Tim D. Plant

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.

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.

Lanthanides Potentiate TRPC5 Currents by an Action at Extracellular Sites Close to the Pore Mouth

Mammalian members of the classical transient receptor potential channel (TRPC) subfamily (TRPC1–7) are Ca2+-permeable cation channels involved in receptor-mediated increases in intracellular Ca2+. Unlike most other TRP-related channels, which are inhibited by La3+ and Gd3+, currents through TRPC4 and TRPC5 are potentiated by La3+. Because these differential effects of lanthanides on TRPC subtypes may be useful for clarifying the role of different TRPCs in native tissues, we characterized the potentiating effect in detail and localized the molecular determinants of potentiation by mutagenesis. Whole cell currents through TRPC5 were reversibly potentiated by micromolar concentrations of La3+or Gd3+, whereas millimolar concentrations were inhibitory. By comparison, TRPC6 was blocked to a similar extent by La3+ or Gd3+ at micromolar concentrations and showed no potentiation. Dual effects of lanthanides on TRPC5 were also observed in outside-out patches. Even at micromolar concentrations, the single channel conductance was reduced by La3+, but reduction in conductance was accompanied by a dramatic increase in channel open probability, leading to larger integral currents. Neutralization of the negatively charged amino acids Glu543and Glu595/Glu598, situated close to the extracellular mouth of the channel pore, resulted in a loss of potentiation, and, for Glu595/Glu598 in a modification of channel inhibition. We conclude that in the micromolar range, the lanthanide ions La3+ and Gd3+ have opposite effects on whole cell currents through TRPC5 and TRPC6 channels. The potentiation of TRPC4 and TRPC5 by micromolar La3+ at extracellular sites close to the pore mouth is a promising tool for identifying the involvement of these isoforms in receptor-operated cation conductances of native cells.

TRPC4 and TRPC5: receptor-operated Ca2+-permeable nonselective cation channels

The seven mammalian channels from the classical (TRPC) subfamily of transient receptor potential (TRP) channels are thought to be receptor-operated cation channels activated in a phospholipase C (PLC)-dependent manner. Based on sequence similarity, TRPC channels can be divided into four subgroups. Group 4 comprises TRPC4 and TRPC5, and is most closely related to group 1 (TRPC1). The functional properties observed following heterologous expression of TRPC4 or TRPC5 in mammalian cells are contradictory and, therefore, controversial. In our hands, and in several independent studies, both channels, probably as homotetramers, form receptor-operated, Ca2+-permeable, nonselective cation channels activated independently of inositol 1,4,5-trisphosphate (InsP(3)) receptor activation or Ca2+ store-depletion. As heteromultimers with TRPC1, TRPC4 and TRPC5 form receptor-operated, Ca2+-permeable, nonselective cation channels with biophysical properties distinct from homomeric TRPC4 or TRPC5. In other studies, TRPC4 and TRPC5 have been shown to be store-operated channels, with moderate to high Ca2+ permeabilities. At present there is no clear explanation for these major differences in functional properties. To date, little is known as to which native cation channels are formed by TRPC4 and TRPC5. Endothelial cells from TRPC4(-/-) mice lack a highly Ca2+-permeable, store-dependent current, and data support a role for TRPC4 in endothelium-mediated vasorelaxation. A similar current in adrenal cortical cells is reduced by TRPC4 antisense. From similarities in the properties of the currents and expression of appropriate isoforms in the tissues, it is likely that heteromultimers of TRPC1 and TRPC4 or TRPC5 form receptor-operated nonselective cation channels in central neurones, and that TRPC4 contributes to nonselective cation channels in intestinal smooth muscle.

Dopamine-Regulated MicroRNA MiR-181a Controls GluA2 Surface Expression in Hippocampal Neurons

ABSTRACT The dynamic expression of AMPA-type glutamate receptors (AMPA-R) at synapses is a key determinant of synaptic plasticity, including neuroadaptations to drugs of abuse. Recently, microRNAs (miRNAs) have emerged as important posttranscriptional regulators of synaptic plasticity, but whether they target glutamate receptors to mediate this effect is not known. Here we used microarray screening to identify miRNAs that regulate synaptic plasticity within the nucleus accumbens, a brain region critical to forming drug-seeking habits. One of the miRNAs that showed a robust enrichment at medium spiny neuron synapses was miR-181a. Using bioinformatics tools, we detected a highly conserved miR-181a binding site within the mRNA encoding the GluA2 subunit of AMPA-Rs. Overexpression and knockdown of miR-181a in primary neurons demonstrated that this miRNA is a negative posttranscriptional regulator of GluA2 expression. Additionally, miR-181a overexpression reduced GluA2 surface expression, spine formation, and miniature excitatory postsynaptic current (mEPSC) frequency in hippocampal neurons, suggesting that miR-181a could regulate synaptic function. Moreover, miR-181a expression was induced by dopamine signaling in primary neurons, as well as by cocaine and amphetamines, in a mouse model of chronic drug treatment. Taken together, our results identify miR-181a as a key regulator of mammalian AMPA-type glutamate receptors, with potential implications for the regulation of drug-induced synaptic plasticity.

Receptor-operated cation channels formed by TRPC4 and TRPC5

TRPC4 and TRPC5 form cation channels that contribute to phospholipase C-dependent Ca2+ entry following stimulation of G-protein-coupled receptors or receptor tyrosine kinases. Surprisingly, in different studies, TRPC4 and TRPC5 have been shown to form either store-operated channels with a relatively high Ca2+ permeability, or nonselective cation channels activated independently of store depletion. In this review, we summarize and discuss data on the regulation and permeability properties of TRPC4 and TRPC5, and data on native channels that might be composed of these isoforms.

Endothelin potentiates TRPV1 via ETA receptor-mediated activation of protein kinase C

BackgroundEndothelin-1 (ET-1) both stimulates nociceptors and sensitizes them to noxious stimuli, an effect probably mediated by the ETA receptor (ETAR) expressed in sensory neurons. The cellular mechanisms of this ET-1-mediated effect are only poorly understood. TRPV1, the heat-, pH- and capsaicin-sensitive cation channel already known to be modulated by a number of cellular mediators released in response to noxious stimuli and during inflammation, is a potential target for the action of ET-1.ResultsWe studied the effects of ET-1 on TRPV1 in sensory neurons from the dorsal root ganglion (DRG) and in HEK293 cells coexpressing TRPV1 and the ETAR. Specific 125I-ET-1 binding sites (817 ± 92 fmol/mg) were detected in membrane preparations of DRG with an ETAR/ETBR ratio of 60:40. In an immunofluorescence analysis, coexpression of TRPV1 and the ETAR was found in a subpopulation of primary sensory neurons. ET-1 strongly potentiated capsaicin-induced TRPV1 currents in some neurons, and in HEK293 cells co-expressing TRPV1 and the ETAR. Weaker potentiation was observed in HEK293 cells coexpressing TRPV1 and the ETBR. ETAR activation also increased responses to low pH and heat. In HEK293 cells, strong potentiation of TRPV1 like that induced by ET-1 via the ETAR could be induced by PKC activation, but not with activators of the adenylyl cyclase or the PKA pathway. Furthermore, inhibition of PKC with bisindolylmaleimide X (BIM X) or mutation of the PKC phosphorylation site S800 completely prevented ETAR-mediated potentiation.ConclusionWe conclude that ET-1 potentiates TRPV1 by a PKC-dependent mechanism and that this could play a major role in the algogenic and hyperalgesic effects of ET-1 described in previous studies.

Potentiation of TRPC5 by protons.

Mammalian members of the classical transient receptor potential channel subfamily (TRPC) are Ca2+-permeable cation channels involved in receptor-mediated increases in intracellular Ca2+. TRPC4 and TRPC5 form a group within the TRPC subfamily and are activated in a phospholipase C-dependent manner by an unidentified messenger. Unlike most other Ca2+-permeable channels, TRPC4 and -5 are potentiated by micromolar concentrations of La3+ and Gd3+. This effect results from an action of the cations at two glutamate residues accessible from the extracellular solution. Here, we show that TRPC4 and -5 respond to changes in extracellular pH. Lowering the pH increased both G protein-activated and spontaneous TRPC5 currents. Both effects were already observed with small reductions in pH (from 7.4 to 7.0) and increased up to pH 6.5. TRPC4 was also potentiated by decreases in pH, whereas TRPC6 was only inhibited, with a pIC50 of 5.7. Mutation of the glutamate residues responsible for lanthanoid sensitivity of TRPC5 (E543Q and E595Q) modified the potentiation of TRPC5 by acid. Further evidence for a similarity in the actions of lanthanoids and H+ on TRPC5 is the reduction in single channel conductance and dramatic increase in channel open probability in the presence of either H+ or Gd3+ that leads to larger integral currents. In conclusion, the high sensitivity of TRPC5 to H+ indicates that, in addition to regulation by phospholipase C and other factors, the channel may act as a sensor of pH that links decreases in extracellular pH to Ca2+ entry and depolarization.

Endothelin-1 potentiates capsaicin-induced TRPV1 currents via the endothelin A receptor.

Endothelin-1 (ET-1) both stimulates nociceptors and sensitizes them to painful stimuli. The cellular mechanisms of the ET-1–mediated effects are only poorly understood. TRPV1, the heat-, proton-, and capsaicin-sensitive cation channel already known to be modulated by a number of cellular mediators released by painful stimuli and during inflammation, is a potential target for the action of ET-1. In immunocytochemistry of rat lumbar dorsal root ganglion using TRPV1- and ETA receptor–specific antibodies, both proteins were found to be co-expressed in small sensory neurons. To provide evidence that ET-1 can modulate TRPV1 activity via the ETA receptor, we used HEK 293 cells transiently co-expressing a fusion protein of TRPV1 and the yellow fluorescent protein (TRPV1-YFP) and the ETA receptor. In whole-cell patch clamp recordings of HEK293 cells co-expressing TRPV1-YFP and the ETA receptor, capsaicin (10 nM) elicited small currents, which were markedly potentiated when capsaicin (10 nM) and ET-1 (100 nM) were applied simultaneously. The data indicate that ET-1 potentiates TRPV1 activity via the ETA receptor and that this process is likely to play a crucial role in the pain-producing and pain-potentiating effects of ET-1. Thus, ETA receptor antagonists may be of importance in painful states with increased circulating ET-1 levels, as found in cancer and in chronic inflammation.

Interdomain interactions control Ca2+-dependent potentiation in the cation channel TRPV4.

Several Ca2+-permeable channels, including the non-selective cation channel TRPV4, are subject to Ca2+-dependent facilitation. Although it has been clearly demonstrated in functional experiments that calmodulin (CaM) binding to intracellular domains of TRP channels is involved in this process, the molecular mechanism remains elusive. In this study, we provide experimental evidence for a comprehensive molecular model that explains Ca2+-dependent facilitation of TRPV4. In the resting state, an intracellular domain from the channel N terminus forms an autoinhibitory complex with a C-terminal domain that includes a high-affinity CaM binding site. CaM binding, secondary to rises in intracellular Ca2+, displaces the N-terminal domain which may then form a homologous interaction with an identical domain from a second subunit. This represents a novel potentiation mechanism that may also be relevant in other Ca2+-permeable channels.