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M Gola

Publications and source records attributed to M Gola.

At least 19 recordsLinked to original sources
PubMed2003

Negative regulation of CFTR activity by extracellular ATP involves P2Y2 receptors in CFTR-expressing CHO cells.

Extracellular nucleotides exert autocrine/ paracrine effects on ion transport by activating P2 receptors. We studied the effects of extracellular ATP and UTP on the cystic fibrosis transmembrane conductance regulator (CFTR) channel stably expressed in Chinese Hamster Ovary cells (CHO-BQI cells). CFTR activity was measured using the (125I) iodide efflux technique and whole-cell patch-clamp recording in response to either forskolin or xanthine derivatives. Using RT-PCR and intracellular calcium concentration ([Ca2+]i) measurement, we showed that CHO-BQI cells express P2Y2 but not P2Y4 receptors. While ATP and UTP induced similar increases in [Ca2+]i, pre-addition by one of these two agonists desensitized the response for the other, suggesting that ATP- and UTP-induced [Ca2+]i increases were mediated by a common receptor, which was identified as the P2Y2 subtype. CFTR activity was reduced by ATP and UTP but not by ADP or adenosine applications. This inhibitory effect of ATP on CFTR activity was not due to a change in cAMP level. Furthermore, CFTR activation by forskolin or IBMX failed to promote [Ca2+]i increase, suggesting that CFTR activation did not generate an ATP release large enough to stimulate P2Y2 receptors. Taken together, our results show that endogenous P2Y2 receptor activation downregulates CFTR activity in a cAMP-independent manner in CHO cells.

Adenosine Triphosphate↗
PubMed2001

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 Ca(2+) 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.

Adoptive Transfer↗
PubMed2000

The soma and neurites of primary afferent neurons in the guinea-pig intestine respond differentially to deformation.

1. Intrinsic primary afferent neurons in the small intestine are exposed to distortion of their processes and of their cell bodies. Recordings of mechanosensitivity have previously been made from these neurons using intracellular microelectrodes, but this form of recording has not permitted detection of generator potentials from the processes, or of responses to cell body distortion. 2. We have developed a technique to record from enteric neurons in situ using patch electrodes. The mechanical stability of the patch recordings has allowed recording in cell-attached and whole cell configuration during imposed movement of the neurons. 3. Pressing with a fine probe initiated generator potentials (14 +/- 9 mV) from circumscribed regions of the neuron processes within the same myenteric ganglion, at distances from 100 to 500 microm from the cell body that was patched. Generator potentials persisted when synaptic transmission was blocked with high Mg2+, low Ca2+ solution. 4. Soma distortion, by pressing down with the whole cell recording electrode, inhibited action potential firing. Consistent with this, moderate intra-electrode pressure (10 mbar; 1 kPa) increased the opening probability of large-conductance (BK) potassium channels, recorded in cell-attached mode, but suction was not effective. In outside-out patches, suction, but not pressure, increased channel opening probability. Mechanosensitive BK channels have not been identified on other neurons. 5. The BK channels had conductances of 195 +/- 25 pS. Open probability was increased by depolarization, with a half-maximum activation at a patch potential of 20 mV and a slope factor of 10 mV. Channel activity was blocked by charybdotoxin (20 nM). 6. Stretch that increased membrane area under the electrode by 15 % was sufficient to double open probability. Similar changes in membrane area occur when the intestine changes diameter and wall tension under physiological conditions. Thus, the intestinal intrinsic primary afferent neurons are detectors of neurite distortion and of compression of the soma, these stimuli having opposite effects on neuron excitability.

Action Potentials↗
PubMed1999

Mapping the functional anatomy of BgK on Kv1.1, Kv1.2, and Kv1.3. Clues to design analogs with enhanced selectivity.

BgK is a peptide from the sea anemone Bunodosoma granulifera, which blocks Kv1.1, Kv1.2, and Kv1.3 potassium channels. Using 25 analogs substituted at a single position by an alanine residue, we performed the complete mapping of the BgK binding sites for the three Kv1 channels. These binding sites included three common residues (Ser-23, Lys-25, and Tyr-26) and a variable set of additional residues depending on the particular channel. Shortening the side chain of Lys-25 by taking out the four methylene groups dramatically decreased the BgK affinity to all Kv1 channels tested. However, the analog K25Orn displayed increased potency on Kv1.2, which makes this peptide a selective blocker for Kv1.2 (K(D) 50- and 300-fold lower than for Kv1.1 and Kv1.3, respectively). BgK analogs with enhanced selectivity could also be made by substituting residues that are differentially involved in the binding to some of the three Kv1 channels. For example, the analog F6A was found to be >500-fold more potent for Kv1.1 than for Kv1.2 and Kv1.3. These results provide new information about the mechanisms by which a channel blocker distinguishes individual channels among closely related isoforms and give clues for designing analogs with enhanced selectivity.

Amino Acid Substitution↗
PubMed1999

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.

Animals↗
PubMed1999

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-alpha-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.

Animals↗
PubMed1999

Cystic fibrosis transmembrane conductance regulator (CFTR) confers glibenclamide sensitivity to outwardly rectifying chloride channel (ORCC) in Hi-5 insect cells.

Increasing evidence is now accumulating for the involvement of the cystic fibrosis transmembrane conductance regulator (CFTR) in the control of the outwardly rectifying chloride channel (ORCC). We have examined the sensitivity of ORCC to the sulfonylurea drug glibenclamide in Hi-5 (Trichoplusia ni) insect cells infected with recombinant baculovirus expressing either wild-type CFTR, DeltaF508-CFTR or E. coli beta galactosidase cDNA and in control cells either infected with virus alone or uninfected. Iodide efflux and single channel patch-clamp experiments confirmed that forskolin and 1-methyl-3-isobutyl xanthine (IBMX) or 7-methyl-1,3 dipropyl xanthine (DPMX) activate CFTR channels (unitary conductance: 9.1 +/- 1.6 pS) only in cells expressing CFTR. In contrast, we identified 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS)-sensitive ORCC in excised membrane patches in any of the cells studied, with similar conductance (22 +/- 2.5 pS at -80 mV; 55 +/- 4.1 pS at +80 mV) and properties. In the presence of 500 microm SITS, channel open probability (Po) of ORCC was reversibly reduced to 0.05 +/- 0.01 in CFTR-cells, to 0.07 +/- 0.02 in non-CFTR expressing cells and to 0.05 +/- 0.02 in DeltaF508-cells. In Hi-5 cells that did not express CFTR, glibenclamide failed to inhibit ORCC activity even at high concentrations (100 microm), whereas 500 microm SITS reversibly inhibited ORCC. In contrast in cells expressing CFTR or DeltaF508, glibenclamide dose dependently (IC50 = 17 microm, Hill coefficient 1.2) and reversibly inhibited ORCC. Cytoplasmic application of 100 microm glibenclamide reversibly reduced Po from 0.88 +/- 0.03 to 0.09 +/- 0.02 (wash: Po = 0.85 +/- 0.1) in CFTR cells and from 0.89 +/- 0.05 to 0.08 +/- 0.05 (wash: Po = 0.87 +/- 0.1) in DeltaF508 cells. In non-CFTR expressing cells, glibenclamide (100 microm) was without effect on Po (control: Po = 0. 89 +/- 0.09, glib.: Po = 0.86 +/- 0.02; wash: Po = 0.87 +/- 0.05). These data strongly suggest that the expression of CFTR confers glibenclamide sensitivity to the ORCC in Hi-5 cells.

1-Methyl-3-isobutylxanthine↗
PubMed1999

Epidemiological survey of contact dermatitis in Italy (1984-1993) by GIRDCA (Gruppo Italiano Ricerca Dermatiti da Contatto e Ambientali).

BACKGROUND: The Gruppo Italiano Ricerca Dermatiti da Contatto e Ambientali (GIRDCA) resolved to obtain the most relevant data regarding contact dermatitis (CD) in Italy by means of a multicenter epidemiological study, involving research units (RU) throughout the country. The survey was performed with the collaboration of Generale Per l'Informatica, Rome (GEPIN) over the periods 1984 to 1988 and 1989 to 1993. The analytical study (of a transverse kind) was aimed at supplying prevalence measures. MATERIALS AND METHODS: The number of subjects taking part in the survey was 42,839. All patients underwent patch testing with the GIRDCA standard series and were included in the survey only if the final diagnosis was either CD or eczematous dermatitis caused by contact. The main anamnestic, clinical data, and allergological test results of all patients were codified into a chart, and subsequently transcribed into a data bank. The data were then processed cumulatively and were statistically analysed by a chi-square for trend test. RESULTS: The four most frequent diagnoses (which, when considered together, make up 39,496 cases, or about 92% of all cases) proved to be nonoccupational allergic contact dermatitis (ACD), nonoccupational irritant contact dermatitis (ICD), occupational ACD, and occupational ICD. The haptens most frequently causing positive reactions in the total number of cases over the first 5 years were, in order of frequency: nickel sulphate, potassium dichromate, cobalt chloride, fragrance mix, balsam of Peru, and, in the second 5-year period, nickel sulphate, cobalt chloride, potassium dichromate, fragrance mix, and thimerosal. Occupational CD (ACD and ICD) was present in 11,694 cases overall, corresponding to approximately 27% of the total number of cases examined, and approximately 29% of all CD (including forms of nonoccupational CD). Regarding distribution by sex, a substantial equivalence of males and females for ACD, and a prevalence of females with ICD can be observed. Five occupations were reputed to be responsible for over 60% of total cases of occupational CD (housewives, bricklayers, workers in the metallurgic and mechanical industries, hairdressers, and healthcare personnel). Regarding the pathogenesis, a clearly dominant percentage of ACD may be observed among bricklayers and hairdressers, and of ICD among housewives. The haptens most frequently noted over the entire decade as the cause of positive reaction in occupational ACD were, in order of frequency, potassium dichromate, nickel sulphate, cobalt chloride, p-phenylenediamine and thiuram mix. Nonoccupational CD (ACD and ICD) was present in 27,802 cases overall, corresponding to about 65% of all cases under examination and over 70% of all CD (also including forms of occupational CD). In regard to distribution by sex, a clear dominance of females, as opposed to males, can be noted for ACD and also (although to a lesser extent) for ICD. The main products and materials (ie, components and relative substances) that are pinpointed as being responsible for nonoccupational ACD, were, in order of frequency, clothing accessories, cosmetics, topical pharmaceuticals, and clothing. The haptens most frequently recognized as the cause of positive reactions were, in order of frequency, nickel sulphate, fragrance mix, cobalt chloride, balsam of Peru, potassium dichromate, ethylenediamine, and diaminodiphenylmethane.

Dermatitis, Allergic Contact↗
PubMed1999

Pharmacological properties of trimebutine and N-monodesmethyltrimebutine.

Trimebutine [2-dimethylamino-2-phenylbutyl-3,4,5-trimethoxybenzoate hydrogen maleate (TMB)] has been demonstrated to be active for relieving abdominal pain in humans. To better understand its mechanism of action, we have tested TMB; nor-TMB, its main metabolite in humans; and their respective stereoisomers for their affinity toward sodium channels labeled by [3H]batrachotoxin, their effect on sodium, potassium, and calcium currents in rat dorsal root ganglia neurons, and their effect on veratridine-induced glutamate release from rat spinal cord slices. TMB has also been tested in an animal model of local anesthesia. TMB (Ki = 2.66 +/- 0.15 microM) and nor-TMB (Ki = 0.73 +/- 0.02 microM) displaced [3H]batrachotoxin from its binding site with affinities similar to that of bupivacaine (Ki = 7.1 +/- 0.9 microM). nor-TMB was found to block veratridine-induced glutamate release with an IC50 value of 8.5 microM, which is very similar to that of bupivacaine (IC50 = 8.2 microM); the effect of TMB was limited to 50% inhibition at 100 microM. TMB and nor-TMB blocked sodium currents in sensory neurons from rat dorsal root ganglia (IC50 = 0.83 +/- 0.09 and 1.23 +/- 0.19 microM, respectively), whereas no effect was observed on calcium currents at the same concentrations. A limited effect was observed on potassium currents (IC50 = 23 +/- 6 at 10 microM) for TMB. In vivo, when tested in the rabbit corneal reflex, TMB displayed a local anesthetic activity 17-fold more potent than that of lidocaine.

Animals↗
PubMed1999

Distribution in rat brain of binding sites of kaliotoxin, a blocker of Kv1.1 and Kv1.3 alpha-subunits.

The distribution of the binding sites for kaliotoxin (KTX), a blocker of voltage-dependent K(+) channels, was studied with quantitative autoradiography in adult rat brain and during postnatal brain maturation. Iodinated KTX bound specifically to tissue sections with a high affinity (K(d) = 82 pM) and a maximal binding capacity of 13.4 fmol/mg protein. The distribution of KTX binding sites within the central nervous system was heterogeneous. The highest densities were found in the neocortex, hypothalamus, dentate gyrus, bed nucleus of the stria terminalis, and parabrachial nuclei. The lowest level was observed in the white matter. From postnatal day 5 onward, KTX binding sites were detectable only in the hindbrain. The density of KTX binding sites in whole brain drastically increased after postnatal day 15 to achieve adult levels at postnatal day 60 in the whole brain. Bath application of KTX to Xenopus laevis oocytes blocked recombinant Kv1.3 and Kv1.1 channels potently and Kv1.2 channels less potently, with respective K(d) values of 0.1, 1.5, and 25 nM. KTX affinities for each of these channels expressed in mammalian cells were about 10-fold lower. A comparison of the distribution of KTX binding sites with that of Kv1 channel polypeptides, together with the pharmacology of KTX block, suggests that the principal targets for KTX in rat brain are K(+) channels containing Kv1.1 and Kv1.3 alpha-subunits.

Aging↗
PubMed1998

Encoding properties induced by a persistent voltage-gated muscarinic sodium current in rabbit sympathetic neurones.

1. A time- and voltage-dependent Na(+)-selective current termed INa,M is activated by muscarinic agonists or splanchnic nerve stimulation in sympathetic neurones of rabbit coeliac and superior mesenteric ganglia. The firing patterns induced by INa,M were investigated in patch-clamped neurones within intact ganglia, and compared with those generated by a neuronal model including INa,M. 2. INa,M was characterized by voltage-dependent low threshold activation and high-threshold inactivation functions. The overlapping functions produced a persistent U-shaped current between -100 and -20 mV, which peaked at the cell resting potential. The activation and inactivation kinetics were fitted to single exponentials with time constants of approximately 100 and 400 ms, respectively. 3. Activating INa,M with muscarinic agonists or nerve stimulation depolarized and fired the neurones. The depolarization was paralleled by an apparent increase in input membrane resistance. The model showed that this paradox resulted from the turning off of INa,M during resistance tests, which also accounted for the all-or-none slow hyperpolarizing responses to current pulses. 4. INa,M gave the neurones an N-shaped I-V relationship capable of producing complex firing patterns. Under given conditions, carbachol-treated neurones could either fire regularly or remain silent at approximately -80 mV, i.e. they displayed bistability. Transitions from one state to the other were triggered with short current pulses. The transitions resulted from the turning on and off of INa,M. 5. Firing reduced INa,M, an effect abolished by blocking Ca2+ channels or adding BAPTA (40 mM) to the pipette. The Ca(2+)-related negative regulation of INa,M may have mediated endogenous bursting activity. Burst firing was generated by the model upon introducing Ca2+ regulation of INa,M. 6. The results demonstrate that INa,M gives prevertebral sympathetic neurones a wide repertoire of firing patterns: pacemaker-like properties, bistability and burst firing capability. They suggest that the INa,M-related encoding properties may provide sympathetic neurotransmission with new potentialities.

Animals↗
PubMed1998

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

1. 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. 2. 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. 3. The CFTR chloride channel activity was blocked by glibenclamide (100 microM) but not by DIDS (100 microM). 4. 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. 5. 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. 6. 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.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1998

[Efficacy of delayed-release lanreotide in the medical therapy of acromegaly].

BACKGROUND: The efficacy of 6 months therapy with slow-release lanreotide (30 mg i.m. every 10-14 days) in 8 acromegalic patients has been studied. METHODS: These patients had been previously treated (for 62 +/- 5.7 months) with octreotide (100 micrograms t.i.d.) and therefore presented, at the beginning of the study, normal mean GH (3.5 +/- 1.1 ng/ml) and IGF-1 (301.7 +/- 32.9 ng/ml) plasma levels. After a week of wash-out, mean GH (5.5 +/- 1.3 ng/ml) and IGF-1 (523.8 +/- 26.7 ng/ml) plasma levels showed a significant increase (p < 0.01) compared to the values observed during the treatment with octreotide. All 8 acromegalic patients then started the treatment with lanreotide, 30 mg i.m. After 14 days, mean GH plasma levels (4.2 +/- 1.3 ng/ml) did not significantly differ (p = NS) from those observed in the same group of patients during treatment with octreotide, whilst plasma IGF-1 levels (477 +/- 43 ng/ml) were significantly higher (p < 0.05). Four patients, in which mean plasma GH values resulted < 5 ng/ml, continued the therapy with lanreotide every 14 days. In the remaining 4 patients, in which plasma GH values were > 5 ng/ml, lanreotide was administered every 10 days. RESULTS: After 3 months of therapy, 6 out of the 8 patients presented persistent GH levels < 5 ng/ml during the day, with IGF-1 levels comparable to those observed during treatment with octreotide. The other 2 subjects presented plasma GH levels > 5 ng/ml during the day, with increased plasma levels of IGF-1. This latter group of patients was resubmitted treatment with octreotide, 100 micrograms t.i.d. After 6 months of therapy, all 6 patients presented GH and IGF-1 plasma levels comparable to those observed during treatment with octreotide. CONCLUSIONS: These data show that slow-release lanreotide can be a valid therapeutic alternative to octreotide in the medical treatment of acromegalic patients.

Acromegaly↗
PubMed1997

A potassium-channel toxin from the sea anemone Bunodosoma granulifera, an inhibitor for Kv1 channels. Revision of the amino acid sequence, disulfide-bridge assignment, chemical synthesis, and biological activity.

The potassium channel toxin secreted by the sea anemone Bunodosoma granulifera (BgK) is a 37-amino-acid peptide containing three disulfide bridges. Because a synthetic peptide corresponding to the reported sequence of BgK was found not to fold properly, the sequence was determined again. The new sequence differed from the previous one in the C-terminal tetrapeptide, which contains two cysteines involved in disulfide bridging. The revised sequence is: V C R D W F K E T A C R H A K S L G N C R T S Q K Y R A N C A K T C E L C. The toxin BgK was synthesized according to the new sequence and folded successfully. Disulfide bridges were assigned by peptide mapping on both natural and synthetic forms to be between Cys2-Cys37, Cys11-Cys30 and Cys20-Cys34. The toxin contains a C-terminal free carboxylate as shown by comparing the native toxin with two synthetic peptides containing the C-terminus in either the carboxylate or carboxamido form. Synthetic BgK inhibits binding of 125I-alpha-dendrotoxin to rat brain synaptosomal membranes, similarly to natural BgK (nanomolar range). No activity was observed on maxi-K+ channels incorporated into planar lipid bilayers. The ability of BgK to block voltage-dependent K+ channels was determined from recordings of whole cell currents in Xenopus oocytes injected with cRNA encoding three cloned Kv1 channels (Kv1.1, Kv1.2, Kv1.3) and one Kv3 (Kv3.1) channel. The Shaker-related Kv1 channels are equally affected by BgK, while the Shaw-related channel Kv3.1 is insensitive up to 0.125 microM toxin. Indeed, half blockage of the current through the three Kv1 channels tested occurred in the same concentration range (Kd = 6 nM for Kv1.1, 15 nM for Kv1.2, 10 nM for Kv1.3). The specificity of BgK for the Shaker-related K+ channels indicates that BgK is able to discriminate a large group of neuronal Kv1 channels in situ. The sequence, the disulfide bridge pattern, the secondary structure and the biological activity of BgK demonstrated that the sea anemone toxins, i.e. BgK, ShK and Kaliseptine, constitute novel molecular probes useful for investigating K+ channel properties.

Amino Acid Sequence↗
PubMed1997

Exotoxin-insensitive G proteins mediate synaptically evoked muscarinic sodium current in rabbit sympathetic neurones.

1. The involvement of G proteins in the transduction pathway that links muscarinic receptors to the low-threshold voltage-dependent sodium current (INa,M) was studied in neurones from intact sympathetic prevertebral ganglia using the whole-cell configuration of the patch-clamp technique. Experiments were performed in the presence of the nicotinic receptor antagonists hexamethonium (50 microM) and d-tubocurarine (50 microM). 2. INa,M was activated by either bath-applying muscarinic agonists or stimulating the preganglionic splanchnic nerves. Synaptically and agonist-mediated INa,M did not display significant run-down or changes in their properties in cells tested, irrespective of whether the pipette solutions contained GTP. 3. Dialysis of sympathetic neurones with GDP beta S (500-750 microM) decreased the amplitude of INa,M by approximately 65% compared with control neurones within 30 min. 4. In the absence of muscarinic receptor stimulation, intracellular dialysis with GTP gamma S (500 microM) for 10 min slowly and slightly (20-25%) activated INa,M. GTP gamma S dialysis markedly slowed down the decay of INa,M after its transient activation with carbachol pulses (10-20 s) or nerve stimulation (3-5 s). The INa,M activation became fully irreversible 2.9 min after the start of GTP gamma S dialysis. Dialysing cells with the G protein activator AIF4-led to a rapid but transient activation of INa,M. 5. Synaptically and agonist-evoked INa,M were not affected in ganglia treated with 0.5-1 microgram ml-1 pertussis toxin (PTX) for 7-24 h at 37 degrees C. Control experiments showed that this treatment severely reduced the PTX-sensitive inhibition of N-type calcium currents induced by carbachol (CCh) and noradrenaline. Application of NEM (N-ethylmaleimide) for 2 min depressed the INa,M evoked in response to bath-applied CCh by only 27%. 6. Incubating ganglia with 5-10 micrograms ml-1 of cholera toxin for 7 h had no effect on the carbachol-induced INa,M but greatly potentiated (approximately 250%) the synaptically evoked INa,M, presumably via a presynaptic mechanism. 7. These results show that the coupling between muscarinic receptors and NaM channels is mediated by pertussis toxin- and cholera toxin-insensitive G proteins, possibly of the Gq/11 or G12 class.

Animals↗
PubMed1997

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.

1-Methyl-3-isobutylxanthine↗