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PubMed2005

Water transport by Na+-coupled cotransporters of glucose (SGLT1) and of iodide (NIS). The dependence of substrate size studied at high resolution.

The relation between substrate and water transport was studied in Na+-coupled cotransporters of glucose (SGLT1) and of iodide (NIS) expressed in Xenopus oocytes. The water transport was monitored from changes in oocyte volume at a resolution of 20 pl, more than one order of magnitude better than previous investigations. The rate of cotransport was monitored as the clamp current obtained from two-electrode voltage clamp. The high resolution data demonstrated a fixed ratio between the turn-over of the cotransporter and the rate of water transport. This applied to experiments in which the rate of cotransport was changed by isosmotic application of substrate, by rapid changes in clamp voltage, or by poisoning. Transport of larger substrates gave rise to less water transport. For the rabbit SGLT1, 378+/-20 (n=18 oocytes) water molecules were cotransported along with the 2 Na+ ions and the glucose-analogue alpha-MDG (MW 194); using the larger sugar arbutin (MW 272) this number was reduced by a factor of at least 0.86+/-0.03 (15). For the human SGLT1 the respective numbers were 234+/-12 (18) and 0.85+/-0.8 (7). For NIS, 253+/-16 (12) water molecules were cotransported for each 2 Na+ and 1 thiocyanate (SCN-, MW 58), with I- as anion (MW 127) only 162+/-11 (19) water molecules were cotransported. The effect of substrate size suggests a molecular mechanism for water cotransport and is opposite to what would be expected from unstirred layer effects. Data were analysed by a model which combined cotransport and osmosis at the membrane with diffusion in the cytoplasm. The combination of high resolution measurements and precise modelling showed that water transport across the membrane can be explained by cotransport of water in the membrane proteins and that intracellular unstirred layers effects are minute.

Animals↗

PubMed2002

Autoimmunity due to molecular mimicry as a cause of neurological disease.

One hypothesis that couples infection with autoimmune disease is molecular mimicry. Molecular mimicry is characterized by an immune response to an environmental agent that cross-reacts with a host antigen, resulting in disease. This hypothesis has been implicated in the pathogenesis of diabetes, lupus and multiple sclerosis (MS). There is limited direct evidence linking causative agents with pathogenic immune reactions in these diseases. Our study establishes a clear link between viral infection, autoimmunity and neurological disease in humans. As a model for molecular mimicry, we studied patients with human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a disease that can be indistinguishable from MS (refs. 5,6,7). HAM/TSP patients develop antibodies to neurons. We hypothesized these antibodies would identify a central nervous system (CNS) autoantigen. Immunoglobulin G isolated from HAM/TSP patients identified heterogeneous nuclear ribonuclear protein-A1 (hnRNP-A1) as the autoantigen. Antibodies to hnRNP-A1 cross-reacted with HTLV-1-tax, the immune response to which is associated with HAM/TSP (refs. 5,9). Immunoglobulin G specifically stained human Betz cells, whose axons are preferentially damaged. Infusion of autoantibodies in brain sections inhibited neuronal firing, indicative of their pathogenic nature. These data demonstrate the importance of molecular mimicry between an infecting agent and hnRNP-A1 in autoimmune disease of the CNS.

Antibodies, Monoclonal↗

PubMed2001

Dynamic sensitivity of ATP-sensitive K(+) channels to ATP.

ATP and MgADP regulate K(ATP) channel activity and hence potentially couple cellular metabolism to membrane electrical activity in various cell types. Using recombinant K(ATP) channels that lack sensitivity to MgADP, expressed in COSm6 cells, we demonstrate that similar on-cell activity can be observed with widely varying apparent submembrane [ATP] ([ATP](sub)). Metabolic inhibition leads to a biphasic change in the channel activity; activity first increases, presumably in response to a fast decrease in [ATP](sub), and then declines. The secondary decrease in channel activity reflects a marked increase in ATP sensitivity and is correlated with a fall in polyphosphoinositides (PPIs), including phosphatidylinositol 4,5-bisphosphate, probed using equilibrium labeling of cells with [(3)H]myo-inositol. Both ATP sensitivity and PPIs rapidly recover following removal of metabolic inhibition, and in both cases recovery is blocked by wortmannin. These data are consistent with metabolism having a dual effect on K(ATP) channel activity: rapid activation of channels because of relief of ATP inhibition and much slower reduction of channel activity mediated by a fall in PPIs. These two mechanisms constitute a feedback system that will tend to render K(ATP) channel activity transiently responsive to a change in [ATP](sub) over a wide range of steady state concentrations.

Adenosine Diphosphate↗

PubMed2005

G-protein coupled activation of potassium channels by endogenous neuropeptides in snail neurons.

Members of the mytilus inhibitory peptide (MIP) family play an important role in the modulation of many physiological processes in molluscs. The signal transduction pathways affected by the MIP effect have not, however, been elucidated. Application of guanosine 5'-[gamma-thio]triphosphate tetralithium salt (GTPgammaS), guanosine 5'-[beta-thio]diphosphate trilithium salt (GDPbetaS), the G-protein inhibitor suramin and pertussis toxin (PTX) demonstrated the involvement of the PTX-insensitive G-protein in the signal transduction pathway mediating MIP effects. Both G-protein alpha(i) and betagamma subunits were identified in D-neurons of Helix pomatia by immunoblotting. Their role in signal transduction was shown in electrophysiological experiments, which supported the notion that, in addition to the Galpha subunit, the betagamma dimer also participates in the neuropeptide-induced activation of K-channels in snail neurons. Finally, neuropeptide-activated responses were inhibited by the activation of adenylyl cyclase and by blockers of the phospholipase pathway. We suggest that bifurcation of the signal transduction takes place at the level of G-protein subunits. The alpha subunit may have a direct effect on adenylyl cyclase, while the betagamma subunit may have a direct effect on phospholipase enzymes.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1997

Inhibition of cyclic AMP-dependent chloride secretion by PP receptors and alpha 2-adrenoceptors in a human colonic epithelial cell line.

The effects of a number of agonists which inhibit intestinal chloride secretion were investigated in Colony-1 (Col-1) cells, a subpopulation derived from the HCA-7 human adenocarcinoma cell line. Neither peptide YY (PYY) or somatostatin 14-28 (SRIF) reduced short-circuit current (SCC) in Col-1 epithelial layers stimulated with vasoactive intestinal polypeptide (VIP), suggesting that their respective receptors are either absent in this cell line, or are not functionally coupled. A second member of the neuropeptide Y family, pancreatic polypeptide (PP), decreased VIP-elevated SCC with an EC50 of 25.6 nM. Maximal PP responses were unaffected by prior addition of PYY, indicating that Col-1 cells may express a PP specific, Y4-like receptor. The alpha 2-adrenoceptor agonist clonidine also attenuated VIP-stimulated SCC (EC50342 nM) through the alpha 2A receptor subtype, since clonidine responses were inhibited by yohimbine and rauwolscine but not altered by previous addition of prazosin. Col-1 cells responded to both apical and basolateral addition of VIP or clonidine; to an extent, this lack of sidedness reflects the ability of drugs to permeate through the Col-1 epithelial layers. Both PP and clonidine also inhibited SCC in unstimulated Col-1 cells or those pretreated with 3-isobutyl-1-methylaxanthine (IBMX) or a submaximal concentration of forskolin, agents which both directly elevate intracellular cAMP. After a maximal concentration of forskolin (10 microM), which increased SCC to a significantly greater extent than either VIP or IBMX, the effects of both agonists were negligible. The absence of PP and clonidine responses under these conditions may have implications for the mechanisms by which these agonists inhibit, chloride secretion in Col-1 epithelia. In addition carbachol reduced SCC stimulated by 10 microM forskolin, in contrast to control carbachol responses which consisted of a rapid decrease followed by a transient elevation in SCC; this observation suggests that Col-1 cells may also be a useful model for studying the interactions between Ca(2+)- and cAMP-dependent mechanisms involved in epithelial ion transport.

Adenocarcinoma↗

PubMed2004

Chemokine receptor CCR6 transduces signals that activate p130Cas and alter cAMP-stimulated ion transport in human intestinal epithelial cells.

Human colon epithelial cells express the G protein-coupled receptor CCR6, the sole receptor for the chemokine CCL20 (also termed MIP-3alpha). CCL20 produced by intestinal epithelial cells is upregulated in response to proinflammatory stimuli and microbial infection, and it chemoattracts leukocytes, including CCR6-expressing immature myeloid dendritic cells, into sites of inflammation. The aim of this study was to determine whether CCR6 expressed by intestinal epithelial cells acts as a functional receptor for CCL20 and whether stimulation with CCL20 alters intestinal epithelial cell functions. The human colon epithelial cell lines T84, Caco-2, HT-29, and HCA-7 were used to model colonic epithelium. Polarized intestinal epithelial cells constitutively expressed CCR6, predominantly on the apical side. Consistent with this, apical stimulation of polarized intestinal epithelial cells resulted in tyrosine phosphorylation of the p130 Crk-associated substrate (Cas), an adaptor/scaffolding protein that localizes in focal adhesions and has a role in regulating cytoskeletal elements important for cell attachment and migration. In addition, CCL20 stimulation inhibited agonist-stimulated production of the second messenger cAMP and cAMP-mediated chloride secretory responses by intestinal epithelial cells. Inhibition was abrogated by pertussis toxin, consistent with signaling through Galphai proteins that negatively regulate adenylyl cyclases and cAMP production. These data indicate that signaling events, occurring via the activation of the apically expressed chemokine receptor CCR6 on polarized intestinal epithelial cells, alter specialized intestinal epithelial cell functions, including electrogenic ion secretion and possibly epithelial cell adhesion and migration.

Blotting, Western↗

PubMed2000

Amplification of odor-induced Ca(2+) transients by store-operated Ca(2+) release and its role in olfactory signal transduction.

A critical role of Ca(2+) in vertebrate olfactory receptor neurons (ORNs) is to couple odor-induced excitation to intracellular feedback pathways that are responsible for the regulation of the sensitivity of the sense of smell, but the role of intracellular Ca(2+) stores in this process remains unclear. Using confocal Ca(2+) imaging and perforated patch recording, we show that salamander ORNs contain a releasable pool of Ca(2+) that can be discharged at rest by the SERCA inhibitor thapsigargin and the ryanodine receptor agonist caffeine. The Ca(2+) stores are spatially restricted; emptying produces compartmentalized Ca(2+) release and capacitative-like Ca(2+) entry in the dendrite and soma but not in the cilia, the site of odor transduction. We deplete the stores to show that odor stimulation causes store-dependent Ca(2+) mobilization. This odor-induced Ca(2+) release does not seem to be necessary for generation of an immediate electrophysiological response, nor does it contribute significantly to the Ca(2+) transients in the olfactory cilia. Rather, it is important for amplifying the magnitude and duration of Ca(2+) transients in the dendrite and soma and is thus necessary for the spread of an odor-induced Ca(2+) wave from the cilia to the soma. We show that this amplification process depends on Ca(2+)-induced Ca(2+) release. The results indicate that stimulation of ORNs with odorants can produce Ca(2+) mobilization from intracellular stores without an immediate effect on the receptor potential. Odor-induced, store-dependent Ca(2+) mobilization may be part of a feedback pathway by which information is transferred from the distal dendrite of an ORN to its soma.

Animals↗

PubMed2001

Functional coupling between 'R-type' Ca2+ channels and insulin secretion in the insulinoma cell line INS-1.

Among voltage-gated Ca2+ channels the non-dihydropyridine-sensitive alpha1E subunit is functionally less well characterized than the structurally related alpha1A (omega-agatoxin-IVA sensitive, P- /Q-type) and alpha1B (omega-conotoxin-GVIA sensitive, N-type) subunits. In the rat insulinoma cell line, INS-1, a tissue-specific splice variant of alpha1E (alpha1Ee) has been characterized at the mRNA and protein levels, suggesting that INS-1 cells are a suitable model for investigating the function of alpha1Ee. In alpha1E-transfected human embryonic kidney (HEK-293) cells the alpha1E-selective peptide antagonist SNX-482 (100 nM) reduces alpha1Ed- and alpha1Ee-induced Ba2+ inward currents in the absence and presence of the auxiliary subunits beta3 and alpha2delta-2 by more than 80%. The inhibition is fast and only partially reversible. No effect of SNX-482 was detected on the recombinant T-type Ca2+ channel subunits alpha1G, alpha1H, and alpha1I showing that the toxin from the venom of Hysterocrates gigas is useful as an alpha1E-selective antagonist. After blocking known components of Ca2+ channel inward current in INS-1 cells by 2 microM (+/-)-isradipine plus 0.5 microM omega-conotoxin-MVIIC, the remaining current is reduced by 100 nM SNX-482 from -12.4 +/- 1.2 pA/pF to -7.6 +/- 0.5 pA/pF (n = 9). Furthermore, in INS-1 cells, glucose- and KCl-induced insulin release are reduced by SNX-482 in a dose-dependent manner leading to the conclusion that alpha1E, in addition to L-type and non-L-type (alpha1A-mediated) Ca2+ currents, is involved in Ca2+ dependent insulin secretion of INS-1 cells.

Alternative Splicing↗

PubMed2001

Amino acid transporter CAATCH1 is also an amino acid-gated cation channel.

CAATCH1 (cation-amino acid transporter/channel) is a recently cloned insect epithelial membrane protein related to mammalian Na(+)-, Cl(-)-coupled neurotransmitter transporters (Feldman, D. H., Harvey, W. R., and Stevens, B. R. (2000) J. Biol. Chem. 275, 24518-24526). In the present study we analyze the relationship between CAATCH1-mediated amino acid transport and ion fluxes by utilizing the Xenopus oocyte expression system in conjunction with electrophysiology and radiotracer uptake. Simultaneous flux measurements reveal that electrical currents and amino acid transport are thermodynamically uncoupled. This observation is supported by measuring significant uptake even in the absence of external alkali cations. Remarkably, CAATCH1-associated Na(+) or K(+) currents are large and do not saturate with voltage nor with cation concentration. These currents reverse in Nernstian fashion, thereby conferring channel activity in CAATCH1. Upon step-changes in the membrane potential, CAATCH1-expressing oocytes exhibit transient currents. Detailed analyses of these transients in the absence and presence of amino acids reveal direct ligand-protein interaction, demonstrating that binding by different amino acids (e.g. proline, threonine, methionine) differentially affects the state probability of CAATCH1 but has no effect on the maximal charge movement (Q(max)). Together these data suggest that CAATCH1 is a multifunction membrane protein that mediates thermodynamically uncoupled amino acid uptake but functions predominantly as an amino acid-gated alkali cation channel.

Amino Acids↗

PubMed1984

Activation of a nicotinic acetylcholine receptor.

We studied activation of the nicotinic acetylcholine (ACh) receptor on cells of a mouse clonal muscle cell line (BC3H1). We analyzed single-channel currents through outside-out patches elicited with various concentrations of acetylcholine (ACh), carbamylcholine (Carb) and suberyldicholine (Sub). Our goal is to determine a likely reaction scheme for receptor activation by agonist and to determine values of rate constants for transitions in that scheme. Over a wide range of agonist concentrations the open-time duration histograms are not described by single exponential functions, but are well-described by the sum of two exponentials, a brief-duration and a long-duration component. At high concentration, channel openings occur in groups and these groups contain an excess number of brief openings. We conclude that there are two open states of the ACh receptor with different mean open times and that a single receptor may open to either open state. The concentration dependence of the numbers of brief and long openings indicates that brief openings do not result from the opening of channels of receptors which have only one agonist molecule bound to them. Closed-time duration histograms exhibit a major brief component at low concentrations. We have used the method proposed by Colquhoun and Sakmann (1981) to analyze these brief closings and to extract estimates for the rates of channel opening (beta) and agonist dissociation (k-2). We find that this estimate of beta does not predict our closed-time histograms at high agonist concentration (ACh: 30-300 microM; Carb: 300-1,000 microM). We conclude that brief closings at low agonist concentrations do not result solely from transitions between the doubly-liganded open and the doubly-liganded closed states. Instead, we postulate the existence of a second closed-channel state coupled to the open state.

Acetylcholine↗

PubMedDate not supplied

Sonic Hedgehog signalling in the developing and adult brain.

Sonic Hedgehog (Shh) belongs to a family of secreted polypeptides implicated in embryonic development. Shh displays inductive, proliferative, neurotrophic and neuroprotective activities on various neural cells and signals through a receptor complex associating Patched (Ptc) and Smoothened (Smo). Shh binding to Ptc leads to downstream activation of target genes, such as transcription factors of the Gli family. We have investigated the distribution of Shh signalling genes in the rat embryo and in the adult, as well as pharmacological properties of Shh peptides. In the ventral neural tube, the distribution of Shh, Ptc and Smo is in agreement with this functional model. In the postnatal cerebellum, Shh expressed by Purkinje cells may act on its target receptor complex localized in the external germinative layer to activate Gli1. Myristoylated ShhN (myrShhN) is more potent than ShhN in stimulating proliferation of rat cerebellar granule cell neuroblasts in culture, as evaluated by [3H]thymidine incorporation, suggesting that amino terminal lipid modification of the molecule plays a crucial role in ShhN biological activity. In the adult brain, Ptc and Smo transcripts are colocalized in a few areas such as the hippocampal granule cells. However, Ptc transcripts are also observed without any detectable Smo expression, such as in the superior colliculus. These observations suggest that in the adult brain, Shh signals through its receptor complex Ptc/Smo, or through Ptc alone. Ptc protein presents a sterol sensing domain which has been identified in several proteins, including TRC8, recently implicated in hereditary renal carcinoma and which is also expressed as a 2.5-kb transcript in several rat brain areas. Altogether, these results suggest other roles for Shh signalling in postnatal and adult brain than those initially established during early embryonic development.

Aging↗

PubMed2000

Ignition of calcium sparks in arterial and cardiac muscle through caveolae.

Ca(2+) sparks are localized intracellular Ca(2+) events released through ryanodine receptors (RyRs) that control excitation-contraction coupling in heart and smooth muscle. Ca(2+) spark triggering depends on precise delivery of Ca(2+) ions through dihydropyridine (DHP)-sensitive Ca(2+) channels to RyRs of the sarcoplasmic reticulum (SR), a process requiring a very precise alignment of surface and SR membranes containing Ca(2+) influx channels and RyRs. Because caveolae contain DHP-sensitive Ca(2+) channels and may colocalize with SR, we tested the hypothesis that caveolae are the structural element necessary for the generation of Ca(2+) sparks. Using methyl-ss-cyclodextrin (dextrin) to deplete caveolae, we found that dextrin dose-dependently decreased the frequency, amplitude, and spatial size of Ca(2+) sparks in arterial smooth muscle cells and neonatal cardiomyocytes. However, temporal characteristics of Ca(2+) sparks were not significantly affected. We ruled out the possibility that the decreases in Ca(2+) spark frequency and size are caused by changes in DHP-sensitive L-type channels, SR Ca(2+) load, or changes in membrane potential. Our results suggest a novel signaling model that explains the formation of Ca(2+) sparks in a caveolae microdomain. The transient elevation in [Ca(2+)](i) at the inner mouth of a single caveolemmal Ca(2+) channel induces simultaneous activation and thus opens several RyRs to generate a local Ca(2+) release event, a Ca(2+) spark. Alterations in the molecular assembly and ultrastructure of caveolae may lead to pathophysiological changes in Ca(2+) signaling. Thus, caveolae may be intimately involved in cardiovascular cell dysfunction and disease.

Aging↗

PubMed1998

Inositol-1,4,5-trisphosphate receptor-mediated Ca mobilization is not required for cerebellar long-term depression in reduced preparations.

Inositol-1,4,5-trisphosphate receptor-mediated Ca mobilization is not required for cerebellar long-term depression in reduced preparations. J. Neurophysiol. 80: 2963-2974, 1998. Cerebellar long-term depression (LTD) is a cellular model system of information storage in which coincident parallel fiber and climbing fiber activation of a Purkinje neuron (PN) gives rise to a sustained attenuation of parallel fiber-PN synaptic strength. Climbing fiber and parallel fiber inputs may be replaced by direct depolarization of the PN and exogenous glutamate pulses, respectively. The parallel fiber-PN synapse has a high-density of mGluR1 receptors that are coupled to phosphoinositide turnover. Several lines of evidence indicated that activation of mGluR1 by parallel fiber stimulation is necessary for the induction of cerebellar LTD. Because phosphoinositide hydrolysis has two initial products, 1, 2-diacylglycerol and inositol-1,4,5-trisphosphate (IP3), we wished to determine whether IP3 signaling via IP3 receptors and consequent Ca mobilization were necessary for the induction of cerebellar LTD. First, ratiometric imaging of free cytosolic Ca was performed on both acutely dissociated and cultured PNs. It was determined that the threshold for glutamate pulses to contribute to LTD induction was below the threshold for producing a Ca transient. Furthermore, the Ca transients produced by depolarization alone and glutamate plus depolarization were not significantly different. Second, the potent and selective IP3 receptor channel blocker xestospongin C was not found to affect the induction of LTD in either acutely dissociated or cultured PNs at a concentration that was sufficient to block mGluR1-evoked Ca mobilization. Third, replacement of mGluR activation by exogenous synthetic diacylglycerol in an LTD induction protocol was successful. Taken together, these results suggest that activation of an IP3 signaling cascade is not required for induction of cerebellar LTD in reduced preparations.

Animals↗

PubMed2000

Is keratoconus a true ectasia? An evaluation of corneal surface area.

BACKGROUND: Keratoconus has long been considered to be an ectasia produced by stromal stretching. Although stretching should result in increased corneal surface area, previous observations of topography during progression of keratoconus have suggested that surface area may actually be conserved. A novel objective surface area measurement based on corneal topography was tested and applied to data from actual corneas under various conditions for comparative analysis. SETTING: The LSU Eye Center clinic videokeratography archives. METHODS: TMS-1 videokeratography files (Tomey Corp, Cambridge, Mass) were obtained from 6 groups of corneas: normal (n = 29), keratoconus from mild to severe states (n = 51), topographically judged keratoconus-suspect conditions (n = 10), postoperative photorefractive keratectomy for myopia (n = 39), with-the-rule corneal astigmatism (n = 17), and keratoglobus (n = 1). Additionally, 3 different spherical test surfaces were analyzed to verify the accuracy of the process. Only maps with no missing data out to ring 29 were used. The cumulative surface area from center to periphery was determined by calculating and summing the area of individual patches along consecutive annular rings. Mean surface area with respect to mean chord radius was plotted for each corneal condition, and curve fitting was used to extend each result to a 5. 85-mm limbus. Means, SEs, and 95% confidence intervals were calculated at intervals for statistical comparisons among all groups. Computer-generated surfaces helped to evaluate the relationship between shape and surface area. RESULTS: When videokeratographic test targets were used, surface area error was less than 2%, which was deemed acceptable. Normal corneas had a mean +/- SE surface area of 120.3 +/- 2.2 mm(2), whereas all keratoconus corneas combined had a mean +/- SE surface area of 116.2 +/- 3.4 mm(2). The difference was not significant at any chord radius (analysis of variance, P<. 05). The keratoglobus cornea was found to have a surface area of 129. 9 mm(2), which was 7.98% greater than normal. An individual with progressive keratoconus exhibited no appreciable trend toward increasing surface area during a 76-month period. The corneas in the other groups resembled normal corneas in their total surface area. CONCLUSIONS: With the exception of the single case of keratoglobus, corneal surface area tended to be conserved near a value of 120 mm(2) for all groups in the study, including corneas with keratoconus. Surface area is remarkably insensitive to curvature change near the vertex. Flattening seen in the periphery of corneas with keratoconus suggests that biomechanical coupling compensates for any increase in curvature occurring in the region of the cone itself. Thus, it seems that keratoconus is not a true ectasia as is keratoglobus, but rather a specialized type of warpage, at least in mild to moderate forms of the disease. Arch Ophthalmol. 2000;118:1179-1186

Algorithms↗

PubMed1999

Alternative sulfonylurea receptor expression defines metabolic sensitivity of K-ATP channels in dopaminergic midbrain neurons.

ATP-sensitive potassium (K-ATP) channels couple the metabolic state to cellular excitability in various tissues. Several isoforms of the K-ATP channel subunits, the sulfonylurea receptor (SUR) and inwardly rectifying K channel (Kir6.X), have been cloned, but the molecular composition and functional diversity of native neuronal K-ATP channels remain unresolved. We combined functional analysis of K-ATP channels with expression profiling of K-ATP subunits at the level of single substantia nigra (SN) neurons in mouse brain slices using an RT-multiplex PCR protocol. In contrast to GABAergic neurons, single dopaminergic SN neurons displayed alternative co-expression of either SUR1, SUR2B or both SUR isoforms with Kir6.2. Dopaminergic SN neurons expressed alternative K-ATP channel species distinguished by significant differences in sulfonylurea affinity and metabolic sensitivity. In single dopaminergic SN neurons, co-expression of SUR1 + Kir6.2, but not of SUR2B + Kir6.2, correlated with functional K-ATP channels highly sensitive to metabolic inhibition. In contrast to wild-type, surviving dopaminergic SN neurons of homozygous weaver mouse exclusively expressed SUR1 + Kir6.2 during the active period of dopaminergic neurodegeneration. Therefore, alternative expression of K-ATP channel subunits defines the differential response to metabolic stress and constitutes a novel candidate mechanism for the differential vulnerability of dopaminergic neurons in response to respiratory chain dysfunction in Parkinson's disease.

ATP-Binding Cassette Transporters↗

PubMed2000

Modeling of membrane excitability in gonadotropin-releasing hormone-secreting hypothalamic neurons regulated by Ca2+-mobilizing and adenylyl cyclase-coupled receptors.

Gonadotropin-releasing hormone (GnRH) secretion from native and immortalized hypothalamic neurons is regulated by endogenous Ca(2+)-mobilizing and adenylyl cyclase (AC)-coupled receptors. Activation of both receptor types leads to an increase in action potential firing frequency and a rise in the intracellular Ca(2+) concentration ([Ca(2+)](i)) and neuropeptide secretion. The stimulatory action of Ca(2+)-mobilizing agonists on voltage-gated Ca(2+) influx is determined by depletion of the intracellular Ca(2+) pool, whereas AC agonist-stimulated Ca(2+) influx occurs independently of stored Ca(2+) and is controlled by cAMP, possibly through cyclic nucleotide-gated channels. Here, experimental records from immortalized GnRH-secreting neurons are simulated with a mathematical model to determine the requirements for generating complex membrane potential (V(m)) and [Ca(2+)](i) responses to Ca(2+)-mobilizing and AC agonists. Included in the model are three pacemaker currents: a store-operated Ca(2+) current (I(SOC)), an SK-type Ca(2+)-activated K(+) current (I(SK)), and an inward current that is modulated by cAMP and [Ca(2+)](i) (I(d)). Spontaneous electrical activity and Ca(2+) signaling in the model are predominantly controlled by I(d), which is activated by cAMP and inhibited by high [Ca(2+)](i). Depletion of the intracellular Ca(2+) pool mimics the receptor-induced activation of I(SOC) and I(SK), leading to an increase in the firing frequency and Ca(2+) influx after a transient cessation of electrical activity. However, increasing the activity of I(d) simulates the experimental response to forskolin-induced activation of AC. Analysis of the behaviors of I(SOC), I(d), and I(SK) in the model reveals the complexity in the interplay of these currents that is necessary to fully account for the experimental results.

Action Potentials↗

PubMed1999

Mechanisms of altered excitation-contraction coupling in canine tachycardia-induced heart failure, I: experimental studies.

Pacing-induced heart failure in the dog recapitulates many of the electrophysiological and hemodynamic abnormalities of the human disease; however, the mechanisms underlying altered Ca2+ handling have not been investigated in this model. We now show that left ventricular midmyocardial myocytes isolated from dogs subjected to 3 to 4 weeks of rapid pacing have prolonged action potentials and Ca2+ transients with reduced peaks, but durations approximately 3-fold longer than controls. To discriminate between action potential effects on Ca2+ kinetics and direct changes in Ca2+ regulatory processes, voltage-clamp steps were used to examine the time constant for cytosolic Ca2+ removal (tauCa). tauCa was prolonged by just 35% in myocytes from failing hearts after fixed voltage steps in physiological solutions (tauCa control, 216+/-25 ms, n=17; tauCa failing, 292+/-23 ms, n=22; P<0.05), but this difference was markedly accentuated when Na+/Ca2+ exchange was eliminated (tauCa control, 282+/-30 ms, n=13; tauCa failing, 576+/-83 ms, n=11; P<0. 005). Impaired sarcoplasmic reticular (SR) Ca2+ uptake and a greater dependence on Na+/Ca2+ exchange for cytosolic Ca2+ removal was confirmed by inhibiting SR Ca2+ ATPase with cyclopiazonic acid, which slowed Ca2+ removal more in control than in failing myocytes. beta-Adrenergic stimulation of SR Ca2+ uptake in cells from failing hearts sufficed only to accelerate tauCa to the range of unstimulated controls. Protein levels of SERCA2a, phospholamban, and Na+/Ca2+ exchanger revealed a pattern of changes qualitatively similar to the functional measurements; SERCA2a and phospholamban were both reduced in failing hearts by 28%, and Na+/Ca2+ exchange protein was increased 104% relative to controls. Thus, SR Ca2+ uptake is markedly downregulated in failing hearts, but this defect is partially compensated by enhanced Na+/Ca2+ exchange. The alterations are similar to those reported in human heart failure, which reinforces the utility of the pacing-induced dog model as a surrogate for the human disease.

Action Potentials↗

PubMed2006

Kir3-based inward rectifier potassium current: potential role in atrial tachycardia remodeling effects on atrial repolarization and arrhythmias.

BACKGROUND: We previously characterized a novel K+ current (IKH) with properties of constitutively active acetylcholine-related current in dog atrium. I(KH) is sensitive to tertiapin-Q (IC50 approximately 10 nmol/L), a highly selective Kir3 current blocker. This study assessed the role of IKH in atrial tachycardia (AT)-remodeled canine left atrium (LA) with the use of tertiapin-Q as a probe. METHODS AND RESULTS: Dogs were subjected to 7 to 13 days of AT (400 bpm). Coronary-perfused LA preparations were studied intact or subjected to cardiomyocyte isolation. IKH was recorded with patch-clamp methods. AT pacing increased time-dependent hyperpolarization-activated current (IKH) at -110 mV from -1.8+/-0.3 (control) to -3.4+/-0.5 pA/pF (AT) and the 100-nmol/L tertiapin-sensitive component from -1.5+/-0.4 (control) to -3.3+/-0.6 pA/pF (AT). Prolonged atrial tachyarrhythmias could be induced with single extrastimuli in AT-remodeled, but not control, preparations, reflecting the atrial fibrillation-promoting effects of AT remodeling. In AT-remodeled preparations, tachyarrhythmia duration averaged 11.0+/-5.2 seconds, with a cycle length of 108+/-6 ms. Tertiapin-Q decreased tachyarrhythmia duration (to 0.6+/-0.1 second; P<0.001) and increased tachyarrhythmia cycle length (to 175+/-10 ms; P<0.001). Atrial action potential duration (APD) was increased 65+/-6% by tertiapin in AT-remodeled hearts versus 19+/-2% (P<0.001) in control. In 2 AT-remodeled preparations, tachyarrhythmia lasted uninterrupted for >20 minutes; tertiapin-Q slowed and then terminated arrhythmia in both. Tertiapin had no effect on left ventricular cardiomyocyte currents or APD. CONCLUSIONS: AT remodeling increases IKH, and a highly selective Kir3 current antagonist, tertiapin-Q, increases APD and suppresses atrial tachyarrhythmias in AT-remodeled preparations without affecting ventricular electrophysiology. Constitutive acetylcholine-related K+ current contributes to AT-remodeling effects in dogs and is a potentially interesting antiarrhythmic target.

Animals↗