The N-terminal homology (ENTH) domain of Epsin 1 is a sensitive reporter of physiological PI(4,5)P2 dynamics.

Phospholipase Cß (PLCß)-induced depletion of phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2) transduces a plethora of signals into cellular responses. Importance and diversity of PI(4,5)P2-dependent processes led to strong need for biosensors of physiological PI(4,5)P2 dynamics applicable in live-cell experiments. Membrane PI(4,5)P2 can be monitored with fluorescently-labelled phosphoinositide (PI) binding domains that associate to the membrane depending on PI(4,5)P2 levels. The pleckstrin homology domain of PLCδ1 (PLCδ1-PH) and the C-terminus of tubby protein (tubbyCT) are two such sensors widely used to study PI(4,5)P2 signaling. However, certain limitations apply to both: PLCδ1-PH binds cytoplasmic inositol-1,4,5-trisphosphate (IP3) produced from PI(4,5)P2 through PLCß, and tubbyCT responses do not faithfully report on PLCß-dependent PI(4,5)P2 dynamics. In searching for an improved biosensor, we fused N-terminal homology domain of Epsin1 (ENTH) to GFP and examined use of this construct as genetically-encoded biosensor for PI(4,5)P2 dynamics in living cells. We utilized recombinant tools to manipulate PI or Gq protein-coupled receptors (GqPCR) to stimulate PLCß signaling and characterized PI binding properties of ENTH-GFP with total internal reflection (TIRF) and confocal microscopy. ENTH-GFP specifically recognized membrane PI(4,5)P2 without interacting with IP3, as demonstrated by dialysis of cells with the messenger through a patch pipette. Utilizing Ci-VSP to titrate PI(4,5)P2 levels, we found that ENTH-GFP had low PI(4,5)P2 affinity. Accordingly, ENTH-GFP was highly sensitive to PLCß-dependent PI(4,5)P2 depletion, and in contrast to PLCδ1-PH, overexpression of ENTH-GFP did not attenuate GqPCR signaling. Taken together, ENTH-GFP detects minute changes of PI(4,5)P2 levels and provides an important complementation of experimentally useful reporters of PI(4,5)P2 dynamics in physiological pathways.

A126 in the active site and TI167/168 in the TI loop are essential determinants of the substrate specificity of PTEN.

PTEN prevents tumor genesis by antagonizing the PI3 kinase/Akt pathway through D3 site phosphatase activity toward PI(3,4)P2 and PI(3,4,5)P3. The structural determinants of this important specificity remain unknown. Interestingly, PTEN shares remarkable homology to voltage-sensitive phosphatases (VSPs) that dephosphorylate D5 and D3 sites of PI(4,5)P2, PI(3,4)P2, and PI(3,4,5)P3. Since the catalytic center of PTEN and VSPs differ markedly only in TI/gating loop and active site motif, we wondered whether these differences explained the variation of their substrate specificity. Therefore, we introduced mutations into PTEN to mimic corresponding sequences of VSPs and studied phosphatase activity in living cells utilizing engineered, voltage switchable PTENCiV, a Ci-VSP/PTEN chimera that retains D3 site activity of the native enzyme. Substrate specificity of this enzyme was analyzed with whole-cell patch clamp in combination with total internal reflection fluorescence microscopy and genetically encoded phosphoinositide sensors. In PTENCiV, mutating TI167/168 in the TI loop into the corresponding ET pair of VSPs induced VSP-like D5 phosphatase activity toward PI(3,4,5)P3, but not toward PI(4,5)P2. Combining TI/ET mutations with an A126G exchange in the active site removed major sequence variations between PTEN and VSPs and resulted in D5 activity toward PI(4,5)P2 and PI(3,4,5)P3 of PTENCiV. This PTEN mutant thus fully reproduced the substrate specificity of native VSPs. Importantly, the same combination of mutations also induced D5 activity toward PI(3,4,5)P3 in native PTEN demonstrating that the same residues determine the substrate specificity of the tumor suppressor in living cells. Reciprocal mutations in VSPs did not alter their substrate specificity, but reduced phosphatase activity. In summary, A126 in the active site and TI167/168 in the TI loop are essential determinants of PTEN's substrate specificity, whereas additional features might contribute to the enzymatic activity of VSPs.

Discovery and functional characterization of a neomorphic PTEN mutation.

Although a variety of genetic alterations have been found across cancer types, the identification and functional characterization of candidate driver genetic lesions in an individual patient and their translation into clinically actionable strategies remain major hurdles. Here, we use whole genome sequencing of a prostate cancer tumor, computational analyses, and experimental validation to identify and predict novel oncogenic activity arising from a point mutation in the phosphatase and tensin homolog (PTEN) tumor suppressor protein. We demonstrate that this mutation (p.A126G) produces an enzymatic gain-of-function in PTEN, shifting its function from a phosphoinositide (PI) 3-phosphatase to a phosphoinositide (PI) 5-phosphatase. Using cellular assays, we demonstrate that this gain-of-function activity shifts cellular phosphoinositide levels, hyperactivates the PI3K/Akt cell proliferation pathway, and exhibits increased cell migration beyond canonical PTEN loss-of-function mutants. These findings suggest that mutationally modified PTEN can actively contribute to well-defined hallmarks of cancer. Lastly, we demonstrate that these effects can be substantially mitigated through chemical PI3K inhibitors. These results demonstrate a new dysfunction paradigm for PTEN cancer biology and suggest a potential framework for the translation of genomic data into actionable clinical strategies for targeted patient therapy.

Regulation of the transient receptor potential channel TRPM3 by phosphoinositides.

The transient receptor potential (TRP) channel TRPM3 is a calcium-permeable cation channel activated by heat and by the neurosteroid pregnenolone sulfate (PregS). TRPM3 is highly expressed in sensory neurons, where it plays a key role in heat sensing and inflammatory hyperalgesia, and in pancreatic ß cells, where its activation enhances glucose-induced insulin release. However, despite its functional importance, little is known about the cellular mechanisms that regulate TRPM3 activity. Here, we provide evidence for a dynamic regulation of TRPM3 by membrane phosphatidylinositol phosphates (PIPs). Phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) and ATP applied to the intracellular side of excised membrane patches promote recovery of TRPM3 from desensitization. The stimulatory effect of cytosolic ATP on TRPM3 reflects activation of phosphatidylinositol kinases (PI-Ks), leading to resynthesis of PIPs in the plasma membrane. Various PIPs directly enhance TRPM3 activity in cell-free inside-out patches, with a potency order PI(3,4,5)P3 > PI(3,5)P2 > PI(4,5)P2 ≈ PI(3,4)P2 >> PI(4)P. Conversely, TRPM3 activity is rapidly and reversibly inhibited by activation of phosphatases that remove the 5-phosphate from PIPs. Finally, we show that recombinant TRPM3, as well as the endogenous TRPM3 in insuloma cells, is rapidly and reversibly inhibited by activation of phospholipase C-coupled muscarinic acetylcholine receptors. Our results reveal basic cellular mechanisms whereby membrane receptors can regulate TRPM3 activity.

Ion channel regulation by phosphoinositides analyzed with VSPs-PI(4,5)P2 affinity, phosphoinositide selectivity, and PI(4,5)P2 pool accessibility.

The activity of many proteins depends on the phosphoinositide (PI) content of the membrane. E.g., dynamic changes of the concentration of PI(4,5)P2 are cellular signals that regulate ion channels. The susceptibility of a channel to such dynamics depends on its affinity for PI(4,5)P2. Yet, measuring affinities for endogenous PIs has not been possible directly, but has relied largely on the response to soluble analogs, which may not quantitatively reflect binding to native lipids. Voltage-sensitive phosphatases (VSPs) turn over PI(4,5)P2 to PI(4)P when activated by depolarization. In combination with voltage-clamp electrophysiology VSPs are useful tools for rapid and reversible depletion of PI(4,5)P2. Because cellular PI(4,5)P2 is resynthesized rapidly, steady state PI(4,5)P2 changes with the degree of VSP activation and thus depends on membrane potential. Here we show that titration of endogenous PI(4,5)P2 with Ci-VSP allows for the quantification of relative PI(4,5)P2 affinities of ion channels. The sensitivity of inward rectifier and voltage-gated K(+) channels to Ci-VSP allowed for comparison of PI(4,5)P2 affinities within and across channel subfamilies and detected changes of affinity in mutant channels. The results also reveal that VSPs are useful only for PI effectors with high binding specificity among PI isoforms, because PI(4,5)P2 depletion occurs at constant overall PI level. Thus, Kir6.2, a channel activated by PI(4,5)P2 and PI(4)P was insensitive to VSP. Surprisingly, despite comparable PI(4,5)P2 affinity as determined by Ci-VSP, the Kv7 and Kir channel families strongly differed in their sensitivity to receptor-mediated depletion of PI(4,5)P2. While Kv7 members were highly sensitive to activation of PLC by Gq-coupled receptors, Kir channels were insensitive even when PI(4,5)P2 affinity was lowered by mutation. We hypothesize that different channels may be associated with distinct pools of PI(4,5)P2 that differ in their accessibility to PLC and VSPs.

Phosphoinositide dynamics in the postsynaptic membrane compartment: Mechanisms and experimental approach.

Phosphoinositides (PIs) are minor constituents of eukaryotic membranes that control a plethora of cellular functions through direct modulation of membrane-associated proteins and through membrane recruitment of enzymes or signaling molecules. It is well established that in neurons PIs play essential roles in the pre-synapse, especially during exocytotic neurotransmitter release and recycling of synaptic vesicles. In contrast, the physiological importance of PIs in postsynaptic membranes is far less understood. The extent and the spatiotemporal characteristics of dynamic changes in the concentrations of PIs caused by synaptic activity are largely unknown. Recent work suggests that postsynaptic PI dynamics are involved in the induction and maintenance of synaptic plasticity, but the general principles are far from clear. This review summarizes current knowledge on the relevance of PIs for postsynaptic processes, focussing on PI signaling in the control of electrical activity and synaptic plasticity. We highlight the state-of-the-art of methods to study PI dynamics and discuss recent technical improvements that should help to define the role of PIs in postsynaptic physiology.

A method to control phosphoinositides and to analyze PTEN function in living cells using voltage sensitive phosphatases.

Voltage sensitive phosphatases (VSPs), including engineered voltage sensitive PTEN, are excellent tools to rapidly and reversibly alter the phosphoinositide (PI) content of the plasma membrane in vivo and study the tumor suppressor PTEN. However, widespread adoption of these tools is hampered by the requirement for electrophysiological instrumentation to control the activity of VSPs. Additionally, monitoring and quantifying the PI changes in living cells requires sophisticated microscopy equipment and image analysis. Here we present methods that bypass these obstacles. First, we explore technically simple means for activation of VSPs via extracellularly applied agents or light. Secondly, we characterize methods to monitor PI(4,5)P2 and PI(3,4,5)P3 levels using fluorescence microscopy or photometry in conjunction with translocation or FRET based PI probes, respectively. We then demonstrate the application of these techniques by characterizing the effect of known PTEN mutations on its enzymatic activity, analyzing the effect of PTEN inhibitors, and detecting in real time rapid inhibition of protein kinase B following depletion of PI(3,4,5)P3. Thus, we established an approach that does not only allow for rapidly manipulating and monitoring PI(4,5)P2 and PI(3,4,5)P3 levels in a population of cells, but also facilitates the study of PTEN mutants and pharmacological targeting in mammalian cells.

A human phospholipid phosphatase activated by a transmembrane control module.

In voltage-sensitive phosphatases (VSPs), a transmembrane voltage sensor domain (VSD) controls an intracellular phosphoinositide phosphatase domain, thereby enabling immediate initiation of intracellular signals by membrane depolarization. The existence of such a mechanism in mammals has remained elusive, despite the presence of VSP-homologous proteins in mammalian cells, in particular in sperm precursor cells. Here we demonstrate activation of a human VSP (hVSP1/TPIP) by an intramolecular switch. By engineering a chimeric hVSP1 with enhanced plasma membrane targeting containing the VSD of a prototypic invertebrate VSP, we show that hVSP1 is a phosphoinositide-5-phosphatase whose predominant substrate is PI(4,5)P(2). In the chimera, enzymatic activity is controlled by membrane potential via hVSP1's endogenous phosphoinositide binding motif. These findings suggest that the endogenous VSD of hVSP1 is a control module that initiates signaling through the phosphatase domain and indicate a role for VSP-mediated phosphoinositide signaling in mammals.

Restoration of ion channel function in deafness-causing KCNQ4 mutants by synthetic channel openers.

BACKGROUND AND PURPOSE: DFNA2 is a frequent hereditary hearing disorder caused by loss-of-function mutations in the voltage-gated potassium channel KCNQ4 (Kv7.4). KCNQ4 mediates the predominant K(+) conductance, I(K,n) , of auditory outer hair cells (OHCs), and loss of KCNQ4 function leads to degeneration of OHCs resulting in progressive hearing loss. Here we explore the possible recovery of channel activity of mutant KCNQ4 induced by synthetic KCNQ channel openers. EXPERIMENTAL APPROACH: Whole cell patch clamp recordings were performed on CHO cells transiently expressing KCNQ4 wild-type (wt) and DFNA2-relevant mutants, and from acutely isolated OHCs. KEY RESULTS: Various known KCNQ channel openers robustly enhanced KCNQ4 currents. The strongest potentiation was observed with a combination of zinc pyrithione plus retigabine. A similar albeit less pronounced current enhancement was observed with native I(K,n) currents in rat OHCs. DFNA2 mutations located in the channel's pore region abolished channel function and these mutant channels were completely unresponsive to channel openers. However, the function of a DFNA2 mutation located in the proximal C-terminus was restored by the combined application of both openers. Co-expression of wt and KCNQ4 pore mutants suppressed currents to barely detectable levels. In this dominant-negative situation, channel openers essentially restored currents back to wt levels, most probably through strong activation of only the small fraction of homomeric wt channels. CONCLUSIONS AND IMPLICATIONS: Our data suggest that by stabilizing the KCNQ4-mediated conductance in OHCs, chemical channel openers can protect against OHC degeneration and progression of hearing loss in DFNA2.

A synthetic prestin reveals protein domains and molecular operation of outer hair cell piezoelectricity.

Prestin, a transporter-like protein of the SLC26A family, acts as a piezoelectric transducer that mediates the fast electromotility of outer hair cells required for cochlear amplification and auditory acuity in mammals. Non-mammalian prestin orthologues are anion transporters without piezoelectric activity. Here, we generated synthetic prestin (SynPres), a chimera of mammalian and non-mammalian prestin exhibiting both, piezoelectric properties and anion transport. SynPres delineates two distinct domains in the protein's transmembrane core that are necessary and sufficient for generating electromotility and associated non-linear charge movement (NLC). Functional analysis of SynPres showed that the amplitude of NLC and hence electromotility are determined by the transport of monovalent anions. Thus, prestin-mediated electromotility is a dual-step process: transport of anions by an alternate access cycle, followed by an anion-dependent transition generating electromotility. The findings define structural and functional determinants of prestin's piezoelectric activity and indicate that the electromechanical process evolved from the ancestral transport mechanism.

Probing the regulation of TASK potassium channels by PI4,5P2 with switchable phosphoinositide phosphatases.

TASK channels are background K+ channels that contribute to the resting conductance in many neurons. A key feature of TASK channels is the reversible inhibition by Gq-coupled receptors, thereby mediating the dynamic regulation of neuronal activity by modulatory transmitters. The mechanism that mediates channel inhibition is not fully understood. While it is clear that activation of Gαq is required, the immediate signal for channel closure remains controversial. Experimental evidence pointed to either phospholipase C (PLC)-mediated depletion of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) as the cause for channel closure or to a direct inhibitory interaction of active Gαq with the channel. Here, we address the role of PI(4,5)P2 for G-protein-coupled receptor (GPCR)-mediated TASK inhibition by using recently developed genetically encoded tools to alter phosphoinositide (PI) concentrations in the living cell.When expressed in CHO cells, TASK-1- and TASK-3-mediated currents were not affected by depletion of plasma membrane PI(4,5)P2 either via the voltage-activated phosphatase Ci-VSP or via chemically triggered recruitment of a PI(4,5)P2-5'-phosphatase. Depletion of both PI(4,5)P2 and PI(4)P via membrane recruitment of a novel engineered dual-specificity phosphatase also did not inhibit TASK currents. In contrast, each of these methods produced robust inhibition of the bona fide PI(4,5)P2-dependent channel KCNQ4. Efficient depletion of PI(4,5)P2 and PI(4)P was further confirmed with a fluorescent phosphoinositide sensor. Moreover, TASK channels recovered normally from inhibition by co-expressed muscarinic M1 receptors when resynthesis of PI(4,5)P2 was prevented by depletion of cellular ATP. These results demonstrate that TASK channel activity is independent of phosphoinositide concentrations within the physiological range. Consequently, Gq-mediated inhibition of TASK channels is not mediated by depletion of PI(4,5)P2.

Controlling the activity of a phosphatase and tensin homolog (PTEN) by membrane potential.

The recently discovered voltage-sensitive phosphatases (VSPs) hydrolyze phosphoinositides upon depolarization of the membrane potential, thus representing a novel principle for the transduction of electrical activity into biochemical signals. Here, we demonstrate the possibility to confer voltage sensitivity to cytosolic enzymes. By fusing the tumor suppressor PTEN to the voltage sensor of the prototypic VSP from Ciona intestinalis, Ci-VSP, we generated chimeric proteins that are voltage-sensitive and display PTEN-like enzymatic activity in a strictly depolarization-dependent manner in vivo. Functional coupling of the exogenous enzymatic activity to the voltage sensor is mediated by a phospholipid-binding motif at the interface between voltage sensor and catalytic domains. Our findings reveal that the main domains of VSPs and related phosphoinositide phosphatases are intrinsically modular and define structural requirements for coupling of enzymatic activity to a voltage sensor domain. A key feature of this prototype of novel engineered voltage-sensitive enzymes, termed Ci-VSPTEN, is the novel ability to switch enzymatic activity of PTEN rapidly and reversibly. We demonstrate that experimental control of Ci-VSPTEN can be obtained either by electrophysiological techniques or more general techniques, using potassium-induced depolarization of intact cells. Thus, Ci-VSPTEN provides a novel approach for studying the complex mechanism of activation, cellular control, and pharmacology of this important tumor suppressor. Moreover, by inducing temporally precise perturbation of phosphoinositide concentrations, Ci-VSPTEN will be useful for probing the role and specificity of these messengers in many cellular processes and to analyze the timing of phosphoinositide signaling.

Aminoglycosides inhibit KCNQ4 channels in cochlear outer hair cells via depletion of phosphatidylinositol(4,5)bisphosphate.

Aminoglycoside antibiotics (AGs) are severely ototoxic. AGs cause degeneration of outer hair cells (OHCs), leading to profound and irreversible hearing loss. The underlying mechanisms are not fully understood. OHC survival critically depends on a specific K+ conductance (I(K,n)) mediated by KCNQ4 (Kv7.4) channels. Dysfunction or genetic ablation of KCNQ4 results in OHC degeneration and deafness in mouse and humans. As a common hallmark of all KCNQ isoforms, channel activity requires phosphatidylinositol(4,5)bisphosphate [PI(4,5)P2]. Because AGs are known to reduce PI(4,5)P2 availability by sequestration, inhibition of KCNQ4 may be involved in the action of AGs on OHCs. Using whole-cell patch-clamp recordings from rat OHCs, we found that intracellularly applied AGs inhibit I(K,n). The inhibition results from PI(4,5)P2 depletion indicated by fluorescence imaging of cellular PI(4,5)P2 and the dependence of inhibition on PI(4,5)P2 availability and on PI(4,5)P2 affinity of recombinant KCNQ channels. Likewise, extracellularly applied AGs inhibited I(K,n) and caused substantial depolarization of OHCs, after rapid accumulation in OHCs via a hair cell-specific apical entry pathway. The potency for PI(4,5)P2 sequestration, strength of I(K,n) inhibition, and resulting depolarization correlated with the known ototoxic potential of the different AGs. Thus, the inhibition of I(K,n) via PI(4,5)P2 depletion and the resulting depolarization may contribute to AG-induced OHC degeneration. The KCNQ channel openers retigabine and zinc pyrithione rescued KCNQ4/I(K,n) activity from AG-induced inhibition. Pharmacological enhancement of KCNQ4 may thus offer a protective strategy against AG-induced ototoxicity and possibly other ototoxic insults.

Ci-VSP is a depolarization-activated phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate 5'-phosphatase.

Phosphoinositides are membrane-delimited regulators of protein function and control many different cellular targets. The differentially phosphorylated isoforms have distinct concentrations in various subcellular membranes, which can change dynamically in response to cellular signaling events. Maintenance and dynamics of phosphoinositide levels involve a complex set of enzymes, among them phospholipases and lipid kinases and phosphatases. Recently, a novel type of phosphoinositide-converting protein (termed Ci-VSP) that contains a voltage sensor domain was isolated. It was already shown that Ci-VSP can alter phosphoinositide levels in a voltage-dependent manner. However, the exact enzymatic reaction catalyzed by Ci-VSP is not known. We used fluorescent phosphoinositide-binding probes and total internal reflection microscopy together with patch-clamp measurements from living cells to delineate substrates and products of Ci-VSP. Upon activation of Ci-VSP by membrane depolarization, membrane association of phosphatidylinositol (PI) (4,5)P2- and PI(3,4,5)P3-specific binding domains decreased, revealing consumption of these phosphoinositides by Ci-VSP. Depletion of PI(4,5)P2 was coincident with an increase in membrane PI(4)P. Similarly, PI(3,4)P2 was generated during depletion of PI(3,4,5)P3. These results suggest that Ci-VSP acts as a 5'-phosphatase of PI(4,5)P2 and PI(3,4,5)P3.

Pharmacological evidence for altered src kinase regulation of I (Ca,L) in patients with chronic atrial fibrillation.

A reduction in L-type Ca(2+) current (I (Ca,L)) contributes to electrical remodeling in chronic atrial fibrillation (AF). Whether the decrease in I (Ca,L) is solely due to a reduction in channel proteins remains controversial. Protein tyrosine kinases (PTK) have been described as potent modulators of I (Ca,L) in cardiomyocytes. We studied alpha(1C) L-type Ca(2+) channel subunit expression and the regulation of I (Ca,L) by PTK in chronic AF using PTK inhibitors: genistein, a nonselective inhibitor of PTK, and 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-3,4-d-pyrimidine (PP1), a selective inhibitor of src kinases. Furthermore, type-1 and type-2A protein phosphatase activity was measured with phosphorylase as substrate in whole-cell lysates derived from atrial tissue of AF patients. Right atrial appendages were obtained from patients undergoing open-heart surgery. Protein levels of alpha(1C) L-type Ca(2+) channel subunit were determined using Western blot analysis and normalized to the protein amounts of calsequestrin as internal control. The protein concentrations of alpha(1C) did not differ between AF and sinus rhythm (SR; alpha(1C)/calsequestrin: 1.0 +/- 0.1 and 1.2 +/- 0.2, respectively, n = 8 patients). In cardiomyocytes from patients in SR (n = 20 patients), genistein and PP1 both evoked similar increases in I (Ca,L) from 3.0 +/- 0.3 to 6.1 +/- 0.8 pA/pF and from 2.8 +/- 0.4 to 6.1 +/- 0.6 pA/pF, respectively. In cells from AF patients (n = 10 patients), basal I (Ca,L) was significantly lower. In this case, genistein lead to the same relative increase in I (Ca,L) as in SR cells (from 1.46 +/- 0.30 to 3.2 +/- 1.0 pA/pF), whereas no increase was elicited by PP1 suggesting impaired regulation of I (Ca,L) by src kinases in AF. Total and type 1 and type 2A-related phosphatase activities were higher in tissue from patients with chronic AF compared to SR (4.8 +/- 0.4, 2.1 +/- 0.2, and 2.7 +/- 0.4 nmol/mg/min and 3.6 +/- 0.4, 1.3 +/- 0.2, and 2.4 +/- 0.3 nmol/mg/min, respectively, n = 7 patients per group). Downregulation of I (Ca,L) in AF is not due to a reduction in L-type Ca(2+) channel protein expression. Indirect evidence for an impaired src kinase regulation of I (Ca,L) together with an increased phosphatase activity suggests that a complex alteration in the kinase/phosphatase balance leads to I (Ca,L) dysregulation in chronic AF.

Effects of inhalational anesthetics on L-type Ca2+ currents in human atrial cardiomyocytes during beta-adrenergic stimulation.

BACKGROUND: Anesthetics may cause cardiac side effects by their action on L-type Ca2+ channels. Direct effects on the channels have not yet been discriminated from an interference with the beta-adrenergic channel regulation. The authors therefore studied the effects of halothane, sevoflurane, and xenon on human cardiac Ca2+ currents during stimulation with isoproterenol. METHODS: Currents through L-type Ca2+ channels were measured with the patch clamp technique in atrial cardiomyocytes obtained from patients undergoing cardiac surgery. Cells were superfused with solutions equilibrated with anesthetics at the desired concentrations. Ca2+ currents during pulses to 10 mV were evaluated with respect to their peak value (I(max)) and to the total moved charge (Q). RESULTS: In the absence and in the presence of isoproterenol (1 microm), sevoflurane (0.29 mm, 1 minimum alveolar concentration [MAC]) significantly depressed Q by 37.8 +/- 7.2% (mean +/- SD) and 40.8 +/- 10.3%, respectively. I(max) was not significantly affected in comparison with control cells never exposed to an anesthetic. Xenon (65%, 1 MAC) did not evoke significant effects. Exposure to halothane (0.39 mm, 1 MAC) during stimulation with isoproterenol significantly reduced Q by 31.3 +/- 23.3% (but not I(max)). After washout of halothane, Q was increased above the level prior to the application of halothane. Moreover, whereas Q promptly declined to baseline levels after washout of isoproterenol in controls, the previous exposure to halothane markedly delayed this decline, leaving Q significantly elevated for several minutes. CONCLUSIONS: Halothane exerts a dual effect on Ca2+ currents. The long-lasting stimulatory effect may contribute to the proarrhythmic potency of the drug that exceeds that of sevoflurane, which only depressed Ca2+ currents.

Expression profile of the transient receptor potential (TRP) family in neutrophil granulocytes: evidence for currents through long TRP channel 2 induced by ADP-ribose and NAD.

An early key event in the activation of neutrophil granulocytes is Ca(2+) influx. Members of the transient receptor potential (TRP) channel family may be held responsible for this. The aim of the present study is to analyse the expression pattern of TRP mRNA and identify characteristic currents unambiguously attributable to particular TRP channels. mRNA was extracted from human neutrophils, isolated by gradient centrifugation and also by magnetically labelled CD15 antibodies. The presence of mRNA was demonstrated using reverse transcriptase-PCR in neutrophils (controlled to be CD5-negative) as well as in human leukaemic cell line 60 (HL-60) cells, for the following TRP species: the long TRPC2 (LTRPC2), the vanilloid receptor 1, the vanilloid receptor-like protein 1 and epithelial Ca(2+) channels 1 and 2. TRPC6 was specific for neutrophils, whereas only in HL-60 cells were TRPC1, TRPC2, TRPC3, melastatin 1 and melastatin-related 1 found. Patch-clamp measurements in neutrophils revealed non-selective cation currents evoked by intracellular ADP-ribose and by NAD(+). Both these modes of activation have been found to be characteristic of LTRPC2. Furthermore, single-channel activity was resolved in neutrophils and it was indistinguishable from that in LTRPC2-transfected HEK-293 cells. The results provide evidence that LTRPC2 in neutrophil granulocytes forms an entry pathway for Na(+) and Ca(2+), which is regulated by ADP-ribose and the redox state.

The TRP family of cation channels: probing and advancing the concepts on receptor-activated calcium entry.

Stimulation of membrane receptors linked to a phospholipase C and the subsequent production of the second messengers diacylglycerol and inositol-1,4,5-trisphosphate (InsP(3)) is a signaling pathway of fundamental importance in eukaryotic cells. Signaling downstream of these initial steps involves mobilization of Ca(2+) from intracellular stores and Ca(2+) influx through the plasma membrane. For this influx, several contrasting mechanisms may be responsible but particular relevance is attributed to the induction of Ca(2+) influx as consequence of depletion of intracellular calcium stores. This phenomenon (frequently named store-operated calcium entry, SOCE), in turn, may be brought about by various signals, including soluble cytosolic factors, interaction of proteins of the endoplasmic reticulum with ion channels in the plasma membrane, and a secretion-like coupling involving translocation of channels to the plasma membrane. Experimental approaches to analyze these mechanisms have been considerably advanced by the discovery of mammalian homologs of the Drosophila cation channel transient receptor potential (TRP). Some members of the TRP family can be expressed to Ca(2+)-permeable channels that enable SOCE; other members form channels activated independently of stores. TRP proteins may be an essential part of endogenous Ca(2+) entry channels but so far expression of most TRP cDNAs has not resulted in restitution of channels found in any mammalian cells, suggesting the requirement for further unknown subunits. A major exception is CaT1, a TRP channel demonstrated to provide Ca(2+)-selective, store-operated currents identical to those characterized in several cell types. Ongoing and future research on TRP channels will be crucial to understand the molecular basis of receptor-mediated Ca(2+) entry, with respect to the structure of the entry channels as well as to the mechanisms of its activation and regulation.