Maurice Gola

Selective Blocking of Voltage-Gated K+ Channels Improves Experimental Autoimmune Encephalomyelitis and Inhibits T Cell Activation

Kaliotoxin (KTX), a blocker of voltage-gated potassium channels (Kv), is highly selective for Kv1.1 and Kv1.3. First, Kv1.3 is expressed by T lymphocytes. Blockers of Kv1.3 inhibit T lymphocyte activation. Second, Kv1.1 is found in paranodal regions of axons in the central nervous system. Kv blockers improve the impaired neuronal conduction of demyelinated axons in vitro and potentiate the synaptic transmission. Therefore, we investigated the therapeutic properties of KTX via its immunosuppressive and symptomatic neurological effects, using experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. The T line cells used to induce adoptive EAE were myelin basic protein (MBP)-specific, constitutively contained mRNA for Kv1.3. and expressed Kv1.3. These channels were shown to be blocked by KTX. Activation is a crucial step for MBP T cells to become encephalitogenic. The addition of KTX during Ag-T cell activation led to a great reduction in the MBP T cell proliferative response, in the production of IL-2 and TNF, and in Ca2+ influx. Furthermore, the addition of KTX during T cell activation in vitro led a decreased encephalitogenicity of MBP T cells. Moreover, KTX injected into Lewis rats impaired T cell function such as the delayed-type hypersensitivity. Lastly, the administration of this blocker of neuronal and lymphocyte channels to Lewis rats improved the symptoms of EAE. We conclude that KTX is a potent immunosuppressive agent with beneficial effects on the neurological symptoms of EAE.

ABC1, an ATP Binding Cassette Transporter Required for Phagocytosis of Apoptotic Cells, Generates a Regulated Anion Flux after Expression in Xenopus laevis Oocytes

The ATP binding cassette transporter ABC1 is a 220-kDa glycoprotein expressed by macrophages and required for engulfment of cells undergoing programmed cell death. Since members of this family of proteins such as P-glycoprotein and cystic fibrosis transmembrane conductance regulator share the ability to transport anions, we have investigated the transport capability of ABC1 expressed in Xenopus oocytes using iodide efflux and voltage-clamp techniques. We report here that ABC1 generates an anion flux sensitive to glibenclamide, sulfobromophthalein, and blockers of anion transporters. The anion flux generated by ABC1 is up-regulated by orthovanadate, cAMP, protein kinase A, and okadaic acid. In other ABC transporters, mutating the conserved lysine in the nucleotide binding folds was found to severely reduce or abolish hydrolysis of ATP, which in turn altered the activity of the transporter. In ABC1, replacement of the conserved lysine 1892 in the Walker A motif of the second nucleotide binding fold increased the basal ionic flux, did not alter the pharmacological inhibitory profile, but abolished the response to orthovanadate and cAMP agonists. Therefore, we conclude that ABC1 is a cAMP-dependent and sulfonylurea-sensitive anion transporter.

Development of substituted Benzo[c]quinolizinium compounds as novel activators of the cystic fibrosis chloride channel.

Chloride channels play an important role in the physiology and pathophysiology of epithelia, but their pharmacology is still poorly developed. We have chemically synthesized a series of substituted benzo[c]quinolizinium (MPB) compounds. Among them, 6-hydroxy-7-chlorobenzo[c]quinolizinium (MPB-27) and 6-hydroxy-10-chlorobenzo[c]quinolizinium (MPB-07), which we show to be potent and selective activators of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. We examined the effect of MPB compounds on the activity of CFTR channels in a variety of established epithelial and nonepithelial cell systems. Using the iodide efflux technique, we show that MPB compounds activate CFTR chloride channels in Chinese hamster ovary (CHO) cells stably expressing CFTR but not in CHO cells lacking CFTR. Single and whole cell patch clamp recordings from CHO cells confirm that CFTR is the only channel activated by the drugs. Ussing chamber experiments reveal that the apical addition of MPB to human nasal epithelial cells produces a large increase of the short circuit current. This current can be totally inhibited by glibenclamide. Whole cell experiments performed on native respiratory cells isolated from wild type and CF null mice also show that MPB compounds specifically activate CFTR channels. The activation of CFTR by MPB compounds was glibenclamide-sensitive and 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid-insensitive. In the human tracheal gland cell line MM39, MPB drugs activate CFTR channels and stimulate the secretion of the antibacterial secretory leukoproteinase inhibitor. In submandibular acinar cells, MPB compounds slightly stimulate CFTR-mediated submandibular mucin secretion without changing intracellular cAMP and ATP levels. Similarly, in CHO cells MPB compounds have no effect on the intracellular levels of cAMP and ATP or on the activity of various protein phosphatases (PP1, PP2A, PP2C, or alkaline phosphatase). Our results provide evidence that substituted benzo[c]quinolizinium compounds are a novel family of activators of CFTR and of CFTR-mediated protein secretion and therefore represent a new tool to study CFTR-mediated chloride and secretory functions in epithelial tissues.

Analysis of whole‐cell currents by patch clamp of guinea‐pig myenteric neurones in intact ganglia

Whole‐cell patch‐clamp recordings taken from guinea‐pig duodenal myenteric neurones within intact ganglia were used to determine the properties of S and AH neurones. Major currents that determine the states of AH neurones were identified and quantified. S neurones had resting potentials of −47 ± 6 mV and input resistances (Rin) of 713 ± 49 MΩ at voltages ranging from −90 to −40 mV. At more negative levels, activation of a time‐independent, caesium‐sensitive, inward‐rectifier current (IKir) decreased Rin to 103 ± 10 MΩ. AH neurones had resting potentials of −57 ± 4 mV and Rin was 502 ± 27 MΩ. Rin fell to 194 ± 16 MΩ upon hyperpolarization. This decrease was attributable mainly to the activation of a cationic h current, Ih, and to IKir. Resting potential and Rin exhibited a low sensitivity to changes in [K+]o in both AH and S neurones. This indicates that both cells have a low background K+ permeability. The cationic current, Ih, contributed about 20 % to the resting conductance of AH neurones. It had a half‐activation voltage of −72 ± 2 mV, and a voltage sensitivity of 8.2 ± 0.7 mV per e‐fold change. Ih has relatively fast, voltage‐dependent kinetics, with on and off time constants in the range of 50–350 ms. AH neurones had a previously undescribed, low threshold, slowly inactivating, sodium‐dependent current that was poorly sensitive to TTX. In AH neurones, the post‐action‐potential slow hyperpolarizing current, IAHP, displayed large variation from cell to cell. IAHP appeared to be highly Ca2+ sensitive, since its activation with either membrane depolarization or caffeine (1 mm) was not prevented by perfusing the cell with 10 mm BAPTA. We determined the identity of the Ca2+ channels linked to IAHP. Action potentials of AH neurones that were elongated by TEA (10 mm) were similarly shortened and IAHP was suppressed with each of the three Ω‐conotoxins GVIA, MVIIA and MVIIC (0.3–0.5 μm), but not with Ω‐agatoxin IVA (0.2 μm). There was no additivity between the effects of the three conotoxins, which indicates the presence of N‐ but not of P/Q‐type Ca2+ channels. A residual Ca2+ current, resistant to all toxins, but blocked by 0.5 mm Cd2+, could not generate IAHP. This patch‐clamp study, performed on intact ganglia, demonstrates that the AH neurones of the guinea‐pig duodenum are under the control of four major currents, IAHP, Ih, an N‐type Ca2+ current and a slowly inactivating Na+ current.

Large conductance Ca(2+)-activated K+ channels are involved in both spike shaping and firing regulation in Helix neurones.

1. The role of BK‐type calcium‐dependent K+ channels (K+Ca) in cell firing regulation was evaluated by performing whole‐cell voltage clamp and patch clamp experiments on the U cell neurones in the snail Helix pomatia. These cells were selected because most of the repolarizing K+ current flowed through K+Ca channels. 2. U cells generated overshooting Ca(2+)‐dependent spikes in Na(+)‐free saline. In response to prolonged depolarizing current, they fired a limited number of spikes of decreasing amplitude, and behaved like fast‐adapting or phasic neurones. 3. Under voltage clamp conditions, the K+Ca current had a slow onset at voltages that induced small Ca2+ entries. By manipulating the Ca2+ entry (either with appropriate voltage programmes or by changing the Ca2+ content of the bath), the K+Ca channel opening was found to be rate limited by the Ca2+ binding step and not by the voltage‐dependent conformational change to the open state. 4. Despite the slow activation rate observed in voltage‐clamped cells, 25‐30% of the available K+Ca current was found to be active during isolated spikes. These data were based on patch clamp, spike‐like voltage clamp and hybrid current clamp‐voltage clamp experiments. 5. The fact that spikes led the slowly rising K+Ca current to shift into a fast activating mode was accounted for by the large surge of Ca2+ current concomitant with spike upstroke. The early calcium surge resulted in local increases in cytosolic calcium, which speeded up the binding of calcium ions to the closed K+Ca channels. From changes in the null Ca2+ current voltage, it was calculated that the submembrane [Ca2+]i increase to 50‐80 microM during the spike. 6. Due to their fast voltage dependence, K+Ca channels appeared to play no role in shaping the interspike trajectory. 7. Even in the fast activating mode, the K+Ca current had a finite rate of rise and was not involved in repolarizing short duration Na(+‐dependent action potentials. The current became more and more active, however, when voltage‐gated K+ channels were progressively inactivated during firing. 8. The fast adaptation exhibited by U cells upon sustained depolarization was not paralleled by a recruitment of K+Ca channels because of the cumulative Ca2+ entries. During a spike burst, the K+Ca current progressively overlapped the depolarizing Ca2+ current, which ultimately stopped the firing. The early opening of K+Ca channels was ascribed to residual Ca2+ accumulation that kept part of the channels in the Ca(2+)‐bound state ready to be opened quickly by cell depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

Structural basis for specificity and potency of xanthine derivatives as activators of the CFTR chloride channel

On the basis of their structure, we compared the ability of 35 xanthine derivatives to activate the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel stably expressed in chinese hamster ovary (CHO) cells using the cell‐attached patch clamp and iodide efflux techniques. Activation of CFTR channels was obtained with 3‐mono, 1,3‐di or 1,3,7‐tri‐substituted alkyl xanthine derivatives (enprofylline, theophylline, aminophylline, IBMX, DPMX and pentoxifylline). By contrast, xanthine derivatives substituted at the C8‐ or N9‐position failed to open CFTR channels. The CFTR chloride channel activity was blocked by glibenclamide (100 μM) but not by DIDS (100 μM). Activation of CFTR by xanthines was not mimicked by the calcium ionophore A23187, adenosine, UTP, ATP or the specific phosphodiesterase inhibitors rolipram, Ro 20‐1724 and milrinone. In addition, we found no correlation between the effect of xanthines on CFTR and on the cellular cyclic AMP or ATP levels. We then synthesized a series of 3,7‐dimethyl‐1‐alkyl xanthine derivatives; among them, 3,7‐dimethyl‐1‐propyl xanthine and 3,7‐dimethyl‐1‐isobutyl xanthine both activated CFTR channels without increasing the intracellular cyclic AMP level, while the structurally related 3,7‐dimethyl‐1‐(2‐propenyl) xanthine and 3,7‐dimethyl‐1‐(oxiranyl methyl) xanthine were inactive. Our findings delineate a novel function for xanthine compounds and identify the molecular features that enable xanthine activation of CFTR. These results may be useful in the development of new molecules for studying the pharmacology of chloride channels.

Possible regulation of CFTR‐chloride channels by membrane‐bound phosphatases in pancreatic duct cells

We have studied CFTR‐Cl− channels in non‐CF CAPAN‐1 and in CFTR‐transfected CFPAC‐PLJ‐CFTR‐6 epithelial cells from human pancreas. Theophylline and IBMX induced the opening of cell‐attached CFTR‐Cl− channels. Theophylline, IBMX and the alkaline phosphatase (AP) inhibitor levamisole enhanced the activity of excised channels and reduced by 70–75% the apical membrane‐associated APs activity. Okadaic acid had no effect on APs and channel activities. A polyclonal anti‐alkaline phosphatase antibody (which detected apical APs) reduced APs activity and activated quiescent excised chloride channels. These results suggest that CFTR channels may be regulated by membrane‐bound phosphatases.

Anion channels in a human pancreatic cancer cell line (Capan-1) of ductal origin

Transepithelial solute transport and bicarbonate secretion are major functions of pancreatic duct cells, and both functions are thought to involve the presence of chloride channels in the apical membrane of the cell. After being isolated from a human pancreatic adenocarcinoma, the Capan-1 cell line conserves most of the properties of ductal cells and thus constitutes a useful system for investigating the role of chloride channels. Using patch-clamp techniques, we identified three different chloride-selective channels in the apical membrane of confluent Capan-1 cells. Two were non-rectifying chloride channels with low (50 pS) and high (350 pS) unitary conductances. Both channels were active in cell-attached recordings, and they were consistently located together in the same patch. Maxi Cl− channels displayed multiple subconductance states, and were reversibly inactivated by either positive or negative voltage changes, which indicates that they were optimally opened at the cell resting potential. The third was an outwardly rectifying chloride channel with a unitary conductance of 38 pS and 70 pS at negative and positive potentials respectively. Rectifying Cl− channels were clustered in discrete loci. They were silent in situ, but became active after patch excision. In inside-out excised patches, the three channels displayed a high selectivity for Cl− over monovalent cations (Na+ and K+) and gluconate. They were blocked by 20–200 μM 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and were insensitive to changes in the Ca2+ concentration. Our results show that the apical membrane of Capan-1 cells contains a high density of chloride channels; these channels may provide pathways for transepithelial solute transport as well as for bicarbonate secretion.

Ca2(+)‐activated K+ current involvement in neuronal function revealed by in situ single‐channel analysis in Helix neurones.

1. The properties of single calcium‐activated potassium channels (or C‐channels) were studied in cell‐attached patches using the patch‐clamp technique. Experiments were performed on identified Ca2(+)‐dependent U cells in juvenile specimens (1‐2 months old) of Helix aspersa. 2. The criteria used to identify C‐channels were based on comparison between macroscopic C‐currents and currents reconstructed from unitary recordings. Both currents had a slow activation rate at large positive potentials which turned into fast activation after large Ca2+ entries. Both currents were blocked by intracellularly injected EGTA. 3. The unitary conductance in normal (5 mM) or reduced (0.5 mM) [K+]o ranged from 24 to 65 pS (mean +/‐ S.D., 48 +/‐ 13; n = 64). With 85‐110 mM [K+]o, which is approximately equal to the internal [K+], the conductance was 64 pS and the reversal potential was approximately 0 mV. 4. C‐channels in U cells were distributed in clusters of three to ten channels (mean 5.05 channels in seventy‐five patches). Calcium channels were present in patches containing clustered C‐channels. C‐channels within clusters behaved independently. 5. With patch electrode containing 8 mM‐calcium, C‐channels opened transiently upon patch depolarization. Reopenings in quiescent depolarized patches were induced by whole‐cell spikes triggered by current pulses applied to an intracellular electrode. Apparent inactivation of C‐channels in depolarized patches was in fact due to a decrease in [Ca2+]i resulting from inactivation of Ca2+ channels. 6. Calcium‐free saline solutions in the patch electrodes prevented C‐channels from opening upon patch depolarization. Entry of calcium through the surrounding membrane induced delayed openings in the patch. Peak opening probability Po occurred 330 +/‐ 30 ms after a brief Ca2+ entry with a lag period of 50‐80 ms. With patch electrodes filled with Ca2(+)‐containing saline solutions and under conditions which maximized C‐channel opening, peak Po was reached in 20‐50 ms. The same value was observed for the whole‐cell C‐current. 7. The peak Po at a given patch potential and in response to a whole‐cell spike was not altered by a previous long‐lasting patch depolarization, or by producing several successive Ca2+ entries. Thus, C‐channels did not appear to be inactivated by depolarization or increase in [Ca2+]i. 8. C‐channels were found to be relatively highly voltage dependent, with an e‐fold increase in Po per 14.9 mV increase in potential.(ABSTRACT TRUNCATED AT 400 WORDS)

Specific distribution of sodium channels in axons of rat embryo spinal motoneurones.

1 The distribution of Na+ channels and development of excitability were investigated in vitro in purified spinal motoneurones obtained from rat embryos at E14, using electrophysiological, immunocytochemical and autoradiographical methods. 2 One hour after plating the motoneurones (DIV0), only somas were present. They expressed a robust delayed rectifier K+ current (IDR) and a fast‐inactivating A‐type K+ current (IA). The rapid neuritic outgrowth was paralleled by the emergence of a fast‐activating TTX‐sensitive sodium current (INa), and by an increase in both K+ currents. 3 The change in the three currents was measured daily, up to DIV8. The large increase in INa observed after DIV2 was accompanied by the onset of excitability. Spontaneous activity was observed as from DIV6. 4 The occurrence of axonal differentiation was confirmed by the fact that (i) only one neurite per motoneurone generated antidromic action potentials; and (ii) 125I‐α‐scorpion toxin binding, a specific marker of Na+ channels, labelled only one neurite and the greatest density was observed in the initial segment. Na+ channels therefore selectively targeted the axon and were absent from the dendrites and somas. 5 The specific distribution of Na+ channels was detectable as soon as the neurites began to grow. When the neuritic outgrowth was blocked by nocodazole, no INa developed. 6 It was concluded that, in spinal embryonic motoneurone in cell culture, Na+ channels, the expression of which starts with neuritic differentiation, are selectively addressed to the axonal process, whereas K+ channels are present in the soma prior to the neuritic outgrowth.