Ação de anestésicos gerais em canais iônicos

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Molecular, 2016.

Le pore oméga, un défaut biophysique commun aux mutations des canaux Nav1.5 causant le développement des arythmies associées à la cardiomyopathie dilatée

Les canaux sodiques dépendants du voltage (Nav) sont des protéines transmembranaires largement exprimées au sein de l’organisme. Ils sont responsables de l’initiation des potentiels d’action au niveau des cellules excitables et régissent ainsi de nombreuses fonctions physiologiques telles que les fonctions cognitives et sensorielles, les fonctions motrices et la fonction cardiaque. Au niveau du coeur, le sous-type Nav1.5 est majoritairement exprimé à la surface des cardiomyocytes. Leurs dysfonctions sont traditionnellement associées à de nombreux troubles électriques cardiaques. Des mutations de ces canaux ont récemment été reliées au développement d’un phénotype clinique complexe associant diverses arythmies et la cardiomyopathie dilatée (DCM), une atteinte morphologique. L’objectif de mon doctorat a donc été l’identification mais aussi la caractérisation d’un potentiel défaut biophysique commun à l’ensemble des mutations Nav1.5 associées au développement de ce phénotype clinique atypique. Premièrement, nous nous sommes intéressés à deux mutations des canaux Nav1.5 retrouvées chez des patients atteints de DCM, et dont les altérations biophysiques ont été décrites comme divergentes. L’étude parallèle de ces deux mutants nous a amenés à identifier une caractéristique commune : la création d’une nouvelle voie de perméation alternative au sein des canaux Nav1.5, le pore oméga. Dans un second temps, nous avons souhaité consolider l’association entre la création du pore oméga et le développement pathologique. Cette seconde étude portant sur deux autres mutants Nav1.5 a permis de confirmer l’apparition d’un pore oméga et ainsi d’accroître la suspicion du caractère délétère de ce pore oméga. Finalement, à l’aide d’une cinquième mutation des canaux Nav1.5, nous avons investigué les conséquences physiopathologiques de la création d’un pore oméga. Cette étude, a clairement démontré les conséquences néfastes d’un tel pore au niveau de l’homéostasie ionique cellulaire. Ces perturbations se répercutent par la suite sur les signaux électriques, les propriétés morphologiques mais aussi fonctionnelles des cardiomyocytes. Les études menées lors de mon doctorat ont ainsi abouti à l’identification du pore oméga comme étant une caractéristique biophysique commune aux mutations des canaux Nav1.5 associées au développement des arythmies et de la dilatation cardiaque.

Elevation of intracellular calcium by muscarinic receptor activation induces a block of voltage-activated rat ether-à-go-go channels in a stably transfected cell line.

We have studied the properties of r-eag voltage-activated potassium channels in a stably transfected human embryonic kidney cell line. It was found that r-eag channels are rapidly and reversibly inhibited by a rise in intracellular calcium from 30 to 300 nM. The inhibition does not appear to depend on the activity of calcium-dependent kinases and phosphatases. The effect of calcium on r-eag channel activity was studied in inside-out membrane patches. Calcium inhibited r-eag channel activity with a mean IC50 of 67 nM. Activation of muscarinic receptors, generating calcium oscillations in the transfected cells, induced a synchronous inhibition of r-eag mediated outward currents. This shows that calcium can mediate r-eag current inhibition following muscarinic receptor activation. The data indicate that r-eag channels are calcium-inhibitable voltage-activated potassium channels.

Independent roles of calcium and voltage-dependent potassium currents in controlling spike frequency adaptation in lateral amygdala pyramidal neurons

The calcium-dependent afterhyperpolarization (AHP) that follows trains of action potentials is responsible for controlling action potential firing patterns in many neuronal cell types. We have previously shown that the slow AHP contributes to spike frequency adaptation in pyramidal neurons in the rat lateral amygdala. In addition, a dendritic voltage-gated potassium current mediated by Kv1.2-containing channels also suppresses action potential firing in these neurons. In this paper we show that this voltage-gated potassium current and the slow AHP act together to control spike frequency adaptation in lateral amygdala pyramidal neurons. The two currents have similar effects on action potential number when firing is evoked either by depolarizing current injections or by synaptic stimulation. However, they differ in their control of firing frequency, with the voltage-gated potassium current but not the slow AHP determining the initial frequency of action potential firing. This dual mechanism of controlling firing patterns is unique to lateral amygdala neurons and is likely to contribute to the very low levels of firing seen in lateral amygdala neurons in vivo.

Signal Transduction, Plasma Membrane Calcium Movements, and Pigment Translocation in Freshwater Shrimp Chromatophores

Crustacean color change results from the differential translocation of chromatophore pigments, regulated by neurosecretory peptides like red pigment concentrating hormone (RPCH) that, in the red ovarian chromatophores of the freshwater shrimp Macrobrachium olfersi, triggers pigment aggregation via increased cytosolic cGMP and Ca(2+) of both smooth endoplasmatic reticulum (SER) and extracellular origin. However, Ca(2+) movements during RPCH signaling and the mechanisms that regulate intracellular [Ca(2+)] are enigmatic. We investigate Ca(2+) transporters in the chromatophore plasma membrane and Ca(2+) movements that occur during RPCH signal transduction. Inhibition of the plasma membrane Ca(2+)-ATPase by La(3+) and indirect inhibition of the Na(+)/Ca(2+) exchanger by ouabain induce pigment aggregation, revealing a role for both in Ca(2+) extrusion. Ca(2+) channel blockade by La(3+) or Cd(2+) strongly inhibits slow-phase RPCH-triggered aggregation during which pigments disperse spontaneously. L-type Ca(2+) channel blockade by gabapentin markedly reduces rapid-phase translocation velocity; N- or P/Q-type blockade by omega-conotoxin MVIIC strongly inhibits RPCH-triggered aggregation and reduces velocity, effects revealing RPCH-signaled influx of extracellular Ca(2+). Plasma membrane depolarization, induced by increasing external K(+) from 5 to 50 mM, produces Ca(2+)-dependent pigment aggregation, whereas removal of K(+) from the perfusate causes pigment hyperdispersion, disclosing a clear correlation between membrane depolarization and pigment aggregation; K(+) channel blockade by Ba(2+) also partially inhibits RPCH action. We suggest that, during RPCH signal transduction, Ca(2+) released from the SER, together with K(+) channel closure, causes chromatophore membrane depolarization, leading to the opening of predominantly N- and/or P/Q-type voltage-gated Ca(2+) channels, and a Ca(2+)/cGMP cascade, resulting in pigment aggregation. J. Exp. Zool. 313A:605-617, 2010. (C) 2010 Wiley-Liss, Inc.

Detrusor overactivity is associated with downregulation of large-conductance calcium- and voltage-activated potassium channel protein

Chang S, Gomes CM, Hypolite JA, Marx J, Alanzi J, Zderic SA, Malkowicz B, Wein AJ, Chacko S. Detrusor overactivity is associated with downregulation of large-conductance calcium-and voltage-activated potassium channel protein. Am J Physiol Renal Physiol 298: F1416-F1423, 2010. First published April 14, 2010; doi: 10.1152/ajprenal.00595.2009.-Large-conductance voltage-and calcium-activated potassium (BK) channels have been shown to play a role in detrusor overactivity (DO). The goal of this study was to determine whether bladder outlet obstructioninduced DO is associated with downregulation of BK channels and whether BK channels affect myosin light chain 20 (MLC(20)) phosphorylation in detrusor smooth muscle (DSM). Partial bladder outlet obstruction (PBOO) was surgically induced in male New Zealand White rabbits. The rabbit PBOO model shows decreased voided volumes and increased voiding frequency. DSM from PBOO rabbits also show enhanced spontaneous contractions compared with control. Both BK channel alpha- and beta-subunits were significantly decreased in DSM from PBOO rabbits. Immunostaining shows BK beta mainly expressed in DSM, and its expression is much less in PBOO DSM compared with control DSM. Furthermore, a translational study was performed to see whether the finding discovered in the animal model can be translated to human patients. The urodynamic study demonstrates several overactive DSM contractions during the urine-filling stage in benign prostatic hyperplasia (BPH) patients with DO, while DSM is very quiet in BPH patients without DO. DSM biopsies revealed significantly less BK channel expression at both mRNA and protein levels. The degree of downregulation of the BK beta-subunit was greater than that of the BK alpha-subunit, and the downregulation of BK was only associated with DO, not BPH. Finally, the small interference (si) RNA-mediated downregulation of the BK beta-subunit was employed to study the effect of BK depletion on MLC(20) phosphorylation. siRNA-mediated BK channel reduction was associated with an increased MLC(20) phosphorylation level in cultured DSM cells. In summary, PBOO-induced DO is associated with downregulation of BK channel expression in the rabbit model, and this finding can be translated to human BPH patients with DO. Furthermore, downregulation of the BK channel may contribute to DO by increasing the basal level of MLC(20) phosphorylation.

Concentration dependence of sodium permeation and sodium ion interactions in the cyclic AMP-gated channels of mammalian olfactory receptor neurons

The dependence of currents through the cyclic nucleotide-gated (CNG) channels of mammalian olfactory receptor neurons (ORNs) on the concentration of NaCl was studied in excised inside-out patches from their dendritic knobs using the patch-clamp technique. With a saturating concentration (100 mu M) of adenosine 3', 5'-cyclic monophosphate (cAMP), the changes in the reversal potential of macroscopic currents were studied at NaCl concentrations from 25 to 300 mM. In symmetrical NaCl solutions without the addition of divalent cations, the current-voltage relations were almost linear, reversing close to O mV. When the external NaCl concentration was maintained at 150 mM and the internal concentrations were varied, the reversal potentials of the cAMP-activated currents closely followed the Na+ equilibrium potential indicating that P-Cl/P-Na approximate to 0. However, at low external NaCl concentrations (less than or equal to 100 mM) there was some significant chloride permeability. Our results further indicated that Na+ currents through these channels: (i) did not obey the independence principle; (ii) showed saturation kinetics with K(m)s in the range of 100-150 mM and (iii) displayed a lack of voltage dependence of conductance in asymmetric solutions that suggested that ion-binding sites were situated midway along the channel. Together, these characteristics indicate that the permeation properties of the olfactory CNG channels are significantly different from those of photoreceptor CNG channels.

Progression of Diet-Induced Diabetes in C57BL6J Mice Involves Functional Dissociation of Ca(2+) Channels From Secretory Vesicles

OBJECTIVE The aim of the study was to elucidate the cellular mechanism underlying the suppression of glucose-induced insulin secretion in mice fed a high-fat diet (HFD) for 15 weeks. RESEARCH DESIGN AND METHODS-C57BL6J mice were fed a HFD or a normal diet (ND) for 3 or 15 weeks. Plasma insulin and glucose levels in vivo were assessed by intraperitoneal glucose tolerance test. Insulin secretion in vitro was studied using static incubations and a perfused pancreas preparation. Membrane currents, electrical activity, and exocytosis were examined by patch-clamp technique measurements. Intracellular calcium concentration ([Ca(2+)](i)) was measured by microfluorimetry. Total internal reflection fluorescence microscope (TIRFM) was used for optical imaging of exocytosis and submembrane depolarization-evoked [Ca(2+)](i). The functional data were complemented by analyses of histology and gene transcription. RESULTS After 15 weeks, but not 3 weeks, mice on HFD exhibited hyperglycemia and hypoinsulinemia. Pancreatic islet content and beta-cell area increased 2- and 1.5-fold, respectively. These changes correlated with a 20-50% reduction of glucose-induced insulin secretion (normalized to insulin content). The latter effect was not associated with impaired electrical activity or [Ca(2+)](i) signaling. Single-cell capacitance and TIRFM measurements of exocytosis revealed a selective suppression (>70%) of exocytosis elicited by short (50 ms) depolarization, whereas the responses to longer depolarizations were (500 ms) less affected. The loss of rapid exocytosis correlated with dispersion of Ca(2+) entry in HFD beta-cells. No changes in gene transcription of key exocytotic protein were observed. CONCLUSIONS HFD results in reduced insulin secretion by causing the functional dissociation of voltage-gated Ca(2+) entry from exocytosis. These observations suggest a novel explanation to the well-established link between obesity and diabetes. Diabetes 59:1192-1201, 2010

Coordinate regulation of gonadotropin-releasing hormone neuronal firing patterns by cytosolic calcium and store depletion

Elevation of cytosolic free Ca2+ concentration ([Ca2+]i) in excitable cells often acts as a negative feedback signal on firing of action potentials and the associated voltage-gated Ca2+ influx. Increased [Ca2+]i stimulates Ca2+-sensitive K+ channels (IK-Ca), and this, in turn, hyperpolarizes the cell and inhibits Ca2+ influx. However, in some cells expressing IK-Ca the elevation in [Ca2+]i by depletion of intracellular stores facilitates voltage-gated Ca2+ influx. This phenomenon was studied in hypothalamic GT1 neuronal cells during store depletion caused by activation of gonadotropin-releasing hormone (GnRH) receptors and inhibition of endoplasmic reticulum (Ca2+)ATPase with thapsigargin. GnRH induced a rapid spike increase in [Ca2+]i accompanied by transient hyperpolarization, followed by a sustained [Ca2+]i plateau during which the depolarized cells fired with higher frequency. The transient hyperpolarization was caused by the initial spike in [Ca2+]i and was mediated by apamin-sensitive IK-Ca channels, which also were operative during the subsequent depolarization phase. Agonist-induced depolarization and increased firing were independent of [Ca2+]i and were not mediated by inhibition of K+ current, but by facilitation of a voltage-insensitive, Ca2+-conducting inward current. Store depletion by thapsigargin also activated this inward depolarizing current and increased the firing frequency. Thus, the pattern of firing in GT1 neurons is regulated coordinately by apamin-sensitive SK current and store depletion-activated Ca2+ current. This dual control of pacemaker activity facilitates voltage-gated Ca2+ influx at elevated [Ca2+]i levels, but also protects cells from Ca2+ overload. This process may also provide a general mechanism for the integration of voltage-gated Ca2+ influx into receptor-controlled Ca2+ mobilization.

Molecular basis of fast inactivation in voltage and Ca2+-activated K+ channels: A transmembrane β-subunit homolog

Voltage-dependent and calcium-sensitive K+ (MaxiK) channels are key regulators of neuronal excitability, secretion, and vascular tone because of their ability to sense transmembrane voltage and intracellular Ca2+. In most tissues, their stimulation results in a noninactivating hyperpolarizing K+ current that reduces excitability. In addition to noninactivating MaxiK currents, an inactivating MaxiK channel phenotype is found in cells like chromaffin cells and hippocampal neurons. The molecular determinants underlying inactivating MaxiK channels remain unknown. Herein, we report a transmembrane β subunit (β2) that yields inactivating MaxiK currents on coexpression with the pore-forming α subunit of MaxiK channels. Intracellular application of trypsin as well as deletion of 19 N-terminal amino acids of the β2 subunit abolished inactivation of the α subunit. Conversely, fusion of these N-terminal amino acids to the noninactivating smooth muscle β1 subunit leads to an inactivating phenotype of MaxiK channels. Furthermore, addition of a synthetic N-terminal peptide of the β2 subunit causes inactivation of the MaxiK channel α subunit by occluding its K+-conducting pore resembling the inactivation caused by the “ball” peptide in voltage-dependent K+ channels. Thus, the inactivating phenotype of MaxiK channels in native tissues can result from the association with different β subunits.

Voltage-controlled gating in a large conductance Ca2+-sensitive K+channel (hslo)

Large conductance calcium- and voltage-sensitive K+ (MaxiK) channels share properties of voltage- and ligand-gated ion channels. In voltage-gated channels, membrane depolarization promotes the displacement of charged residues contained in the voltage sensor (S4 region) inducing gating currents and pore opening. In MaxiK channels, both voltage and micromolar internal Ca2+ favor pore opening. We demonstrate the presence of voltage sensor rearrangements with voltage (gating currents) whose movement and associated pore opening is triggered by voltage and facilitated by micromolar internal Ca2+ concentration. In contrast to other voltage-gated channels, in MaxiK channels there is charge movement at potentials where the pore is open and the total charge per channel is 4–5 elementary charges.

Interactive cloning with the SH3 domain of N-src identifies a new brain specific ion channel protein, with homology to Eag and cyclic nucleotide-gated channels

We have isolated a novel cDNA, that appears to represent a new class of ion channels, by using the yeast two-hybrid system and the SH3 domain of the neural form of Src (N-src) as a bait. The encoded polypeptide, BCNG-1, is distantly related to cyclic nucleotide-gated channels and the voltage-gated channels, Eag and H-erg. BCNG-1 is expressed exclusively in the brain, as a glycosylated protein of ≈132 kDa. Immunohistochemical analysis indicates that BCNG-1 is preferentially expressed in specific subsets of neurons in the neocortex, hippocampus, and cerebellum, in particular pyramidal neurons and basket cells. Within individual neurons, the BCNG-1 protein is localized to either the dendrites or the axon terminals depending on the cell type. Southern blot analysis shows that several other BCNG-related sequences are present in the mouse genome, indicating the emergence of an entire subfamily of ion channel coding genes. These findings suggest the existence of a new type of ion channel, which is potentially able to modulate membrane excitability in the brain and could respond to regulation by cyclic nucleotides.

Two types of calcium response limited to single spines in cerebellar Purkinje cells.

Of fundamental importance in understanding neuronal function is the unambiguous determination of the smallest unit of neuronal integration. It was recently suggested that a whole dendritic branchlet, including tens of spines, acts as the fundamental unit in terms of dendritic calcium dynamics in Purkinje cells. By contrast, we demonstrate that the smallest such unit is the single spine. The results show, by two-photon excited fluorescence laser scanning microscopy, that individual spines are capable of independent calcium activation. Moreover, two distinct spine populations were distinguished by their opposite response to membrane hyperpolarization. Indeed, in a subpopulation of spines calcium entry can also occur through a pathway other than voltage-gated channels. These findings challenge the assumption of a unique parallel fiber activation mode and prompt a reevaluation of the level of functional complexity ascribed to single neurons.

Human vascular to cardiac tissue selectivity of L- and T-type calcium channel antagonists

1 Voltage-operated calcium channel (VOCC) antagonists are effective antihypertensive and antianginal agents but they also depress myocardial contractility. 2 We compared four L-type calcium channel antagonists, felodipine, nifedipine, amlodipine and verapamil and a relatively T-type selective calcium channel antagonist, mibefradil, on human and rat isolated tissue assays to determine their functional vascular to cardiac tissue selectivity (V/C) ratio. 3 The V/C ratio was calculated as the ratio of the IC50 value of the antagonist that reduced (by 50%) submaximally contracted (K+ 62 mM) human small arteries from the aortic vasa vasorum (vascular, V) mounted in a myograph and the IC50 value of the antagonist that reduced (-)-isoprenaline (6 nM) submaximally stimulated human right atrial trabeculae muscle (cardiac, C) mounted in organ chambers. 4 The average pIC(50) Values (-log IC50 M) for the human vascular preparations were felodipine 8.30, nifedipine 7.78, amlodipine 6.64, verapamil 6.26 and mibefradil 6.22. The average pIC(50) values for the cardiac muscle were felodipine 7.21, nifedipine 6.95, verapamil 6.91, amlodipine 5.94, and mibefradil 4.61. 5 The V/C ratio calculated as antilog [pIC(50)V-pIC(50)C] is thus mibefradil 41, felodipine 12, nifedipine 7, amlodipine 5 and verapamil 0.2. 6 In rat small mesenteric arteries the pIC(50) values for the five drugs were similar to the values for human vasa vasorum arteries contracted by K+ 62 mM. However for methoxamine (10 mu M) contraction in the rat arteries the pIC(50) values were lower for felodipine 7.24 and nifedipine 6.23, but similar for verapamil 6.13, amlodipine 6.28 and mibefradil 5.91. 7 In conclusion in the human tissue assays, the putative T-channel antagonist mibefradil shows the highest vascular to cardiac selectivity ratio; some 3 fold higher than the dihydropyridine, felodipine, and some 200 fold more vascular selective than the phenylalkylamine, verapamil. This favourable vascular to cardiac selectivity for mibefradil, from a new chemical class of VOCC antagonist, may be explained by its putative T-channel selectivity.

Odorant-induced currents in intact patches from rat olfactory receptor neurons: Theory and experiment

Odorant-induced currents in mammalian olfactory receptor neurons have proved difficult to obtain reliably using conventional whole-cell recording. By using a mathematical model of the electrical circuit of the patch and rest-of-cell, we demonstrate how cell-attached patch measurements can be used to quantitatively analyze responses to odorants or a high (100 mM) K+ solution. High K+ induced an immediate current flux from cell to pipette, which was modeled as a depolarization of similar to 52 mV, close to that expected from the Nernst equation (56 mV), and no change in the patch conductance. By contrast, a cocktail of cAMP-stimulating odorants induced a current flux from pipette into cell following a significant (4-10 s) delay. This was modeled as an average patch conductance increase of 36 pS and a depolarization of 13 mV, Odorant-induced single channels had a conductance of 16 pS. In cells bathed with no Mg2+ and 0.25 mM Ca2+, odorants induced a current flow from cell to pipette, which was modeled as a patch conductance increase of similar to 115 pS and depolarization of similar to 32 mV, All these results are consistent with cAMP-gated cation channels dominating the odorant response, This approach, which provides useful estimates of odorant-induced voltage and conductance changes, is applicable to similar measurements in any small cells.