TRPC4 and TRPC5: receptor-operated Ca-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 (InsP₃) 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.     

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.     

The mammalian TRPC cation channels

Transient Receptor Potential-Canonical (TRPC) channels are mammalian homologs of Transient Receptor Potential (TRP), a Ca(2+)-permeable channel involved in the phospholipase C-regulated photoreceptor activation mechanism in Drosophila. The seven mammalian TRPCs constitute a family of channels which have been proposed to function as store-operated as well as second messenger-operated channels in a variety of cell types. TRPC channels, together with other more distantly related channel families, make up the larger TRP channel superfamily. This review summarizes recent findings on the structure, regulation and function of the apparently ubiquitous TRPC cation channels.     

Corticolimbic expression of TRPC4 and TRPC5 channels in the rodent brain

The canonical transient receptor potential (TRPC) channels are a family of non-selective cation channels that are activated by increases in intracellular Ca(2+) and G(q)/phospholipase C-coupled receptors. We used quantitative real-time PCR, in situ hybridization, immunoblots and patch-clamp recording from several brain regions to examine the expression of the predominant TRPC channels in the rodent brain. Quantitative real-time PCR of the seven TRPC channels in the rodent brain revealed that TRPC4 and TRPC5 channels were the predominant TRPC subtypes in the adult rat brain. In situ hybridization histochemistry and immunoblotting further resolved a dense corticolimbic expression of the TRPC4 and TRPC5 channels. Total protein expression of HIP TRPC4 and 5 proteins increased throughout development and peaked late in adulthood (6-9 weeks). In adults, TRPC4 expression was high throughout the frontal cortex, lateral septum (LS), pyramidal cell layer of the hippocampus (HIP), dentate gyrus (DG), and ventral subiculum (vSUB). TRPC5 was highly expressed in the frontal cortex, pyramidal cell layer of the HIP, DG, and hypothalamus. Detailed examination of frontal cortical layer mRNA expression indicated TRPC4 mRNA is distributed throughout layers 2-6 of the prefrontal cortex (PFC), motor cortex (MCx), and somatosensory cortex (SCx). TRPC5 mRNA expression was concentrated specifically in the deep layers 5/6 and superficial layers 2/3 of the PFC and anterior cingulate. Patch-clamp recording indicated a strong metabotropic glutamate-activated cation current-mediated depolarization that was dependent on intracellular Ca(2+)and inhibited by protein kinase C in brain regions associated with dense TRPC4 or 5 expression and absent in regions lacking TRPC4 and 5 expression. Overall, the dense corticolimbic expression pattern suggests that these Gq/PLC coupled nonselective cation channels may be involved in learning, memory, and goal-directed behaviors.     

TRPC5 as a candidate for the nonselective cation channel activated by muscarinic stimulation in murine stomach

We investigated which transient receptor potential (TRP) channel is responsible for the nonselective cation channel (NSCC) activated by carbachol (CCh) in murine stomach with RT-PCR and the electrophysiological method. All seven types of TRP mRNA were detected in murine stomach with RT-PCR. When each TRP channel was expressed, the current-voltage relationship of mTRP5 was most similar to that recorded in murine gastric myocytes. mTRP5 showed a conductance order of Cs(+) > K(+) > Na(+), similar to that in the murine stomach. With 0.2 mM GTPgammaS in the pipette solution, the current was activated transiently in both NSCC in the murine stomach and the expressed mTRP5. Both NSCC activated by CCh in murine stomach and mTRP5 were inhibited by intracellularly applied anti-G(q/11) antibody, PLC inhibitor U-73122, IICR inhibitor 2-aminoethoxydiphenylborate, and nonspecific cation channel blockers La(3+) and flufenamate. There were two other unique properties. Both the native NSCC and mTRP5 were activated by 1-oleoyl-2-acetyl-sn-glycerol. Without the activation of NSCC by CCh, the NSCC in murine stomach was constitutively active like mTRP5. From the above results, we suggest that mTRP5 might be a candidate for the NSCC activated by ACh or CCh in murine stomach.     

Trafficking mechanisms and regulation of TRPC channels

TRPC channels are Ca²⁺-permeable cation channels which are regulated downstream from receptor-coupled PIP₂ hydrolysis. These channels contribute to a wide variety of cellular functions. Loss or gain of channel function has been associated with dysfunction and aberrant physiology. TRPC channel functions are influenced by their physical and functional interactions with numerous proteins that determine their regulation, scaffolding, trafficking, as well as their effects on the downstream cellular processes. Such interactions also compartmentalize the Ca²⁺ signals arising from TRPC channels. A large number of studies demonstrate that trafficking is a critical mode by which plasma membrane localization and surface expression of TRPC channels are regulated. This review will provide an overview of intracellular trafficking pathways as well as discuss the current state of knowledge regarding the mechanisms and components involved in trafficking of the seven members of the TRPC family (TRPC1–TRPC7).     

Heteromeric channels formed by TRPC1, TRPC4 and TRPC5 define hippocampal synaptic transmission and working memory

Canonical transient receptor potential (TRPC) channels influence various neuronal functions. Using quantitative high‐resolution mass spectrometry, we demonstrate that TRPC1, TRPC4, and TRPC5 assemble into heteromultimers with each other, but not with other TRP family members in the mouse brain and hippocampus. In hippocampal neurons from Trpc1/Trpc4/Trpc5‐triple‐knockout (Trpc1/4/5^?/?) mice, lacking any TRPC1‐, TRPC4‐, or TRPC5‐containing channels, action potential‐triggered excitatory postsynaptic currents (EPSCs) were significantly reduced, whereas frequency, amplitude, and kinetics of quantal miniature EPSC signaling remained unchanged. Likewise, evoked postsynaptic responses in hippocampal slice recordings and transient potentiation after tetanic stimulation were decreased. In vivo, Trpc1/4/5^?/? mice displayed impaired cross‐frequency coupling in hippocampal networks and deficits in spatial working memory, while spatial reference memory was unaltered. Trpc1/4/5^?/? animals also exhibited deficiencies in adapting to a new challenge in a relearning task. Our results indicate the contribution of heteromultimeric channels from TRPC1, TRPC4, and TRPC5 subunits to the regulation of mechanisms underlying spatial working memory and flexible relearning by facilitating proper synaptic transmission in hippocampal neurons.     

Receptor-mediated regulation of calcium channels and neurotransmitter release

Ca2+ influx into the nerve terminal is normally the trigger for the release of neurotransmitters. Many neurons possess presynaptic receptors whose activation results in changes in the quantity of neurotransmitter released by an action potential. This paper reviews studies that show that presynaptic receptors can regulate the activity of Ca2+ channels in the nerve terminal, resulting in changes in the influx of Ca2+ and in neurotransmitter release. Neurons possess several different types of voltage-sensitive Ca2+ channels. Ca2+ influx through N-type channels appears to trigger transmitter release in many instances. In other cases Ca2+ influx through L channels can influence transmitter release. Neurotransmitters can inhibit N channels through a G protein-mediated transduction mechanism. The G proteins are frequently pertussis toxin substrates. Inhibition of N channels appears to involve changes in their voltage dependence. Neurotransmitters can also regulate neuronal K+ channels. Activation of these K+ channels can lead to a reduction in Ca2+ influx and neurotransmitter release; these effects are also mediated by G proteins. Thus neurotransmitters may often regulate both presynaptic Ca2+ and K+ channels. These two effects may be synergistic mechanisms for the regulation of Ca2+ influx and neurotransmitter release.     

TRPC3 and TRPC4 associate to form a redox-sensitive cation channel. Evidence for expression of native TRPC3-TRPC4 heteromeric channels in endothelial cells

Canonical transient receptor potential proteins (TRPC) have been proposed to form homo- or heteromeric cation channels in a variety of tissues, including the vascular endothelium. Assembly of TRPC multimers is incompletely understood. In particular, heteromeric assembly of distantly related TRPC isoforms is still a controversial issue. Because we have previously suggested TRPC proteins as the basis of the redox-activated cation conductance of porcine aortic endothelial cells (PAECs), we set out to analyze the TRPC subunit composition of endogenous endothelial TRPC channels and report here on a redox-sensitive TRPC3-TRPC4 channel complex. The ability of TRPC3 and TRPC4 proteins to associate and to form a cation-conducting pore complex was supported by four lines of evidence as follows: 1) Co-immunoprecipitation experiments in PAECs and in HEK293 cells demonstrated the association of TRPC3 and TRPC4 in the same complex. 2) Fluorescence resonance energy transfer analysis demonstrated TRPC3-TRPC4 association, involving close proximity between the N terminus of TRPC4 and the C terminus of TRPC3 subunits. 3) Co-expression of TRPC3 and TRPC4 in HEK293 cells generated a channel that displayed distinct biophysical and regulatory properties. 4) Expression of dominant-negative TRPC4 proteins suppressed TRPC3-related channel activity in the HEK293 expression system and in native endothelial cells. Specifically, an extracellularly hemagglutinin (HA)-tagged TRPC4 mutant, which is sensitive to blockage by anti-HA-antibody, was found to transfer anti-HA sensitivity to both TRPC3-related currents in the HEK293 expression system and the redox-sensitive cation conductance of PAECs. We propose TRPC3 and TRPC4 as subunits of native endothelial cation channels that are governed by the cellular redox state.     

The roles of G proteins in the activation of TRPC4 and TRPC5 transient receptor potential channels

TRPC4 and TRPC5 channels are important regulators of electrical excitability in both gastrointestinal myocytes and neurons. Much is known regarding the assembly and function of these channels including TRPC1 as a homotetramer or a heteromultimer and the roles that their interacting proteins play in controlling these events. Further, they are one of the best-studied targets of G protein-coupled receptors and growth factors in general and Gαq protein coupled receptor or epidermal growth factor in particular. However, our understanding of the roles of Gαi/o proteins on TRPC4/5 channels is still rudimentary. We discuss potential roles for Gαi/o proteins in channel activation in addition to their known role in cellular signaling.     

The roles of G proteins in the activation of TRPC4 and TRPC5 transient receptor potential channels

TRPC4 and TRPC5 channels are important regulators of electrical excitability in both gastrointestinal myocytes and neurons. Much is known regarding the assembly and function of these channels including TRPC1 as a homotetramer or a heteromultimer and the roles that their interacting proteins play in controlling these events. Further, they are one of the best-studied targets of G protein-coupled receptors and growth factors in general and Gαq protein coupled receptor or epidermal growth factor in particular. However, our understanding of the roles of Gαi/o proteins on TRPC4/5 channels is still rudimentary. We discuss potential roles for Gαi/o proteins in channel activation in addition to their known role in cellular signaling.     

TRPC1 and TRPC5 form a novel cation channel in mammalian brain

TRP proteins are cation channels responding to receptor-dependent activation of phospholipase C. Mammalian (TRPC) channels can form hetero-oligomeric channels in vitro, but native TRPC channel complexes have not been identified to date. We demonstrate here that TRPC1 and TRPC5 are subunits of a heteromeric neuronal channel. Both TRPC proteins have overlapping distributions in the hippocampus. Coexpression of TRPC1 and TRPC5 in HEK293 cells resulted in a novel nonselective cation channel with a voltage dependence similar to NMDA receptor channels, but unlike that of any reported TRPC channel. TRPC1/TRPC5 heteromers were activated by G(q)-coupled receptors but not by depletion of intracellular Ca(2+) stores. In contrast to the more common view of the TRP family as comprising store-operated channels, we propose that many TRPC heteromers form diverse receptor-regulated nonselective cation channels in the mammalian brain.     

TRPC4 is an essential component of the nonselective cation channel activated by muscarinic stimulation in mouse visceral smooth muscle cells

Classical transient receptor potential channels (TRPCs) are thought to be candidates for the nonselective cation channels (NSCCs) involved in pacemaker activity and its neuromodulation in murine stomach smooth muscle. We aimed to determine the role of TRPC4 in the formation of NSCCs and in the generation of slow waves. At a holding potential of -60 mV, 50 mM carbachol (CCh) induced INSCC of amplitude [500.8+/-161.8 pA (n=8)] at -60 mV in mouse gastric smooth muscle cells. We investigated the effects of commercially available antibodies to TRPC4 on recombinant TRPC4 expressed in HEK cells and CCh-induced NSCCs in gastric smooth muscle cells. TRPC4 currents in HEK cells were reduced from 1525.6+/-414.4 pA (n=8) to 146.4+/-83.3 pA (n=10) by anti-TRPC4 antibody and INSCC amplitudes were reduced from 230.9+/-36.3 pA (n=15) to 49.8+/-11.8 pA (n=9). Furthermore, INSCC in the gastric smooth muscle cells of TRPC4 knockout mice was only 34.4+/-10.4 pA (n=8) at -60 mV. However, slow waves were still present in the knockout mice. Our data suggest that TRPC4 is an essential component of the NSCC activated by muscarinic stimulation in the murine stomach.     

Multiple eicosanoid-activated nonselective cation channels regulate B-lymphocyte adhesion to integrin ligands

Arachidonic acid (AA) is a substrate for a variety of proinflammatory mediators, which are generated by cyclooxygenases (COXs), lipoxygenases (LOXs), and cytochrome P-450 (CYP450) enzymes. COX (e.g., PGs and prostacyclins) and LOX (e.g., leukotrienes) products have well-established proinflammatory roles; however, little is known about the functions of CYP450 products in leukocytes. We previously found that mechanical strain generated by subjecting lymphocytes to hypotonic challenge triggered AA production and that two CYP450 products of AA, 5,6-epoxyeicosatrienoic acid (5,6-EET) and 20-hydroxyeicosatetraenoic acid (20-HETE), as well as a product of LOX, 5-(S)-hydroperoxyeicosatetrenoic acid (5-HPETE), induced Ca²⁺ entry into primary B cells. The main goal of the present studies, therefore, was to define the biophysically properties of eicosanoid-activated channels responsible for Ca²⁺ entry and the physiological consequences of activating these channels, including their role in mechanical signaling. We found that 5,6-EET, 20-HETE, and 5-HPETE each activated distinct Ca²⁺-permeant nonselective cation channels (NSCCs) in primary B cells. These NSCCs each regulate plasma membrane potential and B-cell adhesion to integrin ligands ICAM-1 and VCAM-1. Thus our data demonstrate that proinflammatory mediators produced in response to osmotic and/or physical stress play a direct role in regulating the B-cell membrane potential and their adhesion to specific ECM proteins. These results not only have important implications for understanding normal mechanisms of B-cell activation, differentiation, and trafficking but also point to novel targets for modulating the pathogenesis of B-cell-mediated inflammatory diseases. calcium; arachidonic acid; membrane potential; hypotonicity; cytochrome P-450     

The TRPC3/6/7 subfamily of cation channels

The mammalian transient receptor potential (TRP) proteins consist of a superfamily of Ca2+-permeant non-selective cation channels with structural similarities to Drosophila TRP. The TRP superfamily can be divided into three major families, among them the "canonical TRP" family (TRPC). The seven protein products of the mammalian TRPC family of genes (designated TRPC1-7) share in common the activation through PLC-coupled receptors and have been proposed to encode components of native store-operated channels in different cell types. In addition, the three members of the TRPC3/6/7 subfamily of TRPC channels can be activated by diacylglycerol analogs, providing a possible mechanism of activation of these channels by PLC-coupled receptors. This review summarizes the current knowledge about the mechanism of activation of the TRPC3/6/7 subfamily, as well as the potential role of these proteins as components of native Ca2+-permeant channels.     

Mechanisms of lipid regulation and lipid gating in TRPC channels

TRPC proteins form cation channels that integrate and relay cellular signals by mechanisms involving lipid recognition and lipid-dependent gating. The lipohilic/amphiphilic molecules that function as cellular activators or modulators of TRPC proteins span a wide range of chemical structures. In this context, cellular redox balance is likely linked to the lipid recognition/gating features of TRPC channels. Both classical ligand-protein interactions as well as indirect and promiscuous sensory mechanisms have been proposed. Some of the recognition processes are suggested to involve ancillary lipid-binding scaffolds or regulators as well as dynamic protein–protein interactions determined by bilayer architecture. A complex interplay of protein–protein and protein-lipid interactions is likely to govern the gating and/or plasma membrane recruitment of TRPC channels, thereby providing a distinguished platform for signal integration and coincident signal detection. Both the primary molecular event(s) of lipid recognition by TRPC channels as well as the transformation of these events into distinct gating movements is poorly understood at the molecular level, and it remains elusive whether lipid sensing in TRPCs is conferred to a distinct sensor domain. Recent structural information on the molecular action of lipophilic activators in distantly related members of the TRP superfamily encourages speculations on TRPC gating mechanisms involved in lipid recognition/gating. This review aims to provide an update on the current understanding of the lipid–dependent control of TRPC channels with focus on the TRPC lipid sensing, signal-integration hub and a short discussion of potential links to redox signaling.     

Nucleotide regulation of the voltage-dependent nonselective cation conductance in murine colonic myocytes

ATP is proposed to be a major inhibitory neurotransmitter in the gastrointestinal (GI) tract, causing hyperpolarization and smooth muscle relaxation. ATP activates small-conductance Ca²⁺-activated K⁺ channels that are involved in setting the resting membrane potential and causing inhibitory junction potentials. No reports are available examining the effects of ATP on voltage-dependent inward currents in GI smooth muscle cells. We previously reported two types of voltage-dependent inward currents in murine proximal colonic myocytes: a low-threshold voltage-activated, nonselective cation current (I_VNSCC) and a relatively high-threshold voltage-activated (L-type) Ca²⁺ current (I_L). Here we have investigated the effects of ATP on these currents. External application of ATP (1 mM) did not affect I_VNSCC or I_L in dialyzed cells. ATP (1 mM) increased I_VNSCC and decreased I_L in the perforated whole-cell configuration. UTP and UDP (1 mM) were more potent than ATP on I_VNSCC. ADP decreased I_L but had no effect on I_VNSCC. The order of effectiveness was UTP = UDP > ATP > ADP. These effects were not blocked by pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS), but the phospholipase C inhibitor U-73122 reversed the effects of ATP on I_VNSCC. ATP stimulation of I_VNSCC was also reversed by protein kinase C (PKC) inhibitors chelerythrine chloride or bisindolylmaleimide I. Phorbol 12,13-dibutyrate mimicked the effects of ATP. RT-PCR showed that P2Y₄ is expressed by murine colonic myocytes, and this receptor is relatively insensitive to PPADS. Our data suggest that ATP activates I_VNSCC and depresses I_L via binding of P2Y₄ receptors and stimulation of the phospholipase C/PKC pathway. inhibitory junction potentials; smooth muscle; enteric nervous system     

Single nonselective cation channels and Ca2+-activated K+ channels in aortic endothelial cells

Summary In cultured bovine aortic endothelial cells, elementary K⁺ currents were studied in cell-attached and inside-out patches using the standard patch-clamp technique. Two different cationic channels were found, a large channel with a mean unitary conductance of 150±10 pS and a small channel with a mean unitary conductance of 12.5±1.1 pS. The 150-pS channel proved to be voltag- and Ca²⁺-activatable and seems to be a K⁺ channel. Its open probability increased on membrane depolarization and, at a given membrane potential, was greatly enhanced by elevating the Ca²⁺ concentration at the cytoplasmic side of the membrane from 10^–7 to 10^–4 m. 150-pS channels were not influenced by the patch configuration in that patch excision neither induced rundown nor evoked channel activity in silent cell-attached patches. However, they were only seen in two out of 55 patches. The 12-pS channel was predominant, a nonselective cationic channel with almost the same permeability for K⁺ and Na⁺ whose open probability was minimal near –60 mV but increased on membrane hyperpolarization. An increase in internal Ca²⁺ from 10^–7 to 10^–4 m left the open probability unchanged. Although the K⁺ selectivity of the 150-pS channels remains to be elucidated, it is concluded that they may be involved in controlling Ca²⁺-dependent cellular functions. Under physiological conditions, 12-pS nonselective channels may provide an inward cationic pathway for Na⁺.