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Calcineurin, the conserved protein phosphatase and target of immunosuppressants, is a critical mediator of Ca2+ signaling. Here, to discover calcineurin-regulated processes we examined an understudied isoform, CNAβ1. We show that unlike canonical cytosolic calcineurin, CNAβ1 localizes to the plasma membrane and Golgi due to palmitoylation of its divergent C-terminal tail, which is reversed by the ABHD17A depalmitoylase. Palmitoylation targets CNAβ1 to a distinct set of membrane-associated interactors including the phosphatidylinositol 4-kinase (PI4KA) complex containing EFR3B, PI4KA, TTC7B and FAM126A. Hydrogen-deuterium exchange reveals multiple calcineurin-PI4KA complex contacts, including a calcineurin-binding peptide motif in the disordered tail of FAM126A, which we establish as a calcineurin substrate. Calcineurin inhibitors decrease PI4P production during Gq-coupled GPCR signaling, suggesting that calcineurin dephosphorylates and promotes PI4KA complex activity. In sum, this work discovers a calcineurin-regulated signaling pathway which highlights the PI4KA complex as a regulatory target and reveals that dynamic palmitoylation confers unique localization, substrate specificity and regulation to CNAβ1.

Calcineurin, the conserved protein phosphatase and target of immunosuppressants, is a critical mediator of Ca2+ signaling. To discover novel calcineurin-regulated processes we examined an understudied isoform, CNAβ1. We show that unlike canonical cytosolic calcineurin, CNAβ1 localizes to the plasma membrane and Golgi due to palmitoylation of its divergent C-terminal tail, which is reversed by the ABHD17A depalmitoylase. Palmitoylation targets CNAβ1 to a distinct set of membrane-associated interactors including the phosphatidylinositol 4-kinase (PI4KA) complex containing EFR3B, PI4KA, TTC7B and FAM126A. Hydrogen-deuterium exchange reveals multiple calcineurin-PI4KA complex contacts, including a calcineurin-binding peptide motif in the disordered tail of FAM126A, which we establish as a calcineurin substrate. Calcineurin inhibitors decrease PI4P production during Gq-coupled GPCR signaling, suggesting that calcineurin dephosphorylates and promotes PI4KA complex activity. In sum, this work discovers a new calcineurin-regulated signaling pathway highlighting the PI4KA complex as a regulatory target and revealing that dynamic palmitoylation confers unique localization, substrate specificity and regulation to CNAβ1.

Reliable measurement of ligand binding to cell surface receptors is of outstanding biological and pharmacological importance. Resonance energy transfer–based assays are powerful approaches to achieve this goal, but the currently available methods are hindered by the necessity of receptor tagging, which can potentially alter ligand binding properties. Therefore, we developed a tag-free system to measure ligand‒receptor interactions in live cells using the Gaussia luciferase (GLuc) as a bioluminescence resonance energy transfer donor. GLuc is as small as the commonly applied Nanoluciferase but has enhanced brightness, and its proper substrate is the frequently used coelenterazine. In our assay, bystander bioluminescence resonance energy transfer is detected between a GLuc-based extracellular surface biosensor and fluorescent ligands bound to their unmodified receptors. The broad spectrum of applications includes equilibrium and kinetic ligand binding measurements for both labeled and competitive unlabeled ligands, and the assay can be utilized for different classes of plasma membrane receptors. Furthermore, the assay is suitable for high-throughput screening, as evidenced by the identification of novel α1 adrenergic receptor ligands. Our data demonstrate that GLuc-based biosensors provide a simple, sensitive, and cost-efficient platform for drug characterization and development.

β-Arrestins are key regulators and signal transducers of G protein–coupled receptors (GPCRs). The interaction between receptors and β-arrestins is generally believed to require both receptor activity and phosphorylation by GPCR kinases. In this study, we investigated whether β-arrestins are able to bind second messenger kinase–phosphorylated, but inactive receptors as well. Because heterologous phosphorylation is a common phenomenon among GPCRs, this mode of β-arrestin activation may represent a novel mechanism of signal transduction and receptor cross-talk. Here we demonstrate that activation of protein kinase C (PKC) by phorbol myristate acetate, Gq/11-coupled GPCR, or epidermal growth factor receptor stimulation promotes β-arrestin2 recruitment to unliganded AT1 angiotensin receptor (AT1R). We found that this interaction depends on the stability lock, a structure responsible for the sustained binding between GPCRs and β-arrestins, formed by phosphorylated serine–threonine clusters in the receptor's C terminus and two conserved phosphate-binding lysines in the β-arrestin2 N-domain. Using improved FlAsH-based serine-threonine clusters β-arrestin2 conformational biosensors, we also show that the stability lock not only stabilizes the receptor–β-arrestin interaction, but also governs the structural rearrangements within β-arrestins. Furthermore, we found that β-arrestin2 binds to PKC-phosphorylated AT1R in a distinct active conformation, which triggers MAPK recruitment and receptor internalization. Our results provide new insights into the activation of β-arrestins and reveal their novel role in receptor cross-talk.

The emerging role of mitochondria as signaling organelles raises the question of whether individual mitochondria can initiate heterotypic communication with neighboring organelles. Using fluorescent probes targeted to the endoplasmic-reticulum-mitochondrial interface, we demonstrate that single mitochondria generate oxidative bursts, rapid redox oscillations, confined to the nanoscale environment of the interorganellar contact sites. Using probes fused to inositol 1,4,5-trisphosphate receptors (IP(3)Rs), we show that Ca(2+) channels directly sense oxidative bursts and respond with Ca(2+) transients adjacent to active mitochondria. Application of specific mitochondrial stressors or apoptotic stimuli dramatically increases the frequency and amplitude of the oxidative bursts by enhancing transient permeability transition pore openings. Conversely, blocking interface Ca(2+) transport via elimination of IP(3)Rs or mitochondrial calcium uniporter channels suppresses ER-mitochondrial Ca(2+) feedback and cell death. Thus, single mitochondria initiate local retrograde signaling by miniature oxidative bursts and, upon metabolic or apoptotic stress, may also amplify signals to the rest of the cell.

Plasma membrane (PM) localization of Ras proteins is crucial for transmitting signals upon mitogen stimulation. Post-translational lipid modification of Ras proteins plays an important role in their recruitment to the PM. Electrostatic interactions between negatively charged PM phospholipids and basic amino acids found in K-Ras4B (K-Ras) but not in H-Ras are important for permanent K-Ras localization to the PM. Here, we investigated how acute depletion of negatively charged PM polyphosphoinositides (PPIns) from the PM alters the intracellular distribution and activity of K- and H-Ras proteins. PPIns depletion from the PM was achieved either by agonist-induced activation of phospholipase C β or with a rapamycin-inducible system in which various phosphatidylinositol phosphatases were recruited to the PM. Redistribution of the two Ras proteins was monitored with confocal microscopy or with a recently developed bioluminescence resonance energy transfer-based approach involving fusion of the Ras C-terminal targeting sequences or the entire Ras proteins to Venus fluorescent protein. We found that PM PPIns depletion caused rapid translocation of K-Ras but not H-Ras from the PM to the Golgi. PM depletion of either phosphatidylinositol 4-phosphate (PtdIns4P) or PtdIns(4,5)P2 but not PtdIns(3,4,5)P3 was sufficient to evoke K-Ras translocation. This effect was diminished by deltarasin, an inhibitor of the Ras–phosphodiesterase interaction, or by simultaneous depletion of the Golgi PtdIns4P. The PPIns depletion decreased incorporation of [3H]leucine in K-Ras–expressing cells, suggesting that Golgi-localized K-Ras is not as signaling-competent as its PM-bound form. We conclude that PPIns in the PM are important regulators of K-Ras–mediated signals.

Heterodimerization between angiotensin type 1A receptor (AT1R) and β2-adrenergic receptor (β2AR) has been shown to modulate G protein-mediated effects of these receptors. Activation of G protein-coupled receptors (GPCRs) leads to β-arrestin binding, desensitization, internalization and G protein-independent signaling of GPCRs. Our aim was to study the effect of heterodimerization on β-arrestin coupling. We found that β-arrestin binding of β2AR is affected by activation of AT1Rs. Costimulation with angiotensin II and isoproterenol markedly enhanced the interaction between β2AR and β-arrestins, by prolonging the lifespan of β2AR-induced β-arrestin2 clusters at the plasma membrane. While candesartan, a conventional AT1R antagonist, had no effect on the β-arrestin2 binding to β2AR, TRV120023, a β-arrestin biased agonist, enhanced the interaction. These findings reveal a new crosstalk mechanism between AT1R and β2AR, and suggest that enhanced β-arrestin2 binding to β2AR can contribute to the pharmacological effects of biased AT1R agonists.

Interleukin-2 (IL-2) and IL-15 play pivotal roles in T cell activation, apoptosis, and survival, and are implicated in leukemias and autoimmune diseases. Their heterotrimeric receptors share their β- and γ(c)-chains, but have distinct α-chains. Anti–IL-2Rα (daclizumab) therapy targeting cell surface-expressed receptor subunits to inhibit T cell proliferation has only brought limited success in adult T cell leukemia/lymphoma (ATL) and in multiple sclerosis. We asked whether IL-2R subunits could already preassemble and signal efficiently in the endoplasmic reticulum (ER) and the Golgi. A combination of daclizumab and anti–IL-2 efficiently blocked IL-2–induced proliferation of IL-2–dependent wild-type (WT) ATL cells but not cells transfected with IL-2, suggesting that in IL-2–producing cells signaling may already take place before receptors reach the cell surface. In the Golgi fraction isolated from IL-2–producing ATL cells, we detected by Western blot phosphorylated Jak1, Jak3, and a phosphotyrosine signal attributed to the γ(c)-chain, which occurred at much lower levels in the Golgi of WT ATL cells. We expressed EGFP- and mCherry-tagged receptor chains in HeLa cells to study their assembly along the secretory pathway. Confocal microscopy, Förster resonance energy transfer, and imaging fluorescence cross-correlation spectroscopy analysis revealed partial colocalization and molecular association of IL-2 (and IL-15) receptor chains in the ER/Golgi, which became more complete in the plasma membrane, further confirming our hypothesis. Our results define a paradigm of intracellular autocrine signaling and may explain resistance to antagonistic antibody therapies targeting receptors at the cell surface.

The ER-mitochondrial interface is central to calcium signaling, organellar dynamics, and lipid biosynthesis. The ER and mitochondrial membranes also host sources and targets of reactive oxygen species (ROS), but their local dynamics and relevance remained elusive since measurement and perturbation of ROS at the organellar interface has proven difficult. Employing drug-inducible synthetic ER-mitochondrial linkers, we overcame this problem and demonstrate that the ER-mitochondrial interface hosts a nanodomain of H2O2, which is induced by cytoplasmic [Ca(2+)] spikes and exerts a positive feedback on calcium oscillations. H2O2 nanodomains originate from the mitochondrial cristae, which are compressed upon calcium signal propagation to the mitochondria, likely due to Ca(2+)-induced K(+) and concomitant water influx to the matrix. Thus, ER-mitochondrial H2O2 nanodomains represent a component of inter-organelle communication, regulating calcium signaling and mitochondrial activities.

Deciphering many roles played by inositol lipids in signal transduction and membrane function demands experimental approaches that can detect their dynamic accumulation with subcellular accuracy and exquisite sensitivity. The former criterion is met by imaging of fluorescence biosensors in living cells, whereas the latter is facilitated by biochemical measurements from populations. Here, we introduce BRET-based biosensors able to detect rapid changes in inositol lipids in cell populations with both high sensitivity and subcellular resolution in a single, convenient assay. We demonstrate robust and sensitive measurements of PtdIns4P, PtdIns(4,5)P2 and PtdIns(3,4,5)P3 dynamics, as well as changes in cytoplasmic Ins(1,4,5)P3 levels. Measurements were made during either experimental activation of lipid degradation, or PI 3-kinase and phospholipase C mediated signal transduction. Our results reveal a previously unappreciated synthesis of PtdIns4P that accompanies moderate activation of phospholipase C signaling downstream of both EGF and muscarinic M3 receptor activation. This signaling-induced PtdIns4P synthesis relies on protein kinase C, and implicates a feedback mechanism in the control of inositol lipid metabolism during signal transduction.

The protein MSTO1 has been localized to mitochondria and linked to mitochondrial morphology, but its specific role has remained unclear. We identified a c.22G > A (p.Val8Met) mutation of MSTO1 in patients with minor physical abnormalities, myopathy, ataxia, and neurodevelopmental impairments. Lactate stress test and myopathological results suggest mitochondrial dysfunction. In patient fibroblasts, MSTO1 mRNA and protein abundance are decreased, mitochondria display fragmentation, aggregation, and decreased network continuity and fusion activity. These characteristics can be reversed by genetic rescue. Short‐term silencing of MSTO1 in HeLa cells reproduced the impairment of mitochondrial morphology and dynamics observed in the fibroblasts without damaging bioenergetics. At variance with a previous report, we find MSTO1 to be localized in the cytoplasmic area with limited colocalization with mitochondria. MSTO1 interacts with the fusion machinery as a soluble factor at the cytoplasm‐mitochondrial outer membrane interface. After plasma membrane permeabilization, MSTO1 is released from the cells. Thus, an MSTO1 loss‐of‐function mutation is associated with a human disorder showing mitochondrial involvement. MSTO1 likely has a physiologically relevant role in mitochondrial morphogenesis by supporting mitochondrial fusion.

One of the largest challenges in cell biology is to map the lipid composition of the membranes of various organelles and define the exact location of processes that control the synthesis and distribution of lipids between cellular compartments. The critical role of phosphoinositides, low-abundant lipids with rapid metabolism and exceptional regulatory importance in the control of almost all aspects of cellular functions created the need for tools to visualize their localizations and dynamics at the single cell level. However, there is also an increasing need for methods to determine the cellular distribution of other lipids regulatory or structural, such as diacylglycerol, phosphatidic acid, or other phospholipids and cholesterol. This review will summarize recent advances in this research field focusing on the means by which changes can be described in more quantitative terms.

Lenz-Majewski syndrome (LMS) is a rare disease characterized by complex craniofacial, dental, cutaneous, and limb abnormalities combined with intellectual disability. Mutations in thePTDSS1gene coding one of the phosphatidylserine (PS) synthase enzymes, PSS1, were described as causative in LMS patients. Such mutations render PSS1 insensitive to feedback inhibition by PS levels. Here we show that expression of mutant PSS1 enzymes decreased phosphatidylinositol 4-phosphate (PI4P) levels both in the Golgi and the plasma membrane (PM) by activating the Sac1 phosphatase and altered PI4P cycling at the PM. Conversely, inhibitors of PI4KA, the enzyme that makes PI4P in the PM, blocked PS synthesis and reduced PS levels by 50% in normal cells. However, mutant PSS1 enzymes alleviated the PI4P dependence of PS synthesis. Oxysterol-binding protein-related protein 8, which was recently identified as a PI4P-PS exchanger between the ER and PM, showed PI4P-dependent membrane association that was significantly decreased by expression of PSS1 mutant enzymes. Our studies reveal that PS synthesis is tightly coupled to PI4P-dependent PS transport from the ER. Consequently, PSS1 mutations not only affect cellular PS levels and distribution but also lead to a more complex imbalance in lipid homeostasis by disturbing PI4P metabolism.

Summary Proper control of the mitochondrial Ca2+ uniporter’s pore (MCU) is required to allow Ca2+-dependent activation of oxidative metabolism and to avoid mitochondrial Ca2+ overload and cell death. The MCU’s gatekeeping and cooperative activation is mediated by the Ca2+-sensing MICU1 protein, which has been proposed to form dimeric complexes anchored to the EMRE scaffold of MCU. We unexpectedly find that MICU1 suppresses inhibition of MCU by ruthenium red/Ru360, which bind to MCU’s DIME motif, the selectivity filter. This led us to recognize in MICU1’s sequence a putative DIME interacting domain (DID), which is required for both gatekeeping and cooperative activation of MCU and for cell survival. Thus, we propose that MICU1 has to interact with the D-ring formed by the DIME domains in MCU to control the uniporter.

Formation of intra- and intermolecular disulfide bonds is an essential step in the synthesis of secretory proteins. In eukaryotic cells, this process occurs in the endoplasmic reticulum (ER) and requires an oxidative environment with the action of several chaperones and folding catalysts. During protein folding, Ero1p oxidizes protein disulfide isomerase (PDI), which then directly catalyzes the formation of disulfide bonds in folding proteins. Recent cell-free studies suggest that the terminal electron acceptor in the pathway is molecular oxygen, with the resulting formation of hydrogen peroxide (H(2)O(2)). We report for the first time the measurement of ER H(2)O(2) level in live cells. By targeting a fluorescent protein-based H(2)O(2) sensor to various intracellular compartments, we show that the ER has the highest level of H(2)O(2), and this high concentration is well confined to the lumen of the organelle. Manipulation of the Ero1-Lalpha level--either by overexpression or by siRNA-mediated inhibition--caused parallel changes in luminal H(2)O(2), proving that the activity of Ero1-Lalpha results in H(2)O(2) formation in the ER. We also found that calcium mobilization from intracellular stores induces a decrease in ER H(2)O(2) level, suggesting a complex interplay between redox and calcium signaling in the mammalian ER.

Classical arabinogalactan proteins partially defined by type II O-Hyp-linked arabinogalactans (Hyp-AGs) are structural components of the plant extracellular matrix. Recently we described the structure of a small Hyp-AG putatively based on repetitive trigalactosyl subunits and suggested that AGs are less complex and varied than generally supposed. Here we describe three additional AGs with similar subunits. The Hyp-AGs were isolated from two different arabinogalactan protein fusion glycoproteins expressed in tobacco cells; that is, a 22-residue Hyp-AG and a 20-residue Hyp-AG, both isolated from interferon alpha2b-(Ser-Hyp)(20), and a 14-residue Hyp-AG isolated from (Ala-Hyp)(51)-green fluorescent protein. We used NMR spectroscopy to establish the molecular structure of these Hyp-AGs, which share common features: (i) a galactan main chain composed of two 1-->3 beta-linked trigalactosyl blocks linked by a beta-1-->6 bond; (ii) bifurcated side chains with Ara, Rha, GlcUA, and a Gal 6-linked to Gal-1 and Gal-2 of the main-chain trigalactosyl repeats; (iii) a common side chain structure composed of up to six residues, the largest consisting of an alpha-L-Araf-(1-->5)-alpha-L-Araf-(1-->3)-alpha-L-Araf-(1-->3- unit and an alpha-L-Rhap-(1-->4)-beta-D-GlcUAp-(1-->6)-unit, both linked to Gal. The conformational ensemble obtained by using nuclear Overhauser effect data in structure calculations revealed a galactan main chain with a reverse turn involving the beta-1-->6 link between the trigalactosyl blocks, yielding a moderately compact structure stabilized by H-bonds.

Summary The ER-mitochondrial junction provides a local calcium signaling domain that is critical for both matching energy production with demand and the control of apoptosis. Here, we visualize ER-mitochondrial contact sites and monitor the localized [Ca 2+ ] changes ([Ca 2+ ] ER-mt ) using drug-inducible fluorescent interorganelle linkers. We show that all mitochondria have contacts with the ER, but plasma membrane (PM)-mitochondrial contacts are less frequent because of interleaving ER stacks in both RBL-2H3 and H9c2 cells. Single mitochondria display discrete patches of ER contacts and show heterogeneity in the ER-mitochondrial Ca 2+ transfer. Pericam-tagged linkers revealed IP 3 -induced [Ca 2+ ] ER-mt signals that exceeded 9 μM and endured buffering bulk cytoplasmic [Ca 2+ ] increases. Altering linker length to modify the space available for the Ca 2+ transfer machinery had a biphasic effect on [Ca 2+ ] ER-mt signals. These studies provide direct evidence for the existence of high-Ca 2+ microdomains between the ER and mitochondria and suggest an optimal gap width for efficient Ca 2+ transfer.

Recent studies identified two main components of store-operated calcium entry (SOCE): the endoplasmic reticulum-localized Ca2+ sensor protein, STIM1, and the plasma membrane (PM)-localized Ca2+ channel, Orai1/CRACM1. In the present study, we investigated the phosphoinositide dependence of Orai1 channel activation in the PM and of STIM1 movements from the tubular to PM-adjacent endoplasmic reticulum regions during Ca2+ store depletion. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) levels were changed either with agonist stimulation or by chemically induced recruitment of a phosphoinositide 5-phosphatase domain to the PM, whereas PtdIns4P levels were decreased by inhibition or down-regulation of phosphatidylinositol 4-kinases (PI4Ks). Agonist-induced phospholipase C activation and PI4K inhibition, but not isolated PtdIns(4,5)P(2) depletion, substantially reduced endogenous or STIM1/Orai1-mediated SOCE without preventing STIM1 movements toward the PM upon Ca2+ store depletion. Patch clamp analysis of cells overexpressing STIM1 and Orai1 proteins confirmed that phospholipase C activation or PI4K inhibition greatly reduced I(CRAC) currents. These results suggest an inositide requirement of Orai1 activation but not STIM1 movements and indicate that PtdIns4P rather than PtdIns(4,5)P2 is a likely determinant of Orai1 channel activity.

Intracellular signaling systems of G protein-coupled receptors are well established, but their role in paracrine regulation of adjacent cells is generally considered as a tissue-specific mechanism. We have shown previously that AT(1) receptor (AT(1)R) stimulation leads to diacylglycerol lipase-mediated transactivation of co-expressed CB(1)Rs in Chinese hamster ovary cells. In the present study we detected a paracrine effect of the endocannabinoid release from Chinese hamster ovary, COS7, and HEK293 cells during the stimulation of AT(1) angiotensin receptors by determining CB(1) cannabinoid receptor activity with bioluminescence resonance energy transfer-based sensors of G protein activation expressed in separate cells. The angiotensin II-induced, paracrine activation of CB(1) receptors was visualized by detecting translocation of green fluorescent protein-tagged beta-arrestin2. Mass spectrometry analyses have demonstrated angiotensin II-induced stimulation of 2-arachidonoylglycerol production, whereas no increase of anandamide levels was observed. Stimulation of G(q/11)-coupled M(1), M(3), M(5) muscarinic, V(1) vasopressin, alpha(1a) adrenergic, B(2) bradykinin receptors, but not G(i/o)-coupled M(2) and M(4) muscarinic receptors, also led to paracrine transactivation of CB(1) receptors. These data suggest that, in addition to their retrograde neurotransmitter role, endocannabinoids have much broader paracrine mediator functions during activation of G(q/11)-coupled receptors.

Rapid changes in cytosolic Ca(2+) concentrations [Ca(2+)](i) are the most commonly used signals in biology to regulate a whole host of cellular functions including contraction, secretion and gene activation. A widely utilized form of Ca(2+) influx is termed store-operated Ca(2+) entry (SOCE) owing to its control by the Ca(2+) content of the endoplasmic reticulum (ER). The underlying molecular mechanism of SOCE has eluded identification until recently when two groups of proteins, the ER Ca(2+) sensors stromal interaction molecule (STIM)1 and STIM2 and the plasma-membrane channels Orai1, Orai2 and Orai3, have been identified. These landmark discoveries have enabled impressive progress in clarifying how these proteins work in concert and what developmental and cellular processes require their participation most. As we begin to better understand the biology of the STIM and Orai proteins, the attention to the pharmacological tools to influence their functions quickly follow suit. Here, we briefly summarize recent developments in this exciting area of Ca(2+) signaling.

Inositol lipids and calcium signaling has been inseparable twins during the 1980s when the molecular details of phospholipase C-mediated generation of inositol 1,4,5-trisphosphate (InsP3) and its Ca2+ mobilizing action were discovered. Since then, both the Ca2+- and inositol lipid signaling fields have hugely expanded and the tools allowing dissection of the finest details of their molecular organization also followed closely. Although phosphoinositides regulate many cell functions unrelated to Ca2+ signaling there are still many open questions even in the Ca2+ field that would benefit from single cell monitoring of PtdIns(4,5)P2 or InsP3 changes during agonist stimulation. This chapter is designed to provide practical guidance as well as some theoretical background on measurements of phosphoinositides in live cells using protein domain-GFP chimeras that could be also useful for people working on calcium signaling.

Hepatocyte growth factor (HGF) is important for cell proliferation, differentiation, and related activities. HGF acts through its receptor c-Met, which activates downstream signaling pathways. HGF binds to c-Met at the plasma membrane, where it is generally believed that c-Met signaling is initiated. Here we report that c-Met rapidly translocates to the nucleus upon stimulation with HGF. Ca(2+) signals that are induced by HGF result from phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol 1,4,5-trisphosphate formation within the nucleus rather than within the cytoplasm. Translocation of c-Met to the nucleus depends upon the adaptor protein Gab1 and importin beta1, and formation of Ca(2+) signals in turn depends upon this translocation. HGF may exert its particular effects on cells because it bypasses signaling pathways in the cytoplasm to directly activate signaling pathways in the nucleus.

Type III phosphatidylinositol (PtdIns) 4-kinases (PI4Ks) have been previously shown to support plasma membrane phosphoinositide synthesis during phospholipase C activation and Ca2+signaling. Here, we use biochemical and imaging tools to monitor phosphoinositide changes in the plasma membrane in combination with pharmacological and genetic approaches to determine which of the type III PI4Ks (α or β) is responsible for supplying phosphoinositides during agonist-induced Ca2+signaling. Using inhibitors that discriminate between the α- and β-isoforms of type III PI4Ks, PI4KIIIα was found indispensable for the production of phosphatidylinositol 4-phosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], and Ca2+signaling in angiotensin II (AngII)-stimulated cells. Down-regulation of either the type II or type III PI4K enzymes by small interfering RNA (siRNA) had small but significant effects on basal PtdIns4P and PtdIns(4,5)P2levels in32P-labeled cells, but only PI4KIIIα down-regulation caused a slight impairment of PtdIns4P and PtdIns(4,5)P2resynthesis in AngII-stimulated cells. None of the PI4K siRNA treatments had a measurable effect on AngII-induced Ca2+signaling. These results indicate that a small fraction of the cellular PI4K activity is sufficient to maintain plasma membrane phosphoinositide pools, and they demonstrate the value of the pharmacological approach in revealing the pivotal role of PI4KIIIα enzyme in maintaining plasma membrane phosphoinositides.

Cell-cell communication through connexin43 (Cx43)-based gap junction channels is rapidly inhibited upon activation of various G protein coupled receptors; however, the mechanism is unknown. We show that Cx43-based cell-cell communication is inhibited by depletion of phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5] P-2) from the plasma membrane. Knockdown of phospholipase C beta 3 (PLC beta 3) inhibits PtdIns(4,5) P2 hydrolysis and keeps Cx43 channels open after receptor activation. Using a translocatable 5-phosphatase, we show that PtdIns(4,5) P2 depletion is sufficient to close Cx43 channels. When PtdIns(4,5) P2 is overproduced by PtdIns(4)P 5-kinase, Cx43 channel closure is impaired. We. nd that the Cx43 binding partner zona occludens 1 (ZO-1) interacts with PLC beta 3 via its third PDZ domain. ZO-1 is essential for PtdIns(4,5) P-2-hydrolyzing receptors to inhibit cell-cell communication, but not for receptor PLC coupling. Our results show that PtdIns(4,5) P-2 is a key regulator of Cx43 channel function, with no role for other second messengers, and suggest that ZO-1 assembles PLC beta 3 and Cx43 into a signaling complex to allow regulation of cell-cell communication by localized changes in PtdIns(4,5) P2.

There is hardly a membrane-associated molecular event that is not regulated by phosphoinositides, a minor but critically important class of phospholipids of cellular membranes. The rapid formation, elimination, and conversion of these lipids in specific membrane compartments are ensured by a wealthy number of inositol lipid kinases and phosphatases with unique localization and regulatory properties. The existence of multiple inositol lipid pools have been indicated by metabolic labeling studies, but the level of functional compartmentalization revealed by the identification of numerous protein effectors acted upon by phosphoinositides could not have been foreseen. The changing perception of inositides from just serving as lipid precursors of second messengers to becoming highly dynamic local membrane-bound regulators poses new challenges concerning the detection of their rapid localized changes. Moreover, it is increasingly evident that manipulation of lipids in highly defined compartments would be a highly superior approach to soaking the cells with a particular phosphoinositide when studying the local regulation of the lipid on any effectors. In this review, we will summarize our efforts to improve our tools in studying phosphoinositide dynamics and discuss our views on the values of these methods compared to other options currently used or being explored.

Voltage-dependent anion channel (VDAC) proteins are major components of the outer mitochondrial membrane. VDAC has three isoforms with70% sequence similarity and redundant roles in metabolite and ion transport. However, only Vdac2(-/-) (V2(-/-)) mice are embryonic lethal, indicating a unique and fundamental function of VDAC2 (V2). Recently, a specific V2 requirement was demonstrated for mitochondrial Bak import and truncated Bid (tBid)-induced apoptosis. To determine the relevant domain(s) of V2 involved, VDAC1 (V1) and V2 chimeric constructs were created and used to rescue V2(-/-) fibroblasts. Surprisingly, the commonly cited V2-specific N-terminal extension and cysteines were found to be dispensable for Bak import and high tBid sensitivity. In gain-of-function studies, V2 (123-179) was the minimal sequence sufficient to render V1 competent to support Bak insertion. Furthermore, in loss-of-function experiments, T168 and D170 were identified as critical residues. These motifs are conserved in zebrafish V2 (zfV2) that also rescued V2-deficient fibroblasts. Because high-resolution structures of zfV2 and mammalian V1 have become available, we could superimpose these structures and recognized that the critical V2-specific residues help to create a distinctive open "pocket" on the cytoplasmic surface that could facilitate Bak recruitment.

Reminiscent of bacterial quorum sensing, mammalian mitochondria participate in inter-organelle communication. However, physical structures that enhance or enable interactions between mitochondria have not been defined. Here we report that adjacent mitochondria exhibit coordination of inner mitochondrial membrane cristae at inter-mitochondrial junctions (IMJs). These electron-dense structures are conserved across species, resistant to genetic disruption of cristae organization, dynamically modulated by mitochondrial bioenergetics, independent of known inter-mitochondrial tethering proteins mitofusins and rapidly induced by the stable rapprochement of organelles via inducible synthetic linker technology. At the associated junctions, the cristae of adjacent mitochondria form parallel arrays perpendicular to the IMJ, consistent with a role in electrochemical coupling. These IMJs and associated cristae arrays may provide the structural basis to enhance the propagation of intracellular bioenergetic and apoptotic waves through mitochondrial networks within cells., Mammalian mitochondria are capable of inter-organelle communication, but connections between mitochondria have not been defined. Here, Picard et al. report the presence of inter-mitochondrial junctions, electron-dense regions with coordinated inner membrane cristae that do not depend on mitofusins for their formation.

Rapamycin (rapa)-induced heterodimerization of the FRB domain of the mammalian target of rapa and FKBP12 was used to translocate a phosphoinositide 5-phosphatase (5-ptase) enzyme to the plasma membrane (PM) to evoke rapid changes in phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) levels. Rapa-induced PM recruitment of a truncated type IV 5-ptase containing only the 5-ptase domain fused to FKBP12 rapidly decreased PM PtdIns(4,5)P2 as monitored by the PLCδ1PH-GFP fusion construct. This decrease was paralleled by rapid termination of the ATP-induced Ca2+ signal and the prompt inactivation of menthol-activated transient receptor potential melastatin 8 (TRPM8) channels. Depletion of PM PtdIns(4,5)P2 was associated with a complete blockade of transferrin uptake and inhibition of epidermal growth factor internalization. None of these changes were observed upon rapa-induced translocation of an mRFP-FKBP12 fusion protein that was used as a control. These data demonstrate that rapid inducible depletion of PM PtdIns(4,5)P2 is a powerful tool to study the multiple regulatory roles of this phospholipid and to study differential sensitivities of various processes to PtdIns(4,5)P2 depletion.

The mitochondrial Ca(2+) uniporter has low affinity for Ca(2+), therefore it has been assumed that submicromolar Ca(2+) signals cannot induce mitochondrial Ca(2+) uptake. The close apposition of the plasma membrane or the endoplamic reticulum (ER) to the mitochondria and the limited Ca(2+) diffusion in the cytoplasm result in the formation of perimitochondrial high-Ca(2+) microdomains (HCMDs) capable of activating mitochondrial Ca(2+) uptake. The possibility of mitochondrial Ca(2+) uptake at low submicromolar [Ca(2+)](c) has not yet been generally accepted. Earlier we found in permeabilized glomerulosa, luteal and pancreatic beta cells that [Ca(2+)](m) increased when [Ca(2+)](c) was raised from 60 nM to less than 200 nM. Here we report data obtained from H295R (adrenocortical) cells transfected with ER-targeted GFP. Cytoplasmic Ca(2+) response to angiotensin II was different in mitochondrion-rich and mitochondrion-free domains. The mitochondrial Ca(2+) response to angiotensin II correlated with GFP fluorescence indicating the vicinity of ER. When the cells were exposed to K(+) (inducing Ca(2+) influx), no correlation was found between the mitochondrial Ca(2+) signal and the vicinity of the plasma membrane or the ER. The results presented here provide evidence that mitochondrial Ca(2+) uptake may occur both with and without the formation of HCMDs within the same cell.

The role of mitochondria in cell metabolism and survival is controlled by calcium signals that are commonly transmitted at the close associations between mitochondria and endoplasmic reticulum (ER). However, the physical linkage of the ER-mitochondria interface and its relevance for cell function remains elusive. We show by electron tomography that ER and mitochondria are adjoined by tethers that are approximately 10 nm at the smooth ER and approximately 25 nm at the rough ER. Limited proteolysis separates ER from mitochondria, whereas expression of a short "synthetic linker" (5 nm) leads to tightening of the associations. Although normal connections are necessary and sufficient for proper propagation of ER-derived calcium signals to the mitochondria, tightened connections, synthetic or naturally observed under apoptosis-inducing conditions, make mitochondria prone to Ca2+ overloading and ensuing permeability transition. These results reveal an unexpected dependence of cell function and survival on the maintenance of proper spacing between the ER and mitochondria.

Several pleckstrin-homology (PH) domains with the ability to bind phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3, PIP3] were expressed as green fluorescent protein (GFP) fusion proteins to determine their effects on various cellular responses known to be activated by PIP3. These proteins comprised the PH domains of Akt, ARNO, Btk or GRP1, and were found to show growth-factor-stimulated and wortmannin-sensitive translocation from the cytosol to the plasma membrane in several cell types, indicating their ability to recognize PIP3. Remarkably, although overexpressed Akt-PH–GFP and Btk-PH–GFP were quite potent in antagonizing the PIP3-mediated activation of the Akt protein kinase, such inhibition was not observed with the other PH domains. By contrast, expression of the PH domains of GRP1 and ARNO, but not of Akt or Btk, inhibited the attachment and spreading of freshly seeded cells to culture dishes. Activation of PLCγ by epidermal growth factor (EGF) was attenuated by the PH domains of GRP1, ARNO and Akt, but was significantly enhanced by the Btk PH domain. By following the kinetics of expression of the various GFP-fused PH domains for several days, only the PH domain of Akt showed a lipid-binding-dependent self-elimination, consistent with its interference with the anti-apoptotic Akt signaling pathway. Mutations of selective residues that do not directly participate in PIP3 binding in the GRP1-PH and Akt-PH domain were able to reduce the dominant-negative effects of these constructs yet retain their lipid binding. These data suggest that interaction with and sequestration of PIP3 may not be the sole mechanism by which PH domains interfere with cellular responses and that their interaction with other membrane components, most probably with proteins, allows a more specific participation in the regulation of specific signaling pathways.

Cytosolic Ca2+ ([Ca2+]c) signals triggered by many agonists are established through the inositol 1,4,5-trisphosphate (IP3) messenger pathway. This pathway is believed to use Ca2+-dependent local interactions among IP3 receptors (IP3R) and other Ca2+ channels leading to coordinated Ca2+ release from the endoplasmic reticulum throughout the cell and coupling Ca2+ entry and mitochondrial Ca2+ uptake to Ca2+ release. To evaluate the role of IP3 in the local control mechanisms that support the propagation of [Ca2+]c waves, store-operated Ca2+ entry, and mitochondrial Ca2+ uptake, we used two IP3-binding proteins (IP3BP): 1) the PH domain of the phospholipase C-like protein, p130 (p130PH); and 2) the ligand-binding domain of the human type-I IP3R (IP3R224-605). As expected, p130PH-GFP and GFP-IP3R224-605 behave as effective mobile cytosolic IP3 buffers. In COS-7 cells, the expression of IP3BPs had no effect on store-operated Ca2+ entry. However, the IP3-linked [Ca2+]c signal appeared as a regenerative wave and IP3BPs slowed down the wave propagation. Most importantly, IP3BPs largely inhibited the mitochondrial [Ca2+] signal and decreased the relationship between the [Ca2+]c and mitochondrial [Ca2+] signals, indicating disconnection of the mitochondria from the [Ca2+]c signal. These data suggest that IP3 elevations are important to regulate the local interactions among IP3Rs during propagation of [Ca2+]c waves and that the IP3-dependent synchronization of Ca2+ release events is crucial for the coupling between Ca2+ release and mitochondrial Ca2+ uptake.

The PH domains of OSBP and FAPP1 fused to GFP were used to monitor PI(4)P distribution in COS-7 cells during manipulations of PI 4-kinase (PI4K) activities. Both domains were associated with the Golgi and small cytoplasmic vesicles, and a small fraction of OSBP-PH was found at the plasma membrane (PM). Inhibition of type-III PI4Ks with 10 μM wortmannin (Wm) significantly reduced but did not abolish Golgi localization of either PH domains. Downregulation of PI4KIIα or PI4KIIIβ by siRNA reduced the localization of the PH domains to the Golgi and in the former case any remaining Golgi localization was eliminated by Wm treatment. PLC activation by Ca2+ionophores dissociated the domains from all membranes, but after Ca2+chelation, they rapidly reassociated with the Golgi, the intracellular vesicles and with the PM. PM association of the domains was significantly higher after the Ca2+transient and was abolished by Wm pretreatment. PM relocalization was not affected by down-regulation of PI4KIIIβ or -IIα, but was inhibited by down-regulation of PI4KIIIα, or by 10 μM PAO, which also inhibits PI4KIIIα. Our data suggest that these PH domains detect PI(4)P formation in extra-Golgi compartments under dynamic conditions and that various PI4Ks regulate PI(4)P synthesis in distinct cellular compartments.

The relationship between the ability of isolated pleckstrin homology (PH) domains to bind inositol lipids or soluble inositol phosphates in vitro and to localize to cellular membranes in live cells was examined by comparing the PH domains of phospholipase Cδ1 (PLCδ1) and the recently cloned PLC-like protein p130 fused to the green fluorescent protein (GFP). The prominent membrane localization of PLCδ1PH-GFP was paralleled with high affinity binding to inositol 1,4,5-trisphosphate (InsP3) as well as to phosphatidylinositol 4,5-bisphosphate-containing lipid vesicles or nitrocellulose membrane strips. In contrast, no membrane localization was observed with p130PH-GFP despite its InsP3 and phosphatidylinositol 4,5-bisphosphate-binding properties being comparable with those of PLCδ1PH-GFP. The N-terminal ligand binding domain of the type I InsP3 receptor also failed to localize to the plasma membrane despite its 5-fold higher affinity to InsP3 than the PH domains. By using a chimeric approach and cassette mutagenesis, the C-terminal α-helix and the short loop between the β6–β7 sheets of the PLCδ1PH domain, in addition to its InsP3-binding region, were identified as critical components for membrane localization in intact cells. These data indicate that binding to the inositol phosphate head group is necessary but may not be sufficient for membrane localization of the PLCδ1PH-GFP fusion protein, and motifs located within the C-terminal half of the PH domain provide auxiliary contacts with additional membrane components.

Mitochondria form junctions with the sarco/endoplasmic reticulum (SR/ER), which support many cell functions locally including calcium signaling, organellar dynamics and lipid biosynthesis. The SR/ER and mitochondrial membranes also host sources and targets of reactive oxygen species (ROS) but their local dynamics and relevance remained elusive because measurement and perturbation of ROS at the organellar interface has proven difficult. We will describe fluorescence based strategies to monitor the ROS fluctuations at the SR/ER-mitochondria interface and present evidence for spatially confined ROS fluctuations in association with calcium signal propagation from ER to the mitochondria and transient mitochondrial depolarizations referred as flickers. We will also offer some clues to the functional significance of the ER-mitochondrial nanodomains in regulating calcium signaling and mitochondrial activities.

A range of environmental agents causes tissue injury that has been attributed to reactive oxygen species (ROS). Altered ROS production is commonly originated from the mitochondria and is associated with dysregulation of calcium (Ca2+) homeostasis and mitochondrial apoptosis. However, the causative pathways remain largely unknown because the fundamental roles of mitochondria in cell survival, cell signaling and dynamics are commonly mediated through local communication between mitochondria and other organelles, which have been difficult to directly monitor. Our aim is to develop fluorescent protein-based tools allowing sensitive and specific measurements of ROS and [Ca2+] at the mitochondria-endoplasmic reticulum (ER) interface. Applying these novel tools, we have studied the effects of environmental stress caused by arsenicon local mitochondria-ER communication and its impact for cell signaling, and cell physiology in primary mouse hepatocytes. Either acute addition (30 µM) or prolonged, overnight exposure (3 µM) to sodium arsenite has induced an increase in ROS. Upon prolonged arsenic exposure, these alterations were accompanied by a significant decrease in the cytoplasmic calcium signal to an IP3-linked agonist: a decrease was observed in the fraction of responsive cells, in the amplitude of the response as well as in the sustained phase. Regarding the acute effect of arsenic, we observed a progressive increase of the basal cytoplasmic [Ca2+] with a decrease in the amplitude of the IP3-linked calcium signal. Neither acute nor prolonged arsenic exposure impaired mitochondrial calcium uptake. Nevertheless, we documented increased mitochondrial calcium content in the prolonged arsenic exposure group. In conclusion, the results suggest that an initial component of the arsenic-induced hepatocyte injury is increased ROS, which is associated with impaired local mitochondria-ER calcium communication.

G Protein Coupled Receptors (GPCR) can form dimers or higher ordered oligomers, the process of which can remarkably influence the physiological and pharmacological function of these receptors. Quantitative Bioluminescence Resonance Energy Transfer (qBRET) measurements are the gold standards to prove the direct physical interaction between the protomers of presumed GPCR dimers. For the correct interpretation of these experiments, the expression of the energy donor Renilla luciferase labeled receptor has to be maintained constant, which is hard to achieve in expression systems. To analyze the effects of non-constant donor expression on qBRET curves, we performed Monte Carlo simulations. Our results show that the decrease of donor expression can lead to saturation qBRET curves even if the interaction between donor and acceptor labeled receptors is non-specific leading to false interpretation of the dimerization state. We suggest here a new approach to the analysis of qBRET data, when the BRET ratio is plotted as a function of the acceptor labeled receptor expression at various donor receptor expression levels. With this method, we were able to distinguish between dimerization and non-specific interaction when the results of classical qBRET experiments were ambiguous. The simulation results were confirmed experimentally using rapamycin inducible heterodimerization system. We used this new method to investigate the dimerization of various GPCRs, and our data have confirmed the homodimerization of V2 vasopressin and CaSR calcium sensing receptors, whereas our data argue against the heterodimerization of these receptors with other studied GPCRs, including type I and II angiotensin, β2 adrenergic and CB1 cannabinoid receptors.

Cells deficient in mitochondrial fusion have been shown to have defects linked to the exchange of inner membrane and matrix components. Because outer-mitochondrial membrane (OMM) constituents insert directly from the cytoplasm, a role for fusion in their intermitochondrial transfer was unanticipated. Here, we show that fibroblasts lacking the GTPases responsible for OMM fusion, mitofusins 1 and 2 (MFN1 and MFN2), display more heterogeneous distribution of OMM proteins. Proteins with different modes of OMM association display varying degrees of heterogeneity in Mfn1/2(-/-) cells and different kinetics of transfer during fusion in fusion-competent cells. Proapoptotic Bak exhibits marked heterogeneity, which is normalized upon expression of MFN2. Bak is critical for Bid-induced OMM permeabilization and cytochrome c release, and Mfn1/2(-/-) cells show dysregulation of Bid-dependent apoptotic signaling. Bid sensitivity of Bak-deficient mitochondria is regained upon fusion with Bak-containing mitochondria. Thus, OMM protein distribution depends on mitochondrial fusion and is a locus of apoptotic dysfunction in conditions of fusion deficiency.

Phosphatidylinositol 4-kinases (PI4K) catalyze the first step in the synthesis of phosphatidylinositol 4,5-bisphosphate, an important lipid regulator of several cellular functions. Here we show that the Ca(2+)-binding protein, neuronal calcium sensor-1 (NCS-1), can physically associate with the type III PI4Kbeta with functional consequences affecting the kinase. Recombinant PI4Kbeta, but not its glutathione S-transferase-fused form, showed enhanced PI kinase activity when incubated with recombinant NCS-1, but only if the latter was myristoylated. Similarly, in vitro translated NCS-1, but not its myristoylation-defective mutant, was found associated with recombinant- or in vitro translated PI4Kbeta in PI4Kbeta-immunoprecipitates. When expressed in COS-7 cells, PI4Kbeta and NCS-1 formed a complex that could be immunoprecipitated with antibodies against either proteins, and PI 4-kinase activity was present in anti-NCS-1 immunoprecipitates. Expressed NCS-1-YFP showed co-localization with endogenous PI4Kbeta primarily in the Golgi, but it was also present in the walls of numerous large perinuclear vesicles. Co-expression of a catalytically inactive PI4Kbeta inhibited the development of this vesicular phenotype. Transfection of PI4Kbeta and NCS-1 had no effect on basal PIP synthesis in permeabilized COS-7 cells, but it increased the wortmannin-sensitive [(32)P]phosphate incorporation into phosphatidylinositol 4-phosphate during Ca(2+)-induced phospholipase C activation. These results together indicate that NCS-1 is able to interact with PI4Kbeta also in mammalian cells and may play a role in the regulation of this enzyme in specific cellular compartments affecting vesicular trafficking.

Agonist-induced intracellular Ca(2+) signals following phospholipase C (PLC) activation display a variety of patterns, including transient, sustained, and oscillatory behavior. These Ca(2+) changes have been well characterized, but detailed kinetic analyses of PLC activation in single living cells is lacking, due to the absence of suitable indicators for use in vivo. Recently, green fluorescent protein-tagged pleckstrin homology domains have been employed to monitor PLC activation in single cells, based on (confocal) imaging of their fluorescence translocation from the membrane to the cytosol that occurs upon hydrolysis of phosphatidylinositol bisphosphate. Here we describe fluorescence resonance energy transfer between pleckstrin homology domains of PLCdelta1 tagged with cyan and yellow fluorescent proteins as a sensitive readout of phosphatidylinositol bisphosphate metabolism for use both in cell populations and in single cells. Fluorescence resonance energy transfer requires significantly less excitation intensity, enabling prolonged and fast data acquisition without the cell damage that limits confocal experiments. It also allows experiments on motile or extremely flat cells, and can be scaled to record from cell populations as well as single neurites. Characterization of responses to various agonists by this method reveals that stimuli that elicit very similar Ca(2+) mobilization responses can exhibit widely different kinetics of PLC activation, and that the latter appears to follow receptor activation more faithfully than the cytosolic Ca(2+) transient.

Phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2) pools that bind pleckstrin homology (PH) domains were visualized by cellular expression of a phospholipase C (PLC)δ PH domain–green fluorescent protein fusion construct and analysis of confocal images in living cells. Plasma membrane localization of the fluorescent probe required the presence of three basic residues within the PLCδ PH domain known to form critical contacts with PtdIns(4,5)P2. Activation of endogenous PLCs by ionophores or by receptor stimulation produced rapid redistribution of the fluorescent signal from the membrane to cytosol, which was reversed after Ca2+ chelation. In both ionomycin- and agonist-stimulated cells, fluorescent probe distribution closely correlated with changes in absolute mass of PtdIns(4,5)P2. Inhibition of PtdIns(4,5)P2 synthesis by quercetin or phenylarsine oxide prevented the relocalization of the fluorescent probe to the membranes after Ca2+ chelation in ionomycin-treated cells or during agonist stimulation. In contrast, the synthesis of the PtdIns(4,5)P2 imaged by the PH domain was not sensitive to concentrations of wortmannin that had been found inhibitory of the synthesis of myo-[3H]inositol– labeled PtdIns(4,5)P2. Identification and dynamic imaging of phosphoinositides that interact with PH domains will further our understanding of the regulation of such proteins by inositol phospholipids.

We have examined the structure and function of Ca2+ channels in excitable endocrine cell types, in rat adrenal glomerulosa cells and in two insulin producing cell types, the rat pancreatic beta cell and the INS-1 cell line. In previous studies on glomerulosa cells, we observed low (T-type) and high threshold (L-type) voltage dependent Ca2+ currents in addition to a K+ induced inward rectifying Ca2+ current (Igl). beta cells are known to exhibit T-, L- and N-type currents. We have now found that INS-1 cells also show low threshold (T-type) and high threshold Ca2+ currents. The latter was further resolved by organic inhibitors into L-type and P/Q-type currents and no Igl was detected. The expression of the pore-forming alpha 1 subunit of voltage dependent Ca2+ channels was studied by means of reverse transcription-polymerase chain reaction (RT-PCR), followed by restriction enzyme mapping and/or sequencing. Both in glomerulosa and pancreatic beta cells, the neuroendocrine (D) class of the alpha 1 subunit, known to be responsible for L-type current, represents the majority of the PCR product. Comparable amounts of the neuroendocrine (D) and the neuronal A-type alpha 1 subunits dominate the message in INS-1 cells. Different characteristics of Ca2+ currents in these cell types is discussed in view of the channel repertoire.