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M Gola

Publications and source records attributed to M Gola.

At least 55 records · Page 3Linked to original sources
PubMed1994

The kaliotoxin family enlarged. Purification, characterization, and precursor nucleotide sequence of KTX2 from Androctonus australis venom.

Kaliotoxin (KTX) has been originally described as an inhibitor of the intermediate conductance Ca(2+)-activated K+ channel (Crest, M., Jacquet, G., Gola, M., Zerrouk, H., Benslimane, A., Rochat, H., Mansuelle, P., and Martin-Eauclaire, M.-F. (1992) J. Biol. Chem. 267, 1640-1647). However, the radioiodinated 125I-KTX-(1-37) was also able to bind to the dendrotoxin sensitive voltage-dependent K+ channel (Romi, R., Crest, M., Gola, M., Sampieri, F., Jacquet, G., Zerrouk, H., Mansuelle, P., Sorokine, O., Van Dorsselaer, A., Rochat, H., Martin-Eauclaire, M.-F., and Van Rietschoten, J. (1993) J. Biol. Chem. 268, 26302-26309). By following the ability to compete with 125I-KTX-(1-37) for binding to its receptor on rat brain synaptosomes, a new kaliotoxin-like peptide, KTX2, was isolated from Androctonus australis scorpion venom. It is a 37-amino acid residue peptide, and its sequence shares 76% identity with KTX. The differences between the two peptides concern the NH2-terminal region and the residues 31 and 34 located in the region involved in the channel recognition. These differences may explain the 5-fold decrease of the molluscan Ca(2+)-activated K+ channel blockage by KTX2 (kd = 135 nM) as well as of its binding affinity to rat brain synaptosomes (IC50 = 50 pM), compared with KTX. Specific antibodies raised against KTX-(1-37) were not able to recognize KTX2. Using degenerate primers, a 370-base pair cDNA encoding the KTX2 precursor was amplified by polymerase chain reaction from a cDNA library of A. australis venom glands. It encoded a presumed signal peptide of 22 residues followed by the sequence of the mature peptide.

Amino Acid Sequence↗
PubMed1994

Satellite glial cell responses to neuronal firing in the nervous system of Helix pomatia.

Patch clamp experiments were conducted on satellite glial cells attached to the cell body of neurons in place within the nervous system of the snail Helix pomatia. The glial cells were studied using cell-attached and whole-cell patch clamp configurations while the underlying neurons were under current or voltage clamp control. The resting potential of the glial cells (-69 mV) was more negative than that of the underlying neurons (-53 mV), due to their high K+ selectivity. Densely packed K+ channels were present, some of which were active at the cell resting potential. Neuronal firing elicited a cumulative depolarization of the glial cells. Large K+ currents flowing from V-clamped neurons depolarized the glial layer by up to 30 mV. The glial depolarization was directly correlated with the size of the neuronal K+ current. The glial cells recovered their resting potential within 2-5 sec. The neuronal depolarization induced a delayed (20-30 sec) and persistent (3-4 min) increase in the glial K+ channel opening probability. Likewise, pulses of K+ (20-50 mM)-rich saline activated the glial channels, unless the underlying neuron was held hyperpolarized. In low Ca(2+)-high Mg2+ saline, neuron depolarization and K(+)-rich saline did not activate the glial K+ channels. These data indicate that a calcium-dependent signal released from the neuronal cell body was involved in glial channel regulation. Neuron-induced channel opening may help eliminate the K+ ions flowing from active neurons.

Action Potentials↗
PubMed1994

Active, interactive, and inactive steroid receptor mutants.

In transient co-transfection assays, there is extensive cross-interaction between glucocorticoid receptor (GR) domains. For example, mutation of the conserved Ile residue at position 484 (rat GR map) to cysteine allows a net separation of transactivation and DNA binding. We also observed that the ligand binding domain plays a key role in cooperative transactivation. Furthermore, some carboxy-located mutations markedly alter the response of GR to agonists and antagonists. Finally, different reading frames of the CAG repeat that normally produces an amino-located poly-Gln repeat profoundly affect GR transactivation without altering DNA or ligand binding. This trans-dominant negative phenotype, seen when the CAG repeat yields a poly-Ala stretch, may turn out to be an excellent tool for functional analysis of GR in transgenic organisms.

Amino Acid Sequence↗
PubMed1994

The expression of carbonic anhydrases II and IV in the human pancreatic cancer cell line (Capan 1) is associated with bicarbonate ion channels.

Human pancreatic ductal cells of the Capan 1 cell line differentiate progressively during growth. After the exponential growth phase, the cells elongate and become polarized with their apical poles covered by microvilli and separated from the basolateral pole by tight junctions. In this stationary phase, they form domes, which are thought to result from the exchange of water and electrolytes. In this study, we demonstrated, using patch-clamp techniques, that HCO3- ions exit via the g350 high conductance anionic channel we observed recently at the Capan 1 cell surface. This g350 channel was thought to be either a Cl-/HCO3- antiport or a simple HCO3- channel. The stilbene derivatives 4-acetamido-4 isothiocyano-2-2'-disulfonic acid (SITS) and 4,4' diisothiocyano stilbene-2,2' disulfonic acid (DIDS) reduced both the number of domes and the Cl- and HCO3- flux through the g350 channel. Moreover, using histochemical, immunocytochemical and biochemical methods we showed that Capan 1 cells express a specific pattern of carbonic anhydrases (CA). Two types of CA were detected: the CA II isozyme mainly localized in the cytoplasm, but also found beneath the inner leaflet of the apical plasma membrane, and the CA IV isozyme localized on the outer leaflet of the apical plasma membrane and microvilli. Their molecular masses were 30 (CA II) and 55 kDa (CA IV), respectively. They were expressed continuously during the exponential growth phase, although their activity increased greatly during the stationary phase. Inhibition of dome formation by acetazolamide indicated the existence of a direct relationship between dome formation and CA. Characteristic structures with a central electron-dense core surrounded by a light halo were observed on the surface of cell membrane using histochemical and immunocytochemical methods. These structures were thought to represent a channel, corresponding possibly to CA IV. Our observations suggest that Capan 1 cells, despite their neoplasic transformation, produce HCO3- ions in the same way as normal human pancreatic ductal cells. Capan 1 cells in culture may therefore represent a suitable model for studying pancreatic duct HCO3- secretion at the cellular and molecular levels.

Acetazolamide↗
PubMed1993

Synthesis and characterization of kaliotoxin. Is the 26-32 sequence essential for potassium channel recognition?

Kaliotoxin (KTX), a scorpion toxin characterized as a 37-residue inhibitor of the neuronal high conductance Ca(2+)-activated K+ channels (KCa channels), has been chemically synthetized. Differences were observed between natural toxin and the two peptides, KTX(1-37) and KTX(1-37)-amide. Re-examination of the KTX sequence showed that an extra lysine residue was present at the C-terminal end. The 38-residue synthetic peptide was found identical with natural toxin. All three peptides had comparable activities, with LD50 values of 6-9 pmol/mouse after intracerebroventricular injection, and Kd = 2-8 nM for blockage of the whole cell and unitary molluscan KCa currents. Pairing of the disulfide bonds in synthetic KTX corresponded to that in charybdotoxin and iberiotoxin. A competition assay between 125I-KTX(1-37) and different toxins (KTX, dendrotoxin, charybdotoxin, MCD peptide, and iberiotoxin) for binding to rat brain synaptosomal membranes suggested that KTX interacts also with voltage-gated K+ channels. Shorter peptides, KTX(25-35)-amide and KTX(26-32)-amide, expressed no KTX activity, but were able to compete in binding. They were further shown to antagonize KTX in both its toxicity and blocking activity. The (26-32) sequence of KTX, which is a highly conserved region, may contain a low affinity binding subsite essential for potassium channel recognition.

Amino Acid Sequence↗
PubMed1993

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.

1-Methyl-3-isobutylxanthine↗
PubMed1993

Characterization of cAMP dependent CFTR-chloride channels in human tracheal gland cells.

Human tracheal gland cells are believed to be a major site at the origin of cystic fibrosis. Since this disease is due to mutations in a protein called CFTR, we looked for the activity of CFTR in human tracheal gland cells in culture. We have identified CFTR-like chloride-selective channels as having a linear current voltage relationship and unitary conductance of 7 pS in these cells. In cell-attached patches, theophylline (1 mM), IBMX (1 mM), or a cocktail of dibutyryl cAMP (1 mM) and IBMX (0.1 mM) promoted the opening of channels. The unitary current had a reversal potential close to the cell resting potential. Replacement of choline by K+ or Na+ in the pipette solution was without effect on the current-voltage relationship, the reversal potential or the unitary conductance, which is consistent with the chloride selectivity of the channel. Channels were always found clustered and their opening probability was not noticeably dependent on membrane potential. This work therefore represents the first observation of a CFTR-like channel activity in submucosal gland cells.

Base Sequence↗
PubMed1993

Satellite glial cells in situ within mammalian prevertebral ganglia express K+ channels active at rest potential.

Patch-clamp experiments were performed on satellite glial cells wrapped around sympathetic neurons in the rabbit coeliac ganglion. With the cleaning method used, the glial cells could be kept in place and were directly accessible to the patch-clamp pipettes. Whole-cell recordings showed that glial cells had almost ohmic properties. Their resting potential (-79.1 +/- 1.2 mV) was found to be very nearly the same as the K+ reversal potential and approximately 20 mV more negative than that of the neurons they encapsulated. Unitary currents from ionic channels present in the glial membrane were recorded in the cell-attached configuration with pipettes filled with various amounts of K+, Na+ and gluconate. Only K(+)-selective channels with slight inwardly rectifying properties (in the presence of 150 mM [K+]o) were detected. These channels were active (Po = 0.7-0.8) at the cell resting potential. The channel conductance, but not its opening probability, was dependent on the [K+] in the pipette. Cl(-)-selective channels (outwardly rectifying and large conductance channels) were detected in excised patches. The properties of the K+ channels (increased inward current with [K+] and detectable outward current at low [K+]) are well suited for siphoning the K+ released by active neurons.

Animals↗
PubMed1993

Phosphorylation-regulated low-conductance Cl- channels in a human pancreatic duct cell line.

A low-conductance Cl- channel has been identified in the apical membrane of the human pancreatic duct cell Capan-1 using patch-clamp techniques. Cell-attached channels were activated by the vasoactive intestinal polypeptide (VIP, 0.1 mumol/l), dibutyryl-adenosine 3',5'-cyclic monophosphate (db-cAMP, 1 mmol/l), 8-bromo adenosine 3',5'-cyclic monophosphate (8-Br-cAMP, 1 mmol/l), 3-isobutyl-1-methyl-xanthine (IBMX, 100 mumol/l) and forskolin (10 mumol/l). No channel activity was observed in non-stimulated control cells. In both cell-attached and excised inside-out patches, the channel had a linear current/voltage relationship and a unitary conductance of 9 pS at 23 degrees C and 12 pS at 37 degrees C. Its opening probability was not voltage dependent although pronounced flickering was induced at negative potentials. Anionic substitution led to the selectivity sequence Cl- > I- >> > HCO3- > gluconate. In inside-out excised patches, the channel activity declined spontaneously within a few minutes. Reactivation of silent excised channels was achieved by adding protein kinase A (PKA, in the presence of ATP, cAMP and Mg2+). Conversely, active channels were silenced in the presence of alkaline phosphatase. The PKA-activated Cl- channel was 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS, 100 mumol/l) and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS, 100 mumol/l) insensitive, but was blocked by diphenylamine-2-carboxylic acid (DPC, 100 mumol/l). These results demonstrate that the apical low-conductance Cl- channel in Capan-1 is regulated on-cell by VIP receptors via cAMP and off-cell by PKA and phosphatases.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1993

Single-channel and whole-cell recordings from on-neurone glial cells in Helix pomatia ganglia.

A procedure is described for performing patch-clamp recordings on satellite glial cells kept in place within the nervous ganglia in the mollusc Helix. Glial cell properties were deduced from whole-cell and cell-attached recordings. The glial membrane was found to contain densely packed inwardly rectifying K+ channels. Activation of the neurones, under either current-clamp or voltage-clamp conditions, depolarized the glial cell layer wrapped around the neurones and induced a delayed persistent increase in the K+ channel opening probability. These results suggest that the glial channels opened in response to a signal emanating from the active neurones. This preparation provides a useful means of detecting and analysing neurone-glial interactions at the cell and unitary channel levels.

Animals↗
PubMed1993

Colocalization of active KCa channels and Ca2+ channels within Ca2+ domains in helix neurons.

Large conductance Ca(2+)-activated K+ channels (KCa channels) have to be colocalized with Ca2+ channels to be involved in nerve cell firing regulation. KCa channels were detected in cell-attached patches performed in voltage-clamped Helix neurons. This technique allowed Ca2+ entry to occur either within or around the patch. We observed that the submembrane Ca2+ diffusion was sufficiently limited to prevent the KCa channels from being opened by remote Ca2+ entry. The KCa channels located in areas devoid of Ca2+ channels remained quiescent during cell firing. These crypto channels were detected following either patch excision or intracellular Ca2+ injection. These data provide direct evidence for the existence of functional Ca2+ domains in a nonspecialized neural membrane.

Animals↗
PubMed1993

Electrical and integrative properties of rabbit sympathetic neurones re-evaluated by patch clamping non-dissociated cells.

1. Voltage recordings were performed on non-dissociated sympathetic neurones from rabbit coeliac ganglia using the whole-cell configuration of the patch clamp technique. 2. Cells were classified depending on their firing pattern as silent cells (63%) producing either phasic (24%) or tonic (76%) spike discharge in response to depolarizing currents, and pacemaker cells (37%). 3. All the cells produced large overshooting spikes and prolonged postspike after-hyperpolarization. The peak-to-peak spike amplitude was 113.8 +/- 1 mV. Spikes were shortened and the after-hyperpolarization was suppressed when calcium channel blockers (Cd2+ and La3+) were added. 4. Silent cells have a resting potential of -58.8 +/- 1.5 mV. At potentials ranging from -50 to -90 mV, the input impedance was 490 +/- 27 M omega at 22-24 degrees C and 426 +/- 47 M omega at 35-36 degrees C. The time constant at voltages corresponding to the high input impedance region was 126 +/- 7 ms at 22-24 degrees C and 86 +/- 7 ms at 35-36 degrees C. 5. The firing frequency of the pacemaker cells was 3.2 +/- 0.5 Hz at 35-36 degrees C in the presence of nicotinic blockers. Evidence is given that the firing did not result from cell injury but was induced by an intrinsic pacemaker mechanism. Input impedance of pacemaker neurones was 580 +/- 47 M omega at 22-24 degrees C and 473 +/- 56 M omega at 35-36 degrees C. 6. Most of the pacemaker cells (63%) were motoneurones, since they were antidromically fired by stimulating post-ganglionic nerves. In addition, they received synaptic inputs from both preganglionic fibres (splanchnic nerves) and the periphery (postganglionic nerves). Long-lasting depolarizations were induced in either silent or pacemaker cells by single shocks applied to pre- and postganglionic nerves. 7. Slowly rising voltage ramps revealed the presence of an N-shaped current-voltage relationship in voltage clamped pacemaker cells. The negative slope was located in a subthreshold voltage range, between -83.4 +/- 1.4 and -59.0 +/- 1.8 mV. It was induced by the activation of a low threshold persistent inward current. Although it was tiny (22 +/- 3 pA at its peak level) this current brought the null-current voltage up to -41.0 +/- 1.4 mV, which resulted in continuous firing. 8. Due to the instability introduced by the N-shaped I-V relationship, pacemaker cells can display bistable behaviour characterized by hyperpolarizing responses.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1993

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)

Action Potentials↗
PubMed1992

Kaliotoxin, a novel peptidyl inhibitor of neuronal BK-type Ca(2+)-activated K+ channels characterized from Androctonus mauretanicus mauretanicus venom.

A peptidyl inhibitor of the high conductance Ca(2+)-activated K+ channels (KCa) has been purified to homogeneity from the venom of the scorpion Androctonus mauretanicus mauretanicus. The peptide has been named kaliotoxin (KTX). It is a single 4-kDa polypeptide chain. Its complete amino acid sequence has been determined. KTX displays sequence homology with other scorpion-derived inhibitors of Ca(2+)-activated or voltage-gated K+ channels: 44% homology with charybdotoxin (CTX), 52% with noxiustoxin (NTX), and 44% with iberiotoxin (IbTX). Electrophysiological experiments performed in identified nerve cells from the mollusc Helix pomatia showed that KTX specifically suppressed the whole cell Ca(2+)-activated K+ current. KTX had no detectable effects on voltage-gated K+ current (delayed rectifier and fast transient A current) or on L-type Ca2+ currents. KTX interacts in a one-to-one way with KCa channels with a Kd of 20 nM. Single channel experiments were performed on high conductance KCa channels excised from the above Helix neurons and from rabbit coeliac ganglia sympathetic neurons. KTX acted exclusively at the outer face of the channel. KTX applied on excised outside-out KCa channels induced a transient period of fast-flicker block followed by a persistent channel blockade. The KTX-induced block was not voltage-dependent which suggests differences in the blockade of KCa channels by KTX and by CTX. Comparison of KTX and CTX sequences leads to the identification of a short amino acid sequence (26-33) which may be implicated in the toxin-channel interaction. KTX therefore appears to be a useful tool for elucidating the molecular pharmacology of the high conductance Ca(2+)-activated K+ channel.

Amino Acid Sequence↗
PubMed1992

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 microM 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.

Adenocarcinoma↗
PubMed1992

Single-channel and whole-cell recordings from non-dissociated sympathetic neurones in rabbit coeliac ganglia.

A procedure is described for performing patch-clamp recordings on mammalian sympathetic neurones within intact ganglia. The plasma membrane of superficial neurones was cleaned by blowing (1.5-3 h) a gentle stream of Ringer saline onto ganglia, the connective sheath of which was previously softened by a short protease treatment. This procedure preserved the intraganglionic connectivity so that the neurones could be activated either synaptically or antidromically by stimulating the appropriate nerves. Depending on the duration of the mechanical cleaning step, recordings were performed on either the neurones or the satellite glial cells covering the neuronal cell bodies. The applicability of the various configurations of the patch-clamp technique to studying sympathetic neurones is illustrated by recordings of whole-cell voltage, whole-cell currents and single-channel currents in cell-attached and excised patches. With these techniques, the resolution of the membrane current recordings is higher than with conventional microelectrodes. The results obtained show that mammalian sympathetic neurones have a very high input resistance (0.5 G omega), are electronically compact and may display pacemaker activity. These techniques provide a useful tool for studying the synaptic transmission and neuromodulation mechanisms operating within the sympathetic ganglia.

Animals↗