel operate together to amplify, transmit and generate electric pulses in higher forms of life. Sodium and calcium channels are involved in cell excitation, neuronal transmission, muscle contraction and many functions that relate directly to human diseases. Sodium channels--glycosylated proteins with a relative molecular mass of about 300,000 (ref. 5)--are responsible for signal transduction and amplification, and are chief targets of anaesthetic drugs and neurotoxins. Here we present the three-dimensional structure of the voltage-sensitive sodium channel from the eel Electrophorus electricus. The 19 A structure was determined by helium-cooled cryo-electron microscopy and single-particle image analysis of the solubilized sodium channel. The channel has a bell-shaped outer surface of 135 A in height and 100 A in side length at the square-shaped bottom, and a spherical top with a diameter of 65 A. Several inner cavities are connected to four small holes and eight orifices close to the extracellular and cytoplasmic membrane surfaces. Homologous voltage-sensitive calcium and tetrameric potassium channels, which regulate secretory processes and the membrane potential, may possess a related structure.     

Purified dihydropyridine-binding site from skeletal muscle t-tubules is a functional calcium channel

Many excitable cells contain at least two different voltage-dependent Ca channels (L- and T-type). The cardiac, slow, L-type Ca channel is further modulated by cyclic AMP-dependent phosphorylation, which increases the probability of it being open, and is readily blocked by Ca channel blockers including dihydropyridines and phenylalkylamines. The tritiated congeners of these blockers bind in vitro to sites which have the same pharmacological characteristics as those observed in vivo, that is, stereospecific and allosteric interaction between distinct sites. The dihydropyridine-binding site purified from skeletal muscle t-tubules contains three peptides of relative molecular mass (Mr) 142,000 (142K), 56K and 31K. The cAMP kinase incorporates one mol phosphate per mol of the 142K peptide and binding of (+)PN-200/110, a potent Ca antagonist, is allosterically affected by D-cis-diltiazem and verapamil. The purified dihydropyridine-receptor complex has also been incorporated into phospholipid bilayer membranes. Here, we show for the first time that the complex can be reconstituted to form a functional 20-pS Ca channel that retains the principal regulatory, biochemical and pharmacological properties of membrane-bound L-type Ca channels.     

TRPA1 is a candidate for the mechanosensitive transduction channel of vertebrate hair cells

Mechanical deflection of the sensory hair bundles of receptor cells in the inner ear causes ion channels located at the tips of the bundle to open, thereby initiating the perception of sound. Although some protein constituents of the transduction apparatus are known, the mechanically gated transduction channels have not been identified in higher vertebrates. Here, we investigate TRP (transient receptor potential) ion channels as candidates and find one, TRPA1 (also known as ANKTM1), that meets criteria for the transduction channel. The appearance of TRPA1 messenger RNA expression in hair cell epithelia coincides developmentally with the onset of mechanosensitivity. Antibodies to TRPA1 label hair bundles, especially at their tips, and tip labelling disappears when the transduction apparatus is chemically disrupted. Inhibition of TRPA1 protein expression in zebrafish and mouse inner ears inhibits receptor cell function, as assessed with electrical recording and with accumulation of a channel-permeant fluorescent dye. TRPA1 is probably a component of the transduction channel itself.     

MOD-1 is a serotonin-gated chloride channel that modulates locomotory behaviour in C. elegans

The neurotransmitter and neuromodulator serotonin (5-HT) functions by binding either to metabotropic G-protein-coupled receptors (for example, 5-HT1, 5-HT2, 5-HT4 to 5-HT7), which mediate 'slow' modulatory responses through numerous second messenger pathways, or to the ionotropic 5-HT3 receptor, a non-selective cation channel that mediates 'fast' membrane depolarizations. Here we report that the gene mod-1 (for modulation of locomotion defective) from the nematode Caenorhabditis elegans encodes a new type of ionotropic 5-HT receptor, a 5-HT-gated chloride channel. The predicted MOD-1 protein is similar to members of the nicotinic acetylcholine receptor family of ligand-gated ion channels, in particular to GABA (gamma-aminobutyric acid)- and glycine-gated chloride channels. The MOD-1 channel has distinctive ion selectivity and pharmacological properties. The reversal potential of the MOD-1 channel is dependent on the concentration of chloride ions but not of cations. The MOD-1 channel is not blocked by calcium ions or 5-HT3a-specific antagonists but is inhibited by the metabotropic 5-HT receptor antagonists mianserin and methiothepin. mod-1 mutant animals are defective in a 5-HT-mediated experience-dependent behaviour and are resistant to exogenous 5-HT, confirming that MOD-1 functions as a 5-HT receptor in vivo.