Membrane depolarization combined with Gq-activated G-protein-coupled receptors induce transient receptor potential channel 1 (TRPC1)- dependent potentiation of catecholamine release.

Agonists of the Gq/11-activated G-protein-coupled receptors (GPCRs) combined with strong membrane depolarization (high KCl) induce a synergistic amplification of transmitter release. The molecular basis for the synergy is unknown. Here, we investigated this potentiated transmitter release (PTR) phenomenon at the single cell level by monitoring catecholamine (CA) release in chromaffin cells using amperometry. We found that the 60 mM KCl (K60)-triggered release in bovine chromaffin cells synergizes with bradykinin (BK) or histamine (Hist) to potentiate CA release. PTR was independent of Ca2+ influx through voltage-gated calcium channels (VGCC), but required Ca2+ at the extracellular medium and was abolished by inhibitors of phospholipase Cß (PLCß). The∼four-fold PTR induced in mouse chromaffin cells by BK and K60, was not observed in chromaffin cells prepared from TRPC1 KO mice and was restored by expressing hTRPC1. The synergy between strong voltage perturbation (K60) in the presence of VGCC blockers, and the activation of the Gq/11-PLCß signal-transduction cascade generates unique and fundamental amplified signaling machinery. The concerted activation of two independent cellular pathways could reinforce physiological signals that impinge on regulation of secretory events during repeated sequence of high-frequency excitation, hyperpolarization, and relief of inhibition such as long-term potentiation (LTP), that lead to neuronal synaptic plasticity.

Genetic disruption of G proteins, G(i2)alpha or G(o)alpha, does not abolish inotropic and chronotropic effects of stimulating muscarinic cholinoceptors in atrium.

BACKGROUND AND PURPOSE: Classically, stimulation of muscarinic cholinoceptors exerts negative inotropic and chronotropic effects in the atrium of mammalian hearts. These effects are crucial to the vagal regulation of the heart beat. This effect is assumed to be mediated via GTP binding (G) proteins, because they can be abolished by Pertussis toxin. However, it is unknown which G proteins are involved. EXPERIMENTAL APPROACH: We studied contractility in isolated left or right atrium from genetically manipulated mice with deletion of one of two G proteins, either of the alpha subunit of G(i2) protein (G(i2)alpha) or of the alpha subunit of G(o) protein (G(o)alpha). Preparations were stimulated with carbachol alone or after pretreatment with the beta-adrenoceptor agonist isoprenaline. For comparison, the effects of carbachol on L-type Ca(2+)-channels in isolated ventricular cardiomyocytes were studied. KEY RESULTS: The negative inotropic and chronotropic effects of carbachol alone or in the presence of isoprenaline were identical in atria from knockout or wild-type mice. However, the effect of carbachol on isoprenaline-activated L-type Ca(2+)-channel in isolated ventricular cardiomyocytes was greatly attenuated in both types of knockout mice studied. CONCLUSIONS AND IMPLICATIONS: These data imply that there is either redundancy of G proteins for signal transduction or that Pertussis toxin-sensitive proteins other than G(i2)alpha and G(o)alpha mediate the vagal stimulation in the atrium. Moreover, different G proteins mediate the effect of carbachol in ventricle compared with atrium.

TRPC2: molecular biology and functional importance.

TRPC (canonical transient receptor potential) channels are the closest mammalian homologs of Drosophila TRP and TRP-like channels. TRPCs are rather nonselective Ca2+ permeable cation channels and affect cell functions through their ability to mediate Ca2+ entry into cells and their action to collapse the plasma membrane potentials. In neurons the latter function leads to action potentials. The mammalian genome codes for seven TRPCs of which TRPC2 is the largest with the most restricted pattern of expression and has several alternatively spliced variants. Expressed in model cells, TRPC2 mediates both receptor- and store depletion-triggered Ca2+ entry. TRPC2 is unique among TRPCs in that its complete gene has been lost from the Old World monkey and human genomes, in which its remnants constitute a pseudogene. Physiological roles for TRPC2 have been studied in mature sperm and the vomeronasal sensory system. In sperm, TRPC2 is activated by the sperm's interaction with the oocyte's zona pellucida, leading to entry of Ca2+ and activation of the acrosome reaction. In the vomeronasal sensory organ (VNO), TRPC2 was found to constitute the transduction channel activated through signaling cascade initiated by the interaction of pheromones with V1R and V2R G protein-coupled receptors on the dendrites of the sensory neurons. V1Rs and V2Rs, the latter working in conjunction with class I MHC molecules, activate G(i)- and G(o)-type G proteins which in turn trigger activation of TRPC2, initiating an axon potential that travels to the axonal terminals. The signal is then projected to the glomeruli of the auxiliary olfactory bulb from where it is carried first to the amygdala and then to higher cortical cognition centers. Immunocytochemistry and gene deletion studies have shown that (1) the V2R-G(o)-MHCIb-beta2m pathway mediates male aggressive behavior in response to pheromones; (2) the V1R-G(i2) pathway mediates mating partner recognition, and (3) these differences have an anatomical correlate in that these functional components are located in anatomically distinct compartments of the VNO. Interestingly, these anatomically segregated signaling pathways use a common transduction channel, TRPC2.

Know thy neighbor: a survey of diseases and complex syndromes that map to chromosomal regions encoding TRP channels.

On the basis of their ever-expanding roles, not only in sensory signaling but also in a plethora of other, often Ca(2+)-mediated actions in cell and whole body homeostasis, it is suggested that mutations in TRP channel genes not only cause disease states but also contribute in more subtle ways to simple and complex diseases. A survey is therefore presented of diseases and syndromes that map to one or multiple chromosomal loci containing TRP channel genes. A visual map of the chromosomal locations of TRP channel genes in man and mouse is also presented.

Regression of Peyer's patches in G alpha i2 deficient mice prior to colitis is associated with reduced expression of Bcl-2 and increased apoptosis.

BACKGROUND: G protein deficient (G alpha i2-/-) mice spontaneously develop an inflammatory bowel disease (IBD) closely resembling ulcerative colitis. Previous studies have demonstrated that gut T cells are hyperreactive to the endogenous microflora in most IBD models. AIMS: The aim of this study was to analyse Peyer's patches (PP), the inductive sites for gut mucosal immune responses. SUBJECTS AND METHODS: G alpha i2-/- mice, an animal model for IBD, were analysed using immunological methods with regard to phenotype and function. RESULTS: We found significantly decreased numbers of PP in G alpha i2-/- mice. Even before the onset of colitis, G alpha i2 deficient animals exhibited diminished size of PP, as judged by histology. This involution of PP was associated with strongly increased levels of apoptotic lymphocytes, associated with decreased levels of antiapoptotic intracellular protein Bcl-2. PP T lymphocytes showed highly elevated production of interferon gamma in response to the enteric flora compared with PP T cells from wild-type mice, which produced predominantly interleukin 10. CONCLUSIONS: Thus even before the onset of colitis, the PP in G alpha i2 deficient mice is a Th1 dominated milieu associated with downregulated levels of Bcl-2, resulting in increased apoptosis of lymphocytes leading to regression of PP. We speculate that this Th1 dominated microenvironment in the inductive site for mucosal immune responses contributes to the development of colitis in G alpha i2 deficient mice.

Antibody response to dietary and autoantigens in G alpha i2-deficient mice.

BACKGROUND: Mice with a targeted mutation in the G protein subunit G alpha i2 gene develop a colonic mucosal inflammation, with a highly activated B-cell response. We wanted to investigate whether this increased B-cell activity was directed against dietary antigens and/or various self tissues. METHODS: The level of antibodies specific for dietary (gliadin, soya and fish meal) antigens was measured by ELISA. Reactivity against self antigens was measured by immunohistochemistry on cryo-sectioned mouse and rat tissue. Sera and intestinal lavages were analysed from G alpha i2-/- mice before and after development of colitis and in age-matched wild type litter mates. RESULTS: Titres of antibodies against dietary antigens were significantly enhanced both in serum and in large intestinal lavages from G alpha i2-/- mice with ongoing colitis but not prior to disease, as compared to wild type mice. The autoreactivity to self tissues was significantly increased in G alpha i2-/- mice both before and after development of colitis as compared to litter mate control animals. Self tissue reactivity was directed not only against epithelial cells of the colon, small intestine and gastric glands, but also against smooth muscle cells, hepatocytes, bile duct cells, renal tubule and collecting tubule cells of the kidney. In analogy to human ulcerative colitis, autoantibodies against epithelial cells, bile duct epithelium and neutrophil granulocytes were found. CONCLUSIONS: Earlier increase in levels of autoantibodies (before onset of colitis) than of food antibodies (after onset of colitis) suggests the latter response to be a secondary phenomenon to e.g. a destroyed barrier function.

Trp2 regulates entry of Ca2+ into mouse sperm triggered by egg ZP3.

In many cells, receptor activation initiates sustained Ca2+ entry which is critical in signal transduction. Mammalian transient receptor potential (Trp) proteins, which are homologous to the Drosophila photoreceptor-cell Trp protein, have emerged as candidate subunits of the ion channels that mediate this influx. As a consequence of overexpression, these proteins produce cation currents that open either after depletion of internal Ca2+ stores or through receptor activation. However, determining the role of endogenous Trp proteins in signal transduction is complicated by the absence of selective antagonists. Here we examine Trp function during sperm-egg interaction. The sperm acrosome reaction is a Ca2+-dependent secretory event that must be completed before fertilization. In mammals, exocytosis is triggered during gamete contact by ZP3, a glycoprotein constituent of the egg's extracellular matrix, or zona pellucida (ZP). ZP3 activates trimeric G proteins and phospholipase C and causes a transient Ca2+ influx into sperm through T-type Ca2+ channels. These early responses promote a second Ca2+-entry pathway, thereby producing sustained increases in intracellular Ca2+ concentration ([Ca2+]i) that drive acrosome reactions. Our results show that Trp2 is essential for the activation of sustained Ca2+ influx into sperm by ZP3.

Identification of common binding sites for calmodulin and inositol 1,4,5-trisphosphate receptors on the carboxyl termini of trp channels.

Homologues of Drosophila Trp (transient receptor potential) form plasma membrane channels that mediate Ca(2+) entry following the activation of phospholipase C by cell surface receptors. Among the seven Trp homologous found in mammals, Trp3 has been shown to interact with and respond to IP(3) receptors (IP(3)Rs) for activation. Here we show that Trp4 and other Trp proteins also interact with IP(3)Rs. The IP(3)R-binding domain also interacts with calmodulin (CaM) in a Ca(2+)-dependent manner with affinities ranging from 10 nm for Trp2 to 290 nm for Trp6. In addition, other binding sites for CaM and IP(3)Rs are present in the alpha but not the beta isoform of Trp4. In the presence of Ca(2+), the Trp-IP(3)R interaction is inhibited by CaM. However, a synthetic peptide representing a Trp-binding domain of IP(3)Rs inhibited the binding of CaM to Trp3, -6, and -7 more effectively than that to Trp1, -2, -4, and -5. In inside-out membrane patches, Trp4 is activated strongly by calmidazolium, an antagonist of CaM, and a high (50 microm) but not a low (5 microm) concentration of the Trp-binding peptide of the IP(3)R. Our data support the view that both CaM and IP(3)Rs play important roles in controlling the gating of Trp-based channels. However, the sensitivity and responses to CaM and IP(3)Rs differ for each Trp.

Lack of muscarinic regulation of Ca(2+) channels in G(i2)alpha gene knockout mouse hearts.

The purpose of the present study was to examine the role of G(i2)alpha in Ca(2+) channel regulation using G(i2)alpha gene knockout mouse ventricular myocytes. The whole cell voltage-clamp technique was used to study the effects of the muscarinic agonist carbachol (CCh) and the beta-adrenergic agonist isoproterenol (Iso) on cardiac L-type Ca(2+) currents in both 129Sv wild-type (WT) and G(i2)alpha gene knockout (G(i2)alpha-/-) mice. Perfusion with CCh significantly inhibited the Ca(2+) current in WT cells, and this effect was reversed by adding atropine to the CCh-containing solution. In contrast, CCh did not affect Ca(2+) currents in G(i2)alpha-/- ventricular myocytes. Addition of CCh to Iso-containing solutions attenuated the Iso-stimulated Ca(2+) current in WT cardiomyocytes but not in G(i2)alpha-/- cells. These findings demonstrate that, whereas the Iso-G(s)alpha signal pathway is intact in G(i2)alpha gene knockout mouse hearts, these cells lack the inhibitory regulation of Ca(2+) channels by CCh. Therefore, G(i2)alpha is necessary for the muscarinic regulation of Ca(2+) channels in the mouse heart. Further studies are needed to delineate the possible interaction of G(i) and other cell signaling proteins and to clarify the level of interaction of G protein-coupled regulation of L-type Ca(2+) current in the heart.

Activation of Trp3 by inositol 1,4,5-trisphosphate receptors through displacement of inhibitory calmodulin from a common binding domain.

Mammalian homologues of Drosophila Trp form plasma membrane channels that mediate Ca(2+) influx in response to activation of phospholipase C and internal Ca(2+) store depletion. Previous studies showed that human Trp3 is activated by inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) and identified interacting domains, one on Trp and two on IP(3)R. We now find that Trp3 binds Ca(2+)-calmodulin (Ca(2+)/CaM) at a site that overlaps with the IP(3)R binding domain. Using patch-clamp recordings from inside-out patches, we further show that Trp3 has a high intrinsic activity that is suppressed by Ca(2+)/CaM under resting conditions, and that Trp3 is activated by the following: a Trp-binding peptide from IP(3)R that displaces CaM from Trp3, a myosin light chain kinase Ca(2+)/CaM binding peptide that prevents CaM from binding to Trp3, and calmidazolium, an inactivator of Ca(2+)/CaM. We conclude that inhibition of the inhibitory action of CaM is a key step of Trp3 channel activation by IP(3)Rs.

Most central nervous system D2 dopamine receptors are coupled to their effectors by Go.

We reported previously that Go-deficient mice develop severe neurological defects that include hyperalgesia, a generalized tremor, lack of coordination, and a turning syndrome somewhat reminiscent of unilateral lesions of the dopaminergic nigro-striatal pathway. By using frozen coronal sections of serially sectioned brains of normal and Go-deficient mice, we studied the ability of several G protein coupled receptors to promote binding of GTPgammaS to G proteins and the ability of GTP to promote a shift in the affinity of D2 dopamine receptor for its physiologic agonist dopamine. We found a generalized, but not abolished reduction in agonist-stimulated binding of GTPgammaS to frozen brain sections, with no significant left-right differences. Unexpectedly, the ability of GTP to regulate the binding affinity of dopamine to D2 receptors (as seen in in situ [(35)S]sulpiride displacement curves) that was robust in control mice, was absent in Go-deficient mice. The data suggest that most of the effects of the Gi/Go-coupled D2 receptors in the central nervous system are mediated by Go instead of Gi1, Gi2, or Gi3. In agreement with this, the effect of GTP on dopamine binding to D2 receptors in double Gi1 plus Gi2- and Gi1 plus Gi3-deficient mice was essentially unaffected.

The light response of ON bipolar neurons requires G[alpha]o.

ON bipolar neurons in retina detect the glutamate released by rods and cones via metabotropic glutamate receptor 6 (mGluR6), whose cascade is unknown. The trimeric G-protein G(o) might mediate this cascade because it colocalizes with mGluR6. To test this, we studied the retina in mice negative for the alpha subunit of G(o) (Galpha(o)-/-). Retinal layering, key cell types, synaptic structure, and mGluR6 expression were all normal, as was the a-wave of the electroretinogram, which represents the rod and cone photocurrents. However, the b-wave of the electroretinogram, both rod- and cone-driven components, was entirely missing. Because the b-wave represents the massed response of ON bipolar cells, its loss in the Galpha(o) null mouse establishes that the light response of the ON bipolar cell requires G(o). This represents the first function to be defined in vivo for the alpha subunit of the most abundant G-protein of the brain.

Mechanism of capacitative Ca2+ entry (CCE): interaction between IP3 receptor and TRP links the internal calcium storage compartment to plasma membrane CCE channels.

Activation of cells by agents that stimulate inositol trisphoshate (IP3) formation causes, via IP3 receptor (IP3R) activation, the release of Ca2+ from internal stores and also the entry of Ca2+ via plasma membrane cation channels, referred to as capacitative Ca2+ entry or CCE channels. Trp proteins have been proposed to be the unitary subunits forming CCE channels; however, there is no definitive proof for this hypothesis. We have now identified amino acid sequences of a Trp and of an IP3R that interact to form stable complexes. These complexes appear to form in vivo, as evidenced by co-immunoprecipitation of Trp with IP3R and by the fact that expression of the respective interacting sequences modulates development of CCE brought about by store depletion. The finding that a Trp-interacting sequence of IP3R interferes with natural CCE leads us to conclude that Trp proteins are, indeed, structural members of CCE channels. We conclude further that direct coupling of IP3R to Trp is a physiological mechanism by which cells trigger CCE in response to signals that stimulate phosphoinositide hydrolysis and IP3 formation. Pros and cons of various CCE activation models are discussed.

Expression of the alpha(2)delta subunit interferes with prepulse facilitation in cardiac L-type calcium channels.

We investigated the role of the accessory alpha(2)delta subunit on the voltage-dependent facilitation of cardiac L-type Ca(2+) channels (alpha(1C)). alpha(1C) Channels were coexpressed in Xenopus oocytes with beta(3) and alpha(2)delta calcium channel subunits. In alpha(1C) + beta(3), the amplitude of the ionic current (measured during pulses to 10 mV) was in average approximately 1.9-fold larger after the application of a 200-ms prepulse to +80 mV. This phenomenon, commonly referred to as voltage-dependent facilitation, was not observed when alpha(2)delta was coexpressed with alpha(1C) + beta(3). In alpha(1C) + beta(3), the prepulse produced a left shift ( approximately 40 mV) of the activation curve. Instead, the activation curve for alpha(1C) + beta(3) + alpha(2)delta was minimally affected by the prepulse and had a voltage dependence very similar to the G-V curve of the alpha(1C) + beta(3) channel facilitated by the prepulse. Coexpression of alpha(2)delta with alpha(1C) + beta(3) seems to mimic the prepulse effect by shifting the activation curve toward more negative potentials, leaving little room for facilitation. The facilitation of alpha(1C) + beta(3) was associated with an increase of the charge movement. In the presence of alpha(2)delta, the charge remained unaffected after the prepulse. Coexpression of alpha(2)delta seems to set all the channels in a conformational state from where the open state can be easily reached, even without prepulse.

Requirement of the inositol trisphosphate receptor for activation of store-operated Ca2+ channels.

The coupling mechanism between endoplasmic reticulum (ER) calcium ion (Ca2+) stores and plasma membrane (PM) store-operated channels (SOCs) is crucial to Ca2+ signaling but has eluded detection. SOCs may be functionally related to the TRP family of receptor-operated channels. Direct comparison of endogenous SOCs with stably expressed TRP3 channels in human embryonic kidney (HEK293) cells revealed that TRP3 channels differ in being store independent. However, condensed cortical F-actin prevented activation of both SOC and TRP3 channels, which suggests that ER-PM interactions underlie coupling of both channels. A cell-permeant inhibitor of inositol trisphosphate receptor (InsP3R) function, 2-aminoethoxydiphenyl borate, prevented both receptor-induced TRP3 activation and store-induced SOC activation. It is concluded that InsP3Rs mediate both SOC and TRP channel opening and that the InsP3R is essential for maintaining coupling between store emptying and physiological activation of SOCs.

Modulation of Ca(2+) entry by polypeptides of the inositol 1,4, 5-trisphosphate receptor (IP3R) that bind transient receptor potential (TRP): evidence for roles of TRP and IP3R in store depletion-activated Ca(2+) entry.

Homologues of Drosophilia transient receptor potential (TRP) have been proposed to be unitary subunits of plasma membrane ion channels that are activated as a consequence of active or passive depletion of Ca(2+) stores. In agreement with this hypothesis, cells expressing TRPs display novel Ca(2+)-permeable cation channels that can be activated by the inositol 1,4,5-trisphosphate receptor (IP3R) protein. Expression of TRPs alters cells in many ways, including up-regulation of IP3Rs not coded for by TRP genes, and proof that TRP forms channels of these and other cells is still missing. Here, we document physical interaction of TRP and IP3R by coimmunoprecipitation and glutathione S-transferase-pulldown experiments and identify two regions of IP3R, F2q and F2g, that interact with one region of TRP, C7. These interacting regions were expressed in cells with an unmodified complement of TRPs and IP3Rs to study their effect on agonist- as well as store depletion-induced Ca(2+) entry and to test for a role of their respective binding partners in Ca(2+) entry. C7 and an F2q-containing fragment of IP3R decreased both forms of Ca(2+) entry. In contrast, F2g enhanced the two forms of Ca(2+) entry. We conclude that store depletion-activated Ca(2+) entry occurs through channels that have TRPs as one of their normal structural components, and that these channels are directly activated by IP3Rs. IP3Rs, therefore, have the dual role of releasing Ca(2+) from stores and activating Ca(2+) influx in response to either increasing IP3 or decreasing luminal Ca(2+).

Complete reversal of run-down in rabbit cardiac Ca2+ channels by patch-cramming in Xenopus oocytes; partial reversal by protein kinase A.

The rabbit cardiac Ca2+ channel (alpha1C) expressed in Xenopus oocytes exhibited a complete run-down of ionic currents when cell-attached patches were excised. The alpha1C channel was expressed alone or was coexpressed with the accessory beta2a or beta1b subunit. The catalytic subunit of protein kinase A (PKAc) and MgATP were capable of delaying the run-down of single-channel currents. In 33% of the alpha1C patches, and 26% of the alpha1C+beta2a patches, inclusion of PKAc in the bath solution delayed the run-down for a maximum of 20 min. In experiments where PKAc in the bath was not sufficient to delay the run-down of channel activity, insertion of the patch back into the oocyte (patch-cramming) could restore channel activity. Gating currents were also measured in the alpha1C+beta1b channel and were not subject to any run-down, even after the complete run-down of ionic currents. The results presented here reveal that PKAc is capable of delaying the run-down of currents in a subset of patches. The patch-cramming results suggest that a cytoplasmic factor, in addition to phosphorylation of the channel (by PKAc), may be involved in the maintenance of channel activity.