Paradoxical contribution of SK3 and GIRK channels to the activation of mouse vomeronasal organ

The vomeronasal organ (VNO) is essential for intraspecies communication in many terrestrial vertebrates. The ionic mechanisms of VNO activation remain unclear. We found that the calcium-activated potassium channel SK3 and the G protein-activated potassium channel GIRK are part of an independent pathway for VNO activation. In slice preparations, the potassium channels attenuated inward currents carried by TRPC2 and calcium-activated chloride channels (CACCs). In intact tissue preparations, paradoxically, the potassium channels enhanced urine-evoked inward currents. This discrepancy resulted from the loss of a high concentration of lumenal potassium, which enabled the influx of potassium ions to depolarize the VNO neurons in vivo. Both Sk3 (also known as Kcnn3) and Girk1 (also known as Kcnj3) homozygous null mice showed deficits in mating and aggressive behaviors, and the deficiencies in Sk3(-/-) mice were exacerbated by Trpc2 knockout. Our results suggest that VNO activation is mediated by TRPC2, CACCs and two potassium channels, all of which contributed to the in vivo depolarization of VNO neurons.     

A TRPC5-regulated calcium signaling pathway controls dendrite patterning in the mammalian brain

Transient receptor potential (TRP) channels have been implicated as sensors of diverse stimuli in mature neurons. However, developmental roles for TRP channels in the establishment of neuronal connectivity remain largely unexplored. Here, we identify an essential function for TRPC5, a member of the canonical TRP subfamily, in the regulation of dendrite patterning in the mammalian brain. Strikingly, TRPC5 knockout mice harbor long, highly branched granule neuron dendrites with impaired dendritic claw differentiation in the cerebellar cortex. In vivo RNAi analyses suggest that TRPC5 regulates dendrite morphogenesis in the cerebellar cortex in a cell-autonomous manner. Correlating with impaired dendrite patterning in the cerebellar cortex, behavioral analyses reveal that TRPC5 knockout mice have deficits in gait and motor coordination. Finally, we uncover the molecular basis of TRPC5's function in dendrite patterning. We identify the major protein kinase calcium/calmodulin-dependent kinase II β (CaMKIIβ) as a critical effector of TRPC5 function in neurons. Remarkably, TRPC5 forms a complex specifically with CaMKIIβ, but not the closely related kinase CaMKIIα, and thereby induces the CaMKIIβ-dependent phosphorylation of the ubiquitin ligase Cdc20-APC at the centrosome. Accordingly, centrosomal CaMKIIβ signaling mediates the ability of TRPC5 to regulate dendrite morphogenesis in neurons. Our findings define a novel function for TRPC5 that couples calcium signaling to a ubiquitin ligase pathway at the centrosome and thereby orchestrates dendrite patterning and connectivity in the brain.     

Renoprotection: focus on TRPV1, TRPV4, TRPC6 and TRPM2

Members of the transient receptor potential (TRP) cation channel receptor family have unique sites of regulatory function in the kidney which enables them to promote regional vasodilatation and controlled Ca²⁺ influx into podocytes and tubular cells. Activated TRP vanilloid 1 receptor channels (TRPV1) have been found to elicit renoprotection in rodent models of acute kidney injury following ischaemia/reperfusion. Transient receptor potential cation channel, subfamily C, member 6 (TRPC6) in podocytes is involved in chronic proteinuric kidney disease, particularly in focal segmental glomerulosclerosis (FSGS). TRP vanilloid 4 receptor channels (TRPV4) are highly expressed in the kidney, where they induce Ca²⁺ influx into endothelial and tubular cells. TRP melastatin (TRPM2) non‐selective cation channels are expressed in the cytoplasm and intracellular organelles, where their inhibition ameliorates ischaemic renal pathology. Although some of their basic properties have been recently identified, the renovascular role of TRPV1, TRPV4, TRPC6 and TRPM2 channels in disease states such as obesity, hypertension and diabetes is largely unknown. In this review, we discuss recent evidence for TRPV1, TRPV4, TRPC6 and TRPM2 serving as potential targets for acute and chronic renoprotection in chronic vascular and metabolic disease.     

Ca2+-activated Cl− currents are dispensable for olfaction

Canonical olfactory signal transduction involves the activation of cyclic AMP-activated cation channels that depolarize the cilia of receptor neurons and raise intracellular calcium. Calcium then activates Cl(-) currents that may be up to tenfold larger than cation currents and are believed to powerfully amplify the response. We identified Anoctamin2 (Ano2, also known as TMEM16B) as the ciliary Ca(2+)-activated Cl(-) channel of olfactory receptor neurons. Ano2 is expressed in the main olfactory epithelium (MOE) and in the vomeronasal organ (VNO), which also expresses the related Ano1 channel. Disruption of Ano2 in mice virtually abolished Ca(2+)-activated Cl(-) currents in the MOE and VNO. Ano2 disruption reduced fluid-phase electro-olfactogram responses by only ～40%, did not change air-phase electro-olfactograms and did not reduce performance in olfactory behavioral tasks. In contrast with the current view, cyclic nucleotide-gated cation channels do not need a boost by Cl(-) channels to achieve near-physiological levels of olfaction.     

Down regulation of TRPC1 by shRNA reduces mechanosensitivity in mouse dorsal root ganglion neurons in vitro

Mechanosensitivity is a crucial but poorly understood property of the sensory nervous system. Transient receptor potential (TRP) channels, which have been found to be responsible for the detection of other sensory stimuli such as temperature and pungent chemicals, have been suggested to also recognize stretch or pressure to cell membranes. TRPC1 is one candidate from studies in oocytes but evidence in native sensory neurons has been lacking. Therefore, we have measured an increase in intracellular Ca²⁺ levels upon mechanical activation of native mouse dorsal root ganglion (DRG) neurons in culture using hypoosmolar buffer. Our results show that down regulation of TRPC1 with short hairpin RNA results in a 65% reduction of neurons with stretch activated responses. These results implicate a direct or indirect involvement of TRPC1 in the mechanosensitivity of DRG neurons.     

On the putative role of transient receptor potential cation channels in asthma

The mammalian transient receptor potential (TRP) superfamily consists of 28 mammalian TRP cation channels, which can be subdivided into six main subfamilies: the TRPC (' C anonical'), TRPV (' V anilloid'), TRPM (' M elastatin'), TRPP (' P olycystin'), TRPML (' M uco l ipin') and the TRPA (' A nkyrin') groups. Increasing evidence has accumulated during the previous few years that links TRP channels to the cause of several diseases or to critically influence and/or determine their progress. This review focuses on the possible role of TRP channels in the aetiology of asthmatic lung disease.     

Substance P evokes cation currents through TRP channels in HEK293 cells

Activation of any of the three known tachykinin receptors (NK1R, -2R, or -3R) can cause a rise in [Ca2+]i via a pertussis toxin-insensitive heterotrimeric G protein, Gq/G11, activation of phospholipase C (PLC), and a membrane depolarization. Tachykinins can depolarize neurons by two distinct mechanisms: 1) they reduce a resting K+ current in many neurons or 2) in parasympathetic and vagal primary sensory neurons, they activate a nonspecific cation current (Icat). Transient receptor potential channels (TRPC) are nonspecific cation channels that can be activated by a rise in [Ca2+]i in a PLC-dependent manner. The present work tests whether NK2R can signal TRPC. We applied standard whole cell patch-clamp recordings to HEK293 cells stably transfected with the human TRP3 channels (TRP3C), and transiently transfected with a functional NK2R-EGFP. Bath applied Substance P (SP, 1 microM) induced an Icat in the cells expressing both TRP3C and NK2R. Icat reached its peak value in approximately 3 min (195 +/- 120.0 s, mean +/- SE, n = 20), had a peak density of 11.3 +/- 3.48 pA/pF (n = 24), and was blocked by an NK2R-specific antagonist (SR48968, 100 nM). The Erev value for the SP current was 6.8 +/- 7.66 mV (n = 6), suggestive of a nonspecific cation channel. Icat was not measurable in TRP3C-expressing HEK293 cells without NK2R expression (n = 6) or in wild-type HEK293 cells with NK2R expression (n = 12). These data indicate that NK2R can be functionally coupled to TRP channels in HEK293 cells and suggest that SP-induced cation currents in vagal primary sensory neurons might be mediated by TRPC.     

Functional transient receptor potential canonical type 1 channels in human atrial myocytes

Transient receptor potential (TRP) channels are not well understood in human atrium, and the present study was therefore designed to investigate whether TRPC channels would mediate the nonselective cation current reported previously and are involved in the formation of store-operated Ca²⁺ entry (SOCE) channels in human atrial myocytes using approaches of whole-cell patch voltage-clamp, RT-PCR, Western blotting, co-immunoprecipitation, and confocal scanning approaches, etc. We found that a nonselective cation current was recorded under K⁺-free conditions in human atrial myocytes, and the current was inhibited by the TRP channel blocker La³⁺. Thapsigargin enhanced the current, and its effect was suppressed by La³⁺ and prevented by pipette inclusion of anti-TRPC1 antibody. Endothlin-1 and angiotensin II enhanced the current that could be inhibited by La³⁺. Gene and protein expression of TRPC1 channels were abundant in human atria. In addition, mRNA and protein of STIM1 and Orai1, components of SOCE channels, were abundantly expressed in human atria. Co-immunoprecipitation analysis demonstrated an interaction of TRPC1 with STIM1 and/or Orai1. Ca²⁺ signaling mediated by SOCE channels was detected by a confocal microscopy technique. These results demonstrate the novel evidence that TRPC1 channels not only mediate the nonselective cation current, but also form SOCE channels in human atria as a component. TRPC1 channels can be activated by endothelin-1 or angiotensin II, which may be involved in the atrial electrical remodeling in patients with atrial fibrillation.     

Expression of transient receptor potential (TRP) channel mRNAs in the mouse olfactory bulb

Transient receptor potential (TRP) channels are a large family of cation channels. The 28 TRP channel subtypes in rodent are divided into 6 subfamilies: TRPC1-7, TRPV1-6, TRPM1-8, TRPP2/3/5, TRPML1-3 and TRPA1. TRP channels are involved in peripheral olfactory transduction. Several TRPC channels are expressed in unidentified neurons in the main olfactory bulb (OB), but the expression of most TRP channels in the OB has not been investigated. The present study employed RT-PCR as an initial survey of the expression of TRP channel mRNAs in the mouse OB and in 3 cell types: external tufted, mitral and granule cells. All TRP channel mRNAs except TRPV5 were detected in OB tissue. Single cell RT-PCR revealed that external tufted, mitral and granule cell populations expressed in aggregate 14 TRP channel mRNAs encompassing members of all 6 subfamilies. These different OB neuron populations expressed 7–12 channel mRNAs. Common channel expression was more similar among external tufted and mitral cells than among these cells and granule cells. These results indicate that a large number of TRP channel subtypes are expressed in OB neurons, providing the molecular bases for these channels to regulate OB neuron activity and central olfactory processing.     

Na+ entry and modulation of Na+/Ca2+ exchange as a key mechanism of TRPC signaling

Ion channels formed by canonical transient receptor potential (TRPC) proteins are considered to be key players in cellular Ca²⁺ homeostasis. As permeation of Ca²⁺ through TRPC homo- and/or heteromeric channels has been repeatedly demonstrated, analysis of the physiological role of TRPC proteins was so far based on the concept that these proteins form regulated Ca²⁺ entry channels. The well-recognized lack of cation selectivity of TRPC channels and the ability to generate substantial monovalent conductances that govern membrane potential and cation gradients were barely appreciated as a physiologically relevant issue. Nonetheless, recent studies suggest monovalent, specifically Na⁺ permeation through TRPC cation channels as an important event in TRPC signaling. TRPC-mediated Na⁺ entry may be converted into a distinct pattern of cellular Ca²⁺ signals by interaction with Na⁺/Ca²⁺ exchanger proteins. This review discusses current concepts regarding the link between Na⁺ entry through TRPC channels and cellular Ca²⁺ signaling.     

Physiological mechanisms of TRPC activation

TRPC (canonical transient receptor potential) channels are vertebrate homologs of the Drosophila photoreceptor channel, TRP. Considerable research has been brought to bear on the seven members of this family, especially with regard to their possible role in calcium entry. Unfortunately, the current literature presents a confusing picture, with different laboratories producing widely differing results and interpretations. It appears that ectopically expressed TRPC channels can be activated by phospholipase C products (generally, diacylglycerols), by stimulation of trafficking to the plasma membrane, or by depletion of intracellular Ca²⁺ stores. Here, I discuss the possibility that these diverse experimental findings arise because TRPC channels can, under both experimental as well as physiological conditions, be activated in three distinct ways, possibly depending on their subunit composition and/or signaling complex environment. The TRPCs may be unique among ion-channel subunit families in being able to participate in the assembly and function of multiple types of physiologically important ion channels.     

Dual depolarization responses generated within the same lateral septal neurons by TRPC4-containing channels

In the central nervous system, canonical transient receptor potential (TRPC) channels have been implicated in mediating neuronal excitation induced by stimulating metabotropic receptors, including group 1 metabotropic glutamate receptors (mGluRs). Lateral septal (LS) neurons express high levels of TRPC4 and group I mGluRs. However, to what extent native TRPC4-containing channels (TRPC4-cc) are activated as well as the impact of different levels of TRPC4-cc activation on neuronal excitability remain elusive. Here, we report that stimulating LS neurons with group I mGluR agonist, (S)-3,5-DHPG, causes either an immediate increase in firing rate or an initial burst followed by a pause of firing, which can be correlated with below-threshold-depolarization (BTD) or above-threshold-plateau-depolarization (ATPD), respectively, in whole-cell recordings. The early phase of BTD and the entire ATPD are completely absent in neurons from TRPC4^?/? mice. Moreover, in the same LS neurons, BTD can be converted to ATPD at more depolarized potentials or with a brief current injection, suggesting that BTD and ATPD may represent partial and full activations of TRPC4-cc, respectively. We show that coincident mGluR stimulation and depolarization is required to evoke strong TRPC4-cc current, and Na⁺ and Ca²⁺ influx, together with dynamic changes of intracellular Ca²⁺, are essential for ATPD induction. Our results suggest that TRPC4-cc integrates metabotropic receptor stimulation with intracellular Ca²⁺ signals to generate two interconvertible depolarization responses to affect excitability of LS neurons in distinct fashions.     

TRP channels in Drosophila photoreceptor cells

TRP cation channels are conserved throughout animal phylogeny and include many members that function in sensory physiology. The founding TRP is required for Drosophila phototransduction and has served as a paradigm for unravelling the roles and macromolecular organizations of TRP channels in native tissues. Two other TRPC channels, TRPL and TRPγ, are expressed in photoreceptor cells and form heteromultimers with TRP and with each other. TRP is a member of a supramolecular signalling complex, the signalplex, which includes the PDZ scaffold protein, INAD, and two other core members that remain bound and depend on INAD for localization. Other INAD binding proteins are proposed to interact dynamically with INAD, one of which, TRPL, undergoes light-dependent translocation in photoreceptor cells. Surprisingly, TRP has non-channel functions, including an anchoring role necessary for retaining INAD in the rhabdomeres. Loss of TRP function or constitutive TRP activity results in retinal degeneration, which can be suppressed by disruption or overexpression of the Na⁺/Ca²⁺ exchanger, CalX, respectively. Given that hypoxia-induced constitutive activity of some mammalian TRPs leads to neuronal cell death, interventions that increase Na⁺/Ca²⁺ exchanger or decrease TRP function have the potential to reduce the severity of cell death due to ischaemia.     

Functional role of TRPC channels in the regulation of endothelial permeability

The endothelial cells (ECs) form a semipermeable barrier between the blood and the tissue. An important function of the endothelium is to maintain the integrity of the barrier function of the vessel wall. Ca²⁺ signaling in ECs plays a key role in maintaining the barrier integrity. Transient receptor potential canonical (TRPC) channels are mammalian homologs of Drosophila TRP Ca²⁺-permeable channels expressed in EC. TRPC channels are thought to function as a Ca²⁺ entry channel operated by store-depletion as well as receptor-activated channels in a variety of cell types, including ECs. Inflammatory mediators such as thrombin, histamine, bradykinin, and others increase endothelial permeability by actin polymerization-dependent EC rounding and formation of inter-endothelial gaps, a process critically dependent on the increase in EC cytosolic [Ca²⁺] ([Ca²⁺]_i). Increase in endothelial permeability depends on both intracellular Ca²⁺ release and extracellular Ca²⁺ entry through TRPC channels. This review summarizes recent findings on the role of TRPC channels in the mechanism of Ca²⁺ entry in ECs, and, in particular, the role of TRPC channels in regulating endothelial barrier function.     

Homo- and heteromeric assembly of TRP channel subunits

Mammalian homologues of the Drosophila melanogaster transient receptor potential (TRP) channels are the second largest cation channel family within the superfamily of hexahelical cation channels. Most mammalian TRP channels function as homooligomers and mediate mono- or divalent cation entry upon activation by a variety of stimuli. Because native TRP channels may be multimeric proteins of possibly complex composition, it is difficult to compare cation conductances in native tissues to those of clearly defined homomeric TRP channel complexes in living cells. Therefore, the possibility of heteromeric TRP channel assembly has been investigated in recent years by several groups. As a major conclusion of these studies, most heteromeric TRP channel complexes appear to consist of subunit combinations only within relatively narrow confines of phylogenetic subfamilies. Although the general capability of heteromer formation between closely related TRP channel subunits is now clearly established, we are only beginning to understand whether these heteromeric complexes are of physiological significance. This review summarizes the current knowledge on the promiscuity and specificity of the assembly of channel complexes composed of TRPC-, TRPV- and TRPM-subunits of mammalian TRP channels.     

Store-operated Ca2+ entry in platelets occurs independently of transient receptor potential (TRP) C1

Changes in [Ca²⁺]_i are a central step in platelet activation. In nonexcitable cells, receptor-mediated depletion of intracellular Ca²⁺ stores triggers Ca²⁺ entry through store-operated calcium (SOC) channels. Stromal interaction molecule 1 (STIM1) has been identified as an endoplasmic reticulum (ER)-resident Ca²⁺ sensor that regulates store-operated calcium entry (SOCE), but the identity of the SOC channel in platelets has been controversially debated. Some investigators proposed transient receptor potential (TRP) C1 to fulfil this function based on the observation that antibodies against the channel impaired SOCE in platelets. However, others could not detect TRPC1 in the plasma membrane of platelets and raised doubts about the specificity of the inhibiting anti-TRPC1 antibodies. To address the role of TRPC1 in SOCE in platelets, we analyzed mice lacking TRPC1. Platelets from these mice display fully intact SOCE and also otherwise unaltered calcium homeostasis compared to wild-type. Furthermore, platelet function in vitro and in vivo is not altered in the absence of TRPC1. Finally, studies on human platelets revealed that the presumably inhibitory anti-TRPC1 antibodies have no specific effect on SOCE and fail to bind to the protein. Together, these results provide evidence that SOCE in platelets is mediated by channels other than TRPC1. David Varga-Szabo and Kalwant S. Authi contributed equally to this article.     

ATP modulates intracellular Ca2+ and firing rate through a P2Y1 purinoceptor in cane toad pacemaker cells

Bipolar sensory neurons within the vomeronasal organ (VNO) are thought to mediate the detection of pheromones in vertebrates. In the mouse, VNO neurons respond to pheromones with a rise in intracellular Ca²⁺ that accompanies a depolarization of the cell. Transduction of the pheromone appears to occur through the activation of a phosphatidylinositol signalling pathway, but the ion channels that respond to this signalling pathway have not been identified. In this report patch-clamp recording from hamster vomeronasal sensory neurons was used to identify second-messenger-gated channels that might play a role in transduction. The results demonstrate that VNO neurons show abundant expression of a Ca^2+-activated non-selective (CaNS) cation channel. The CaNS channel does not discriminate between Na⁺ and K⁺ and has a slope conductance of 22 pS. Half-activation of the channel occurs at a Ca²⁺ concentration of 0.5 m m (at -80 mV). The probability of opening ( P _o) of the channel is further augmented at positive potentials, and shows an e-fold voltage dependence per 37 mV. The channel exhibits rapid rundown following patch excision with P _o decreasing from near 1.0 to near 0. The adenine nucleotides ATP and cAMP block the channel with an apparent affinity of 3 and 42 μ m , respectively (-80 mV). Both the activation of the channel by Ca²⁺ and the block of the channel by adenine nucleotides show a mild voltage dependence, which can be accounted for by the voltage dependence of P _o. The properties of this channel make it a candidate to either directly mediate vomeronasal sensory transduction, or to amplify the primary sensory response.     

Detection of estrus by male mice: Synergistic role of olfactory–vomeronasal system

In rodents, olfactory pathway comprises two distinct systems viz, the main olfactory and vomeronasal systems, both differing in anatomy, physiology and function. The precise role of the main olfactory/vomeronasal system in estrus detection is yet to be explored. Therefore, the present investigation was planned to elucidate the role of main olfactory and vomeronasal system in the estrus discriminating ability of male mice. Female urine samples of proestrus, estrus, metestrus, diestrus, ovarectomized, ovarectomized plus estrogen treated and prepubertal mice were used for the present study. In addition, the urine from intact, castrated and castrated with testosterone treated mice was also tested for odour preference by male mice. The male responders were categorized into three groups namely (a) normal, (b) ZnSO₄-irrigated and (c) vomeronasal organ (VNO)-ablated. The behavioural responses such as frequency and duration of visits to urine samples were carried out in a Y-maze apparatus to assess odour preference. The normal mice displayed more frequent visits to estrus urine samples than to non-estrus samples. In contrast, ZnSO₄-irrigated mice showed significant reduction in the frequency of visits towards estrus urine, whereas, the vomeronasal (VNO)-ablated mice did not show any noticeable preference. With regard to the duration of visits the VNO-ablated mice showed significant reduction in visiting time when compared to ZnSO₄-irrigated mice. This finding indicated that the main olfactory system (MOS) was involved primarily in the attraction from a distance, while the VNO played a major role in close proximity (pre-copulatory behaviour). The males spent less time with the urine of same-sex; however, the response was higher with castrated male urine which was reduced on testosterone treatment indicating that a specific odour in intact male causes aversive behaviour in male. This study provides support to the fact that volatile compounds could also be perceived by VNO, probably when the main olfactory system is in functional state. The study implies that the olfactory–vomeronasal system plays a synergistic role in the detection of estrus.