Specific association of the gene product of PKD2 with the TRPC1 channel

The function(s) of the genes (PKD1 and PKD2) responsible for the majority of cases of autosomal dominant polycystic kidney disease is unknown. While PKD1 encodes a large integral membrane protein containing several structural motifs found in known proteins involved in cell–cell or cell–matrix interactions, PKD2 has homology to PKD1 and the major subunit of the voltage-activated Ca2+ channels. We now describe sequence homology between PKD2 and various members of the mammalian transient receptor potential channel (TRPC) proteins, thought to be activated by G protein-coupled receptor activation and/or depletion of internal Ca2+ stores. We show that PKD2 can directly associate with TRPC1 but not TRPC3 in transfected cells and in vitro. This association is mediated by two distinct domains in PKD2. One domain involves a minimal region of 73 amino acids in the C-terminal cytoplasmic tail of PKD2 shown previously to constitute an interacting domain with PKD1. However, distinct residues within this region mediate specific interactions with TRPC1 or PKD1. The C-terminal domain is sufficient but not necessary for the PKD2–TRPC1 association. A more N-terminal domain located within transmembrane segments S2 and S5, including a putative pore helical region between S5 and S6, is also responsible for the association. Given the ability of the TRPC to form functional homo- and heteromultimeric complexes, these data provide evidence that PKD2 may be functionally related to TRPC proteins and suggest a possible role of PKD2 in modulating Ca2+ entry in response to G protein-coupled receptor activation and/or store depletion.

Functional identification and reconstitution of an odorant receptor in single olfactory neurons

The olfactory system is remarkable in its capacity to discriminate a wide range of odorants through a series of transduction events initiated in olfactory receptor neurons. Each olfactory neuron is expected to express only a single odorant receptor gene that belongs to the G protein coupled receptor family. The ligand–receptor interaction, however, has not been clearly characterized. This study demonstrates the functional identification of olfactory receptor(s) for specific odorant(s) from single olfactory neurons by a combination of Ca2+-imaging and reverse transcription–coupled PCR analysis. First, a candidate odorant receptor was cloned from a single tissue-printed olfactory neuron that displayed odorant-induced Ca2+ increase. Next, recombinant adenovirus-mediated expression of the isolated receptor gene was established in the olfactory epithelium by using green fluorescent protein as a marker. The infected neurons elicited external Ca2+ entry when exposed to the odorant that originally was used to identify the receptor gene. Experiments performed to determine ligand specificity revealed that the odorant receptor recognized specific structural motifs within odorant molecules. The odorant receptor-mediated signal transduction appears to be reconstituted by this two-step approach: the receptor screening for given odorant(s) from single neurons and the functional expression of the receptor via recombinant adenovirus. The present approach should enable us to examine not only ligand specificity of an odorant receptor but also receptor specificity and diversity for a particular odorant of interest.

Phentolamine block of KATP channels is mediated by Kir6.2

The ATP-sensitive K+-channel (KATP channel) plays a key role in insulin secretion from pancreatic β cells. It is closed both by glucose metabolism and the sulfonylurea drugs that are used in the treatment of noninsulin-dependent diabetes mellitus, thereby initiating a membrane depolarization that activates voltage-dependent Ca2+ entry and insulin release. The β cell KATP channel is a complex of two proteins: Kir6.2 and SUR1. The former is an ATP-sensitive K+-selective pore, whereas SUR1 is a channel regulator that endows Kir6.2 with sensitivity to sulfonylureas. A number of drugs containing an imidazoline moiety, such as phentolamine, also act as potent stimulators of insulin secretion, but their mechanism of action is unknown. We have used a truncated form of Kir6.2, which expresses independently of SUR1, to show that phentolamine does not inhibit KATP channels by interacting with SUR1. Instead, our results argue that phentolamine may interact directly with Kir6.2 to produce a voltage-independent reduction in channel activity. The single-channel conductance is unaffected. Although the ATP molecule also contains an imidazoline group, the site at which phentolamine blocks is not identical to the ATP-inhibitory site, because phentolamine block of an ATP-insensitive mutant (K185Q) is normal. KATP channels also are found in the heart where they are involved in the response to cardiac ischemia: they also are blocked by phentolamine. Our results suggest that this may be because Kir6.2, which is expressed in the heart, forms the pore of the cardiac KATP channel.

Stimulation of CD95 (Fas) blocks T lymphocyte calcium channels through sphingomyelinase and sphingolipids

Calcium influx through store-operated calcium release-activated calcium channels (CRAC) is required for T cell activation, cytokine synthesis, and proliferation. The CD95 (Apo-1/Fas) receptor plays a role in self-tolerance and tumor immune escape, and it mediates apoptosis in activated T cells. In this paper we show that CD95-stimulation blocks CRAC and Ca2+ influx in lymphocytes through the activation of acidic sphingomyelinase (ASM) and ceramide release. The block of Ca2+ entry is lacking in CD95-defective lpr lymphocytes as well as in ASM-defective cells and can be restored by retransfection of ASM. C2 ceramide, C6 ceramide, and sphingosine block CRAC reversibly, whereas the inactive dihydroceramide has no effect. CD95-stimulation or the addition of ceramide prevents store-operated Ca2+ influx, activation of the transcriptional regulator NFAT, and IL-2 synthesis. The block of CRAC by sphingomyelinase metabolites adds a function to the repertoire of the CD95 receptor inhibiting T cell activation signals.

Dynamic regulation of neuronal NO synthase transcription by calcium influx through a CREB family transcription factor-dependent mechanism

Neuronal nitric oxide (NO) synthase (nNOS) is dynamically regulated in response to a variety of physiologic and pathologic stimuli. Although the dynamic regulation of nNOS is well established, the molecular mechanisms by which such diverse stimuli regulate nNOS expression have not yet been identified. We describe experiments demonstrating that Ca2+ entry through voltage-sensitive Ca2+ channels regulates nNOS expression through alternate promoter usage in cortical neurons and that nNOS exon 2 contains the regulatory sequences that respond to Ca2+. Deletion and mutational analysis of the nNOS exon 2 promoter reveals two critical cAMP/Ca2+ response elements (CREs) that are immediately upstream of the transcription start site. CREB binds to the CREs within the nNOS gene. Mutation of the nNOS CREs as well as blockade of CREB function results in a dramatic loss of nNOS transcription. These findings suggest that nNOS is a Ca2+-regulated gene through the interactions of CREB on the CREs within the nNOS exon 2 promoter and that these interactions are likely to be centrally involved in the regulation of nNOS in response to neuronal injury and activity-dependent plasticity.

Excitation–contraction coupling is unaffected by drastic alteration of the sequence surrounding residues L720–L764 of the α1S II-III loop

The II-III loop of the skeletal muscle dihydropyridine receptor (DHPR) α1S subunit is responsible for bidirectional-signaling interactions with the ryanodine receptor (RyR1): transmitting an orthograde, excitation–contraction (EC) coupling signal to RyR1 and receiving a retrograde, current-enhancing signal from RyR1. Previously, several reports argued for the importance of two distinct regions of the skeletal II-III loop (residues R681–L690 and residues L720–Q765, respectively), claiming for each a key function in DHPR–RyR1 communication. To address whether residues 720–765 of the II-III loop are sufficient to enable skeletal-type (Ca2+ entry-independent) EC coupling and retrograde interaction with RyR1, we constructed a green fluorescent protein (GFP)-tagged chimera (GFP-SkLM) having rabbit skeletal (Sk) DHPR sequence except for a II-III loop (L) from the DHPR of the house fly, Musca domestica (M). The Musca II-III loop (75% dissimilarity to α1S) has no similarity to α1S in the regions R681–L690 and L720–Q765. GFP-SkLM expressed in dysgenic myotubes (which lack endogenous α1S subunits) was unable to restore EC coupling and displayed strongly reduced Ca2+ current densities despite normal surface expression levels and correct triad targeting (colocalization with RyR1). Introducing rabbit α1S residues L720–L764 into the Musca II-III loop of GFP-SkLM (substitution for Musca DHPR residues E724–T755) completely restored bidirectional coupling, indicating its dependence on α1S loop residues 720–764 but its independence from other regions of the loop. Thus, 45 α1S-residues embedded in a very dissimilar background are sufficient to restore bidirectional coupling, indicating that these residues may be a site of a protein–protein interaction required for bidirectional coupling.

TRPC1, a human homolog of a Drosophila store-operated channel.

In many vertebrate and invertebrate cells, inositol 1,4,5-trisphospate production induces a biphasic Ca2+ signal. Mobilization of Ca2+ from internal stores drives the initial burst. The second phase, referred to as store-operated Ca2+ entry (formerly capacitative Ca2+ entry), occurs when depletion of intracellular Ca2+ pools activates a non-voltage-sensitive plasma membrane Ca2+ conductance. Despite the prevalence of store-operated Ca2+ entry, no vertebrate channel responsible for store-operated Ca2+ entry has been reported. trp (transient receptor potential), a Drosophila gene required in phototransduction, encodes the only known candidate for such a channel throughout phylogeny. In this report, we describe the molecular characterization of a human homolog of trp, TRPC1. TRPC1 (transient receptor potential channel-related protein 1) was 40% identical to Drosophila TRP over most of the protein and lacked the charged residues in the S4 transmembrane region proposed to be required for the voltage sensor in many voltage-gated ion channels. TRPC1 was expressed at the highest levels in the fetal brain and in the adult heart, brain, testis, and ovaries. Evidence is also presented that TRPC1 represents the archetype of a family of related human proteins.

Cone photoreceptors respond to their own glutamate release in the tiger salamander.

Pulse-like currents resembling miniature postsynaptic currents were recorded in patch-clamped isolated cones from the tiger salamander retina. The events were absent in isolated cones without synaptic terminals. The frequency of events was increased by either raising the osmotic pressure or depolarizing the cell. It was decreased by the application of either glutamate or the glutamate-transport blockers dihydrokainate and D,L-threo-3-hydroxyaspartate. The events required external Na+ for which Li+ could not substitute. The reversal potential of these currents followed the equilibrium potential for Cl- when internal Cl- concentration was changed. Thus, these miniature currents appear to represent the presynaptic activation of the glutamate receptor with glutamate transporter-like pharmacology, caused by the photoreceptor's own vesicular glutamate release. Using a noninvasive method to preserve the intracellular Cl- concentration, we showed that glutamate elicits an outward current in isolated cones. Fluorescence of the membrane-permeable form of fura-2 was used to monitor Ca2+ entry at the cone terminal as a measure of membrane depolarization. The increase in intracellular Ca2+ concentration, elicited by puff application of 30 mM KCl, was completely suppressed in the presence of 100 microM glutamate. Puff application of glutamate alone had no measurable depolarizing effect. These results suggest that the equilibrium potential for Cl-, ECl, was more negative than the activation range for Ca2+ channels and that glutamate elicited an outward current, hyperpolarizing the cones.

Depletion-activated calcium current is inhibited by protein kinase in RBL-2H3 cells.

Whole-cell patch-clamp recordings and single-cell Ca2+ measurements were used to study the control of Ca2+ entry through the Ca2+ release-activated Ca2+ influx pathway (ICRAC) in rat basophilic leukemia cells. When intracellular inositol 1,4,5-trisphosphate (InsP3)-sensitive stores were depleted by dialyzing cells with high concentrations of InsP3, ICRAC inactivated only slightly in the absence of ATP. Inclusion of ATP accelerated inactivation 2-fold. The inactivation was increased further by the ATP analogue adenosine 5'-[gamma-thio]triphosphate, which is readily used by protein kinases, but not by 5'-adenylyl imidodiphosphate, another ATP analogue that is not used by kinases. Neither cyclic nucleotides nor inhibition of calmodulin or tyrosine kinase prevented the inactivation. Staurosporine and bisindolylmaleimide, protein kinase C inhibitors, reduced inactivation of ICRAC, whereas phorbol ester accelerated inactivation of the current. These results demonstrate that a protein kinase-mediated phosphorylation, probably through protein kinase C, inactivates ICRAC. Activation of the adenosine receptor (A3 type) in RBL cells did not evoke much Ca2+ influx or systematic activation of ICRAC. After protein kinase C was blocked, however, large ICRAC was observed in all cells and this was accompanied by large Ca2+ influx. The ability of a receptor to evoke Ca2+ entry is determined, at least in part, by protein kinase C. Antigen stimulation, which triggers secretion through a process that requires Ca2+ influx, activated ICRAC. The regulation of ICRAC by protein kinase will therefore have important consequences on cell functioning.

Potassium channels and membrane potential in the modulation of intracellular calcium in vascular endothelial cells

K+ Channels and Membrane Potential in Endothelial Cells. The endothelium plays a vital role in the control of vascular functions, including modulation of tone; permeability and barrier properties; platelet adhesion and aggregation; and secretion of paracrine factors. Critical signaling events in many of these functions involve an increase in intracellular free Ca2+ concentration ([Ca2+](i)). This rise in [Ca2+](i) occurs via an interplay between several mechanisms, including release from intracellular stores, entry from the extracellular space through store depletion and second messenger-mediated processes, and the establishment of a favorable electrochemical gradient. The focus of this review centers on the role of potassium channels and membrane potential in the creation of a favorable electrochemical gradient for Ca2+ entry. In addition, evidence is examined for the existence of various classes of potassium channels and the possible influence of regional variation in expression and experimental conditions.

Antisense-mediated inhibition of the plasma membrane Calcium-ATPase suppresses proliferation of MCF-7 cells

Alterations in Ca2+ signaling may contribute to tumorigenesis and the mechanism of action of some anticancer drugs. The plasma membrane calcium-ATPase (PMCA) is a crucial controller of intracellular Ca2+ signaling. Altered PMCA expression occurs in the mammary gland during lactation and in breast cancer cell lines. Despite this, the consequences of PMCA inhibition in breast cancer cell lines have not been investigated. In this work, we used Tet-off PMCA antisense-expressing MCF-7 cells to assess the effects of PMCA inhibition in a human breast cancer cell line. At a level of PMCA inhibition that did not completely prevent PMCA-mediated Ca2+ efflux and did not induce cell death, a dramatic inhibition of cellular proliferation was observed. Fluorescence-activated cell sorting analysis indicated that PMCA antisense involves changes in cell cycle kinetics but not cell cycle arrest. We concluded that modulation of PMCA has important effects in regulating the proliferation of human breast cancer MCF-7 cells.

Calcium transport and signaling in the mammary gland: Targets for breast cancer

The mammary gland is subjected to extensive calcium loads during lactation to support the requirements of milk calcium enrichment. Despite the indispensable nature of calcium homeostasis and signaling in regulating numerous biological functions, the mechanisms by which systemic calcium is transported into milk by the mammary gland are far from completely understood. Furthermore, the implications of calcium signaling in terms of reaulating proliferation, differentiation and apoptosis in the breast are currently uncertain. Deregulation of calcium homeostasis and signaling is associated with mammary gland pathophysiology and as such, calcium transporters, channels and binding proteins represent potential drug targets for the treatment of breast cancer. (c) 2005 Elsevier B.V. All rights reserved.

Synaptic activation of transient receptor potential channels by metabotropic glutamate receptors in the lateral amygdala

Classical mammalian transient receptor potential channels form non-selective cation channels that open in response to activation of phospholipase C-coupled metabotropic receptors, and are thought to play a key role in calcium homeostasis in non-excitable cells. Within the nervous system transient receptor potential channels are widely distributed but their physiological roles are not well understood. Here we show that in the rat lateral amygdala transient receptor potential channels mediate an excitatory synaptic response to glutamate. Activation of group l etabotropic glutamate receptors on pyramidal neurons in the lateral amygdala with either exogenous or synaptically released glutamate evokes an inward current at negative potentials with a current voltage relationship showing a region of negative slope and steep outward rectification. This current is blocked by inhibiting G protein function with GTP-beta-S, by inhibiting phospholipase C or by infusing transient receptor potential antibodies into lateral amygdala pyramidal neurons. Using RT-PCR and Western blotting we show that transient receptor potential 1, transient receptor potential 4 and transient receptor potential 5 are present in the lateral amygdala. Single cell PCR confirms the presence of transient receptor potential 1 and transient receptor potential 5 in pyramidal neurons and we show by co-immunoprecipitation that transient receptor potential 1 and transient receptor potential 5 co-assemble as a heteromultimers in the amygdala. These results show that in lateral amygdala pyramidal neurons synaptically released glutamate activates transient receptor potential channels, which we propose are likely to be heteromultimeric channels containing transient receptor potential 1 and transient receptor potential 5/transient receptor potential 4. (c) 2005 Published by Elsevier Ltd on behalf of IBRO.

Calcium response of KCl-excited populations of ventricular myocytes from the European sea bass (Dicentrarchus labrax): a promising approach to integrate cell-to-cell heterogeneity in studying the cellular basis of fish cardiac performance

Climate change challenges the capacity of fishes to thrive in their habitat. However, through phenotypic diversity, they demonstrate remarkable resilience to deteriorating conditions. In fish populations, inter-individual variation in a number of fitness-determining physiological traits, including cardiac performance, is classically observed. Information about the cellular bases of inter-individual variability in cardiac performance is scarce including the possible contribution of excitation-contraction (EC) coupling. This study aimed at providing insight into EC coupling-related Ca2+ response and thermal plasticity in the European sea bass (Dicentrarchus labrax). A cell population approach was used to lay the methodological basis for identifying the cellular determinants of cardiac performance. Fish were acclimated at 12 and 22 A degrees C and changes in intracellular calcium concentration ([Ca2+](i)) following KCl stimulation were measured using Fura-2, at 12 or 22 A degrees C-test. The increase in [Ca2+](i) resulted primarily from extracellular Ca2+ entry but sarcoplasmic reticulum stores were also shown to be involved. As previously reported in sea bass, a modest effect of adrenaline was observed. Moreover, although the response appeared relatively insensitive to an acute temperature change, a difference in Ca2+ response was observed between 12- and 22 A degrees C-acclimated fish. In particular, a greater increase in [Ca2+](i) at a high level of adrenaline was observed in 22 A degrees C-acclimated fish that may be related to an improved efficiency of adrenaline under these conditions. In conclusion, this method allows a rapid screening of cellular characteristics. It represents a promising tool to identify the cellular determinants of inter-individual variability in fishes' capacity for environmental adaptation.

A study of the electrical basis for the inhibition and excitation in autonomically innervated smooth muscle

The aim of this thesis was to investigate the electrical and mechanical responses to inhibitory non-adrenergic noncholinergic (NANC) nerve stimulation in the bovine retractor penis muscle (BRP) and compare them with those to an inhibitory extract made from this muscle. The extract may contain the NANC inhibitory transmitter of the BRP and possibly of other smooth muscles. Because of species differences in the electrical response to NANC nerves in the rat and rabbit anococcygeus the effects of the extract on these tissues was also investigated. Prior to the investigation of the extract, both the excitatory and inhibitory responses to field stimulation in the BRP, and the effects of passive membrane potential displacement were studied using conventional intra- or extracellular (sucrose gap) recording techniques. The majority of cells in the BRP were electrically quiescent independent of the resting tone. The most frequent (in approximately 25% of preparations) form of spontaneous activity, oscillations in membrane potential and tone, may represent a pacemaker activity. The BRP had cable properties; the time constant and space constant indicated a high membrane resistance. In the absence of tone, field stimulation of the BRP evoked excitatory junction potentials (ejps) in every cell impaled and contractions, graded with the strength, frequency and number of pulses; spikes were not observed. Guanethidine (1-3 x 10-5M) abolished the ejps and contractions, confirming their adrenergic origin. Noradrenaline added exogenously depolarised and contracted the muscle. These effects were blocked by the a-adrenoceptor antagonists, phentolamine and prazosin. However, phentolamine (2.5x 10-6M) inhibited the contraction without reducing the ejp significantly. These effects may be independent of adrenoceptor blockade or the ejp may be mediated by a substance other than noradrenaline (e.g. ATP) released from adrenergic nerves. Prazosin (1.4 x lO-6M) failed to block either the ejp or contraction, indicating the possible existence of two types of adrenoceptor in the BRP; one activated by neuronally-released and the other by exogenously-added noradrenaline. ATP, a contaminant in the extract, also depolarised and contracted the BRP. Physostigmine reduced whilst atropine enhanced the ejps and contractions without similarly affecting the response to exogenous noradrenaline. This confirmed the presence of a cholinergic inhibitory innervation acting on the excitatory adrenergic fibres (Klinge and Sjostrand, 1977). TEA (1 x lO-4M) enhanced the ejp and contraction. Higher concentrations (0.5 to 10 x 10-3M) depolarised, increased the tone and evoked electrical and mechanical oscillations but no spikes. The depolarisation and contraction to exogenous noradrenaline were not enhanced, indicating that TEA acts on the adrenergic nerves. Some post-synaptic effect to block K+ channels also seems likely. The relationship between ejp amplitude and membrane potential in the double sucrose gap was linear and indicated a reversal potential more positive than -30mV. Electrotonic pulse amplitude decreased during the ejp, indicating an increased membrane conductance. Ejps and contractions were reduced following the replacement of the NaCl of the Krebs solution with sodium glutamate. This may be due to the effects of glutamate itself (e.g. Ca2+ chelation) rather than reduction in the membrane Cl- gradient. Tone usually developed spontaneously and was accompanied by membrane depolarisation (from -53 to -45mV) which may open voltage-dependent channels, causing Ca2+ entry and/or its release from intracellular binding sites. Field stimulation produced inhibitory potentials (ijps) and relaxations graded with the strength and number of pulses but showing little frequency dependence. Rebound depolarisation and contraction often followed the ijp and relaxation. Tetrodotoxin (3 x IO-6M), but not adrenergic or cholinergic antagonists, abolished the ijp and relaxation, confirming their non-adrenergic non-cholinergic neurogenic nature. The extract, prepared and acid-activated as described by Gillespie, Hunter and Martin (1981), hyperpolarised and relaxed the BRP, as did sodium nitroprusside and adenosine triphosphate (ATP). Unlike the activated extract or sodium nitroprusside, desensitisation to ATP occurred rapidly and without any change in the inhibitory electrical or mechanical responses to field stimulation. The ijp and relaxation in the BRP were insensitive to apamin but abolished by oxyhaemoglobin (4-8 x 10-6M), as were the responses to extract and sodium nitroprusside. In TEA (10-2M), field stimulation evoked relaxations with no accompanying electrical change. The ijp may be unconnected with or additional to another mechanism producing relaxation. The relationship between membrane potential and ijp in the BRP was non-linear. Ijp amplitude was initially increased during membrane potential displacement from -45mV to approximately -60mV. Thereafter (-60 to -l03mV) the ijp was reduced. Ijps were abolished at -27 and -103mV; reversal was not observed. The hyperpolarisation to extract was also enhanced during passive displacement of the membrane potential to more negative values (-57mV). Membrane resistance increased during the ijp. The extract produced inconsistent changes in membrane resistance, possibly because of the presence of more than one active component. K+ withdrawal failed to enhance the ijp or hyperpolarisation to extract and 20mM K+ did not abolish the the ijp at membrane potentials exceeding EK (-49mV). Thus, the ijp or hyperpolarisation to extract are unlikely to be mediated by an increased K+ conductance. Reducing the Cl- abolished the hyperpolarisation to field stimulation and extract. This occurred more quickly than the anticipated reduction in the Cl- gradient and may be due to Ca2+ chelation by the anion substitute (glutamate or benzenesulphonate) or blockade of the resting conductance which is normally inactivated by the transmitter. Ouabain (1-5x 10-5M), which reduces both the Na+ and Cl- gradients, abolished the ijp, implicating either of these ions as the ionic species involved. In the rat and rabbit anococcygeus, field stimulation and extract each reduced guanethidine-induced tone. This was unaccompanied in the majority of cells in the rat by any significant electrical response. In the remaining cells, inhibition of the membrane potential oscillations occurred. The rabbit anococcygeus differed in that inhibition of the electrical oscillations was observed in every cell exhibiting this behaviour. However, the majority of cells in the rabbit were electrically quiescent and showed only small hyperpolarisations to field stimulation and no electrical response to extract. Apamin (1 x 10-7M) failed to block the electrical and mechanical response to field stimulation in the rabbit but did inhibit transiently that to extract. The latter effect may be due to the initial excitatory effects of apamin. The similarities between the electrical effects of the extract and those of inhibitory nerve stimulation in the BRP, rat and rabbit anococcygeus muscles are generally consistent with their being mediated by the same active component. Moreover, the ijp in the BRP shows properties which have not been reported in other non-adrenergic noncholinergically innervated smooth muscles.