Persistent GABAA/C responses to gabazine, taurine and beta-alanine in rat hypoglossal motoneurons.

In hypoglossal motoneurons, a sustained anionic current, sensitive to a blocker of ρ-containing GABA receptors, (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) and insensitive to bicuculline, was previously shown to be activated by gabazine. In order to better characterize the receptors involved, the sensitivity of this atypical response to pentobarbital (30µM), allopregnanolone (0.3µM) and midazolam (0.5µM) was first investigated. Pentobarbital potentiated the response, whereas the steroid and the benzodiazepine were ineffective. The results indicate the involvement of hybrid heteromeric receptors, including at least a GABA receptor ρ subunit and a γ subunit, accounting for the pentobarbital-sensitivity. The effects of the endogenous ß amino acids, taurine and ß-alanine, which are released under various pathological conditions and show neuroprotective properties, were then studied. In the presence of the glycine receptor blocker strychnine (1µM), both taurine (0.3-1mM) and ß-alanine (0.3mM) activated sustained anionic currents, which were partly blocked by TPMPA (100µM). Thus, both ß amino acids activated ρ-containing GABA receptors in hypoglossal motoneurons. Bicuculline (20µM) reduced responses to taurine and ß-alanine, but small sustained responses persisted in the presence of both strychnine and bicuculline. Responses to ß-alanine were slightly increased by allopregnanolone, indicating a contribution of the bicuculline- and neurosteroid-sensitive GABAA receptors underlying tonic inhibition in these motoneurons. Since sustained activation of anionic channels inhibits most mature principal neurons, the ρ-containing GABA receptors permanently activated by taurine and ß-alanine might contribute to some of their neuroprotective properties under damaging overexcitatory situations.

Oestradiol rapidly enhances spontaneous glycinergic synaptic inhibition of hypoglossal motoneurones.

Whereas oestradiol is well-known to facilitate excitatory glutamatergic synaptic transmission, its effects on fast inhibitory neurotransmission are not as well established. Possible acute modulation of the spontaneous glycinergic synaptic activity by oestradiol was investigated in voltage-clamped hypoglossal motoneurones by whole-cell patch-clamp recording in rat brainstem slices. The spontaneous glycinergic synaptic activity was continuously recorded in each neurone under control conditions, during 12-20 min of perfusion with 17beta-oestradiol and during washing. When oestradiol was diluted in ethanol, the control solution contained the same amount of ethanol. At 100 nM, oestradiol markedly increased the frequency of the total spontaneous glycinergic activity. Similar experiments were performed after blockade of action potentials by tetrodotoxin, aiming to isolate miniature glycinergic synaptic currents. Oestradiol increased the frequency of glycinergic miniatures in most slices, in some cases within less than 1 min. In some slices, oestradiol also favoured the occurrence of glycinergic miniatures of large amplitude. These effects were slowly reversible during washing. At 1 nm, oestradiol still increased the frequency of glycinergic miniatures. The results were confirmed in the absence of ethanol by using water-soluble cyclodextrin-encapsulated oestradiol. In these experiments, the control solution contained the same amount of (2-hydroxypropyl)-beta-cyclodextrin as the oestradiol-containing solution. In addition, prolonged control recordings were performed without applying oestradiol to check the stability of the glycinergic synaptic activity during prolonged whole-cell recordings. The results show, for the first time, that, within a few minutes, oestradiol can enhance the spontaneous synaptic release of a major inhibitory transmitter, glycine.

The estrogen receptor modulator tamoxifen enhances spontaneous glycinergic synaptic inhibition of hypoglossal motoneurons.

Tamoxifen (Tam), a widely used anticancer agent, is now also used for healthy women with risk of breast cancer. Furthermore, it is the prototype of the selective estrogen receptor modulator family, with promise for neuroprotection. However, possible effects on neurotransmission have been little explored. Recently, Tam was shown to potentiate chloride responses to low concentrations of exogenous glycine in cultured spinal neurons from rat embryo. The present study investigates the possible modulation by Tam of the spontaneous synaptic glycinergic activity recorded from voltage-clamped hypoglossal motoneurons, using the whole-cell patch-clamp technique in brainstem slices from juvenile rat. Miniature currents were isolated with tetrodotoxin. Tam increased the mean amplitude of glycinergic miniature currents, by 68-79% at 2 microM (in nine of 10 cells) and by 47% at 0.5 microM (in four of nine cells). Furthermore, Tam markedly increased the frequency of glycinergic miniatures, by a factor reaching 15 in some neurons, even in the presence of the Ca2+ channel blocker Cd2+. Tam also increased the frequency of the total spontaneous glycinergic activity without tetrodotoxin. The increase in miniature amplitude is consistent with the increase in postsynaptic glycine receptor sensitivity previously reported. The increase in frequency indicates an additional presynaptic effect. Addition of exogenous glycine could also increase the frequency of glycinergic miniatures. Thus, one of the presynaptic effects of Tam might be potentiation of the basal activity of presynaptic glycine receptors facilitating glycine release. Possible risks related to modulation of glycinergic neurotransmission by Tam should be considered when recommending its use in healthy individuals.

Modulation of glycine responses by dihydropyridines and verapamil in rat spinal neurons.

Although glycine receptors (GlyRs) are responsible for the main spinal inhibitory responses in adult vertebrates, in the embryo they have been reported to mediate depolarizing responses, which can sometimes activate dihydropyridine-sensitive L-type calcium channels. However, these channels are not the only targets of dihydropyridines (DHPs), and we questioned whether GlyRs might be directly modulated by DHPs. By whole-cell recording of cultured spinal neurons, we investigated modulation of glycine responses by the calcium channel antagonists, nifedipine, nitrendipine, nicardipine and (R)-Bay K 8644, and by the calcium channel, agonist (S)-Bay K 8644. At concentrations between 1 and 10 microM, all these DHPs could block glycine responses, even in the absence of extracellular Ca2+. The block was stronger at higher glycine concentrations, and increased with time during each glycine application. Nicardipine blocked GABAA responses from the same neurons in a similar manner. In addition to their blocking effects, nitrendipine and nicardipine potentiated the peak responses to low glycine concentrations. Both effects of extracellular nitrendipine on glycine responses persisted when the drug was present in the intracellular solution. Thus, these modulations are related neither to calcium channel modulation nor to possible intracellular effects of DHPs. Another type of calcium antagonist, verapamil (10-50 microM), also blocked glycine responses. Our results suggest that some of the effects of calcium antagonists, including the neuroprotective and anticonvulsant effects of DHPs, might result partly from their interactions with ligand-gated chloride channels.

Glycine receptor beta subunits play a critical role in potentiation of glycine responses by ICS-205,930.

The sensitivity of various types of recombinant glycine receptors (GlyRs) to ICS-205,930 was studied by fast perfusion in Xenopus laevis oocytes. This compound has previously been shown to potentiate glycine responses in rat spinal neurons between 10 nM and 1 microM, independently of its 5-HT(3) antagonist properties. In contrast, submicromolar concentrations of ICS-205,930 failed to affect responses of homomeric GlyRs formed from human alpha1 or alpha2 subunits, and micromolar concentrations (1-20 microM) acted differentially on the two types of homomeric receptors, potentiating the responses to glycine (10-20 microM) of alpha1 homomeric GlyRs and inhibiting the responses of alpha2 homomeric GlyRs. GlyRs beta subunits markedly influenced the modulations induced by ICS-205,930. In oocytes expressing alpha1/beta or alpha2/beta heteromeric GlyRs, low concentrations of ICS-205,930 (20 nM-1 microM) induced a potentiation of glycine responses that was counteracted by an inhibitory effect at higher concentrations. Thus, GlyRs beta subunits reduce by 2 orders of magnitude the concentration range potentiating alpha1-containing GlyRs and are required for potentiation of alpha2-containing GlyRs. These results reveal a new high-affinity potentiating site on GlyRs, to which beta subunits participate. The difference in ICS sensitivity between alpha1 and alpha2 GlyRs cannot be explained by their difference in TM2 segment and extracellular domains partly conserved between glycine and 5-HT(3) receptors are probably involved in the interaction of some 5-HT(3) antagonists with GlyRs.

Glycinergic potentiation by some 5-HT(3) receptor antagonists: insight into selectivity.

The ability of various 5-HT(3) receptor antagonists to potentiate spinal glycine responses was investigated. Whereas (3-alpha-tropanyl)-1H-indole-3-carboxylate (ICS 205930), (3-alpha-tropanyl)-3,5-dichlorobenzoate (MDL 72222) and 1-methyl-N-(3-alpha-tropanyl)-1H-indazole-3-carboxamide (LY 278584) exhibited this property, even in identified motoneurones, several other chemically similar 5-HT(3) receptor antagonists did not. Introducing a methyl group on the nitrogen of the azabicyclo moiety of ICS 205930 greatly reduced the ability to potentiate glycine responses. Neither endo-1-methyl-N-(9-methyl-9-azabicyclo[3.3. 1]non-3-yl)-indazole-3-carboxamide (granisetron), differing from LY 278584 by an additional carbon in this cycle, nor 2beta-carbomethoxy-3beta-benzoyloxytropane (cocaine), 1,2,3, 9-tetrahydro-9-methyl-3-[(2-methyl-1H-imidazol-1-yl)-methyl]-4H-carba zol-4-one (ondansetron) and (S)-4-amino-N-(1-azabicyclo[2.2. 2]oct-3-yl)-5-chloro-2-methoxy-benzamide ((S)-zacopride) could potentiate glycine responses. A pharmacophore model of the glycinergic potentiators was generated by molecular modelling using MDL 72222 as a template. According to this model, an aromatic ring, a carbonyl group and a tropane nitrogen atom are required for glycinergic potentiation, as previously described for 5-HT(3) receptor antagonism. However, the steric allowance at the glycine receptor site and the tridimensional arrangement of the pharmacophoric elements appear to be more restricted.

Functional cystic fibrosis transmembrane conductance regulator tagged with an epitope of the vesicular stomatis virus glycoprotein can be addressed to the apical domain of polarized cells.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-activated chloride channel apically localized in epithelial cells. In cystic fibrosis patients, the gene encoding this N-linked glycoprotein is mutated. About 70% of CF patients express a mutated form of CFTR, deleted at the phenylalanine residue at position 508 (deltaF508). CFTR-deltaF508 fails to exit the endoplasmic reticulum; it remains incompletely glycosylated and is rapidly degraded. To optimize CFTR detection for membrane localization studies and biochemical studies, we tagged wild-type and deltaF508 CFTR with the VSV-G epitope at their carboxy-terminal ends. We have generated pig kidney epithelial cell clones (LLCPK1) expressing VSV-G-tagged human wild-type and deltaF508-CFTR. In CFTR-expressing cells, the transfected protein is maturated and transported to the apical membrane where it is concentrated. The cells exhibit a strong anion channel activity after stimulation by cAMP, as demonstrated by a halide sensitive fluorescent dye assay (6-methoxy-N-ethylquinominium, SPQ), and whole-cell patch-clamp approach. This activity of CFTR-VSV-G is indistinguishable from the wild-type CFTR. In contrast, in cells expressing tagged deltaF508-CFTR or in non-transfected cells, no anion channel activity could be detected after stimulation by cAMP. In deltaF508-CFTR-VSV-G-expressing cells, the mutated CFTR remained in the incompletely glycosylated form and was localized in the endoplasmic reticulum. These cell lines reproduce the cellular fate of wild-type and mutated CFTR-deltaF508. To our knowledge, they are the first differentiated epithelial cell lines stably expressing tagged CFTR and CFTR-deltaF508 in which cellular processing and functional activity of these two proteins are reproduced. Thus the addition of the VSV-G epitope does not impair the localization and function of CFTR, and these cell lines can be used to examine CFTR function in vitro.

Synaptic control of glycine and GABA(A) receptors and gephyrin expression in cultured motoneurons.

We have evaluated the influence of the secretory phenotype of presynaptic boutons on the accumulation of postsynaptic glycine receptors (GlyRs), type A GABA receptors (GABA(A)Rs), and gephyrin clusters. The cellular distribution of these components was analyzed on motoneurons cultured either alone or with glycinergic and/or GABAergic neurons. In motoneurons cultured alone, we observed gephyrin clusters at nonsynaptic sites and in front of cholinergic boutons, whereas glycine and GABA(A) receptors formed nonsynaptic clusters. These receptors are functionally and pharmacologically similar to those found in cultures of all spinal neurons. Motoneurons receiving GABAergic innervation from dorsal root ganglia neurons displayed postsynaptic clusters of gephyrin and GABA(A)Rbeta but not of GlyRalpha/beta subunits. In motoneurons receiving glycinergic and GABAergic innervation from spinal interneurons, gephyrin, GlyRalpha/beta, and GABA(A)Rbeta formed mosaics at synaptic loci. These results indicate that (1) the transmitter phenotype of the presynaptic element determines the postsynaptic accumulation of specific receptors but not of gephyrin and (2) the postsynaptic accumulation of gephyrin alone cannot account for the formation of GlyR-rich microdomains.

Mode of action of ICS 205,930, a novel type of potentiator of responses to glycine in rat spinal neurones.

The effect of a novel potentiator of glycine responses, ICS 205,930, was studied by whole-cell recordings from spinal neurones, and compared with that of other known potentiators, in an attempt to differentiate their sites of action. The ability of ICS 205,930 (0.2 microM) to potentiate glycine responses persisted in the presence of concentrations of Zn2+ (5-10 microM) that were saturating for the potentiating effect of this ion. Preincubation with 10 microM Zn2+ before application of glycine plus Zn2+ had an inhibitory effect, which did not result from Zn2+ entry into the neurone, since it persisted with either 10 mM internal EGTA or 10 microM internal Zn2+. To test whether the potentiating effects of ICS 205,930 and Zn2+ interact, both compounds were applied without preincubation. The potentiating effect of ICS 205,930 was similar for responses to glycine and for responses to glycine plus Zn2+, provided the concentrations of agonist were adjusted so as to induce control responses of identical amplitudes. ICS 205,930 remained able to potentiate glycine responses in the presence of ethanol (200 mM). ICS 205,930 also retained its potentiating effect in the presence of the anaesthetic propofol (30 90 microM), which strongly potentiated glycine responses but, in contrast with ICS 205,930, also markedly increased the resting conductance. The anticonvulsant chlormethiazole (50-100 microM) neither potentiated glycine responses nor prevented the effect of ICS 205,930, even though it increased the resting conductance and potentiated GABA(A) responses. The mechanism of action of ICS 205,930 appears to be different from those by which Zn2+, propofol or ethanol potentiate glycine responses.

Potentiation of chloride responses to glycine by three 5-HT3 antagonists in rat spinal neurones.

1. Modulations of Cl- responses to glycine by 5-hydroxytryptamine ligands were studied in cultured spinal neurones, by the whole-cell recording technique. 2. Three 5-HT3 antagonists were found to potentiate reversibly responses to low concentrations of glycine. Potentiations were induced by micromolar concentrations of LY-278,584 (1-10 microM) and by concentrations of MDL-72222 or ICS-205,930 between 10 nM and 1 microM. 3. Potentiations were observed over the whole voltage range without any change in the reversal potential of the glycine responses and without affecting the resting conductance. 4. The degree of potentiation was variable among cells. It increased with the concentration of the modulator, but only up to 100 nM for MDL-72222 and ICS-205,930. 5. The potentiation appeared to result from an increase in the affinity for glycine of glycine receptors. 6. Neither the blockade of glycine uptake by Na+ removal, nor the excision of membrane patches prevented the potentiation. 7. At high concentrations (10 microM), both MDL-72222 and ICS-205,930 had, in contrast, a blocking effect on glycine responses. 8. Potentiation by LY-278,584 and a dose-dependent modulation by MDL-72222 were also observed for taurine responses. 9. The effects on glycine responses of various ligands of 5-HT3 receptors (including agonists) are discussed. The ability of LY-278,584, MDL-72222 and ICS-205,930 to potentiate glycine responses appears to be independent of their known 5-HT3 receptor antagonist properties. It would be interesting to look for chemically related drugs that would be specific potentiators of glycine responses.

Voltage-dependent block of NMDA responses by 5-HT agonists in ventral spinal cord neurones.

1. Modulation by 5-hydroxytryptamine receptor agonists of the NMDA responses of ventral spinal cord neurones was studied by use of the whole-cell patch-clamp technique. 2. In a Mg-free solution containing tetrodotoxin and glycine, 5-hydroxytryptamine (5-HT, 10-100 microM) reduced the NMDA response, the block increasing with hyperpolarization. Kainate responses were little affected. 3. Some classical agonists of 5-HT receptors induced similar blocking effects. At 10 microM, both a selective agonist of 5-HT2 receptors, (+/-)-2,5-dimethoxy-4 iodo amphetamine (DOI), and a selective agonist of some 5-HT1 receptors, (+/-)-8-hydroxy-2(n-dipropyl amino) tetralin (8-OH-DPAT), induced pronounced blocking effects, of 48% and 33% respectively at -100 mV, whereas another 5-HT1 agonist, 5-carboxamidotryptamine (5-CT) was ineffective. At 100 microM, 5-methoxytryptamine (5-MeOT) induced a complete block of the NMDA responses recorded at -100 mV. The order of potency was: 5-MeOT congruent to DOI > 8-OH-DPAT > 5-HT > 5-CT. 4. Neither spiperone nor ketanserin (1 microM) prevented the blocking effect of 5-HT or DOI. 5. Prolonged preincubations with 5-HT did not block the response if NMDA was applied without 5-HT. When 5-HT agonists were applied both by preincubation and with NMDA, the degree of block increased during the NMDA application. 6. Lowering the NMDA concentration (from 100 to 20 microM) slightly decreased the blocking effect of 5-MeOT. 7. External Mg2+ ions (1 mM) also reduced the blocking effects of 5-HT and 5-MeOT. 8. The blocking effects described appear to be independent of classical 5-HT receptors. Their voltage-dependence suggests a mechanism of open channel block consistent with all the results obtained.

Modulation of recombinant alpha 6 beta 2 gamma 2 GABAA receptors by neuroactive steroids.

The sensitivity of the recombinant alpha 6 beta 2 gamma 2 GABAA receptor expressed in HEK 293 cells to neuroactive steroids was studied. The steroids 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-OH-DHP), pregnenolone sulfate and 3 alpha-OH-DHP sulfate have different modulatory effects on [3H]muscimol or [3H]Ro15-4513 binding to the alpha 6 beta 2 gamma 2 than to the alpha 1 beta 2 gamma 2 receptor. Binding of both radioactive ligands to the alpha 6 beta 2 gamma 2 receptor was maximally potentiated with each steroid used (10 nM) and decreased with further increases in steroid concentration. Using whole-cell recording, the GABA response of clusters of transfected HEK 293 cells was strongly potentiated by 3 or 10 nM 3 alpha-OH-DHP. In contrast, this response was reduced by 100 nM 3 alpha-OH-DHP. This latter effect appears to be related to the acceleration of the GABA response desensitization, observed in isolated cells. 3 alpha-OH-DHP (10 or 100 nM) was able to activate a response in the absence of GABA. It is proposed that the interaction of neuroactive steroids with the alpha 6 beta 2 gamma 2 receptor involves at least two distinct binding sites. One of them might be located close to the GABA binding site.

Dual modulation of the L-type calcium current of rat osteoblastic cells by parathyroid hormone: opposite effects of protein kinase C and cyclic nucleotides.

Using whole-cell voltage-clamp recording of rat osteoblastic cells, we show that PTH-(1-34), known to stimulate protein kinase C (PKC) and adenylate cyclase, has a dual effect on the L-type calcium current. It induces a long-lasting increase and a superimposed reversible decrease, which can be separated by repeating hormone applications. The stimulatory effect is the only effect induced by the (3-34) fragment, able to stimulate PKC but unable to stimulate adenylate cyclase. The L current is stimulated by an active phorbol ester and is reduced by permeable analogues of cyclic AMP. Thus, the effect of PTH-(1-34) can be explained by the opposite effects of PKC and cyclic AMP. Dibutyryl cyclic GMP reduces the L current even more potently than dibutyryl cyclic AMP. The above modulations are all voltage-insensitive. These results led us to reinvestigate the effects of some vitamin D3 metabolites known to stimulate PKC and/or guanylate cyclase, and previously reported to affect the voltage-sensitivity of the L current. We only detected voltage-insensitive effects.

Activation of hyperpolarization and atypical osmosensitivity of a Cl- current in rat osteoblastic cells.

During whole-cell recording of rat osteoblastic cells with high-Cl- internal solutions, 10 sec hyperpolarizing jumps from 0 mV induce a slow inward current relaxation, which is shown to be carried by hyperpolarization-activated Cl- channels. This relaxation increases and becomes faster with stronger hyperpolarizations. It is insensitive to Cs+ ions but is blocked in a voltage-dependent manner by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) 1 mM and is reduced by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) 0.1 mM. Cd2+ ions are potent blockers of this current, blocking completely above 300 microM. The amplitude of the Cl- current activated by a given hyperpolarization increases during the first 10-20 min of whole-cell recording. This evolution and the fact that some recently cloned Cl- channels have been reported to be activated both by hyperpolarization and by external hyposmolarity led us to investigate the effects of external osmolarity. Reducing the external osmolarity induces a large Cl- current. However, this hyposmolarity-induced Cl- current and the hyperpolarization-activated Cl- current are shown to be distinct; 1,9-dideoxy forskolin selectively blocks the hyposmolarity-activated current. We show that the hyperpolarization-activated Cl- current is osmosensitive, but in an unusual way: it is reduced by external hyposmolarity and is increased by external hyperosmolarity. Furthermore, these modulations are more pronounced for small hyperpolarizations. The osmosensitivity of the hyperpolarization-activated Cl- current suggests a mechanosensitivity (activation by positive external pressure) that is likely to be physiologically important to bone cells.

Concentration-dependent modulations of potassium and calcium currents of rat osteoblastic cells by arachidonic acid.

We show that the voltage-gated K+ and Ca2+ currents of rat osteoblastic cells are strongly modulated by arachidonic acid (AA), and that these modulations are very sensitive to the AA concentration. At 2 or 3 microM, AA reduces the amplitude and accelerates the inactivation of the K+ current activated by depolarization; at higher concentrations (> or = 5 microM), AA still blocks this K+ current, but also induces a very large noninactivating K+ current. At 2 or 3 microM, AA enhances the T-type Ca2+ current, close to its threshold of activation, whereas at 10 microM, it blocks that current. AA (1-10 microM) also blocks the dihydropyridine-sensitive L-type Ca2+ current. Thus, the effect of AA on Ca2+ entry through voltage-gated Ca2+ channels can change qualitatively with the AA concentration: at 2 or 3 microM, AA will favor Ca2+ entry through T channels, both by lowering the voltage-gated K+ conductance and by increasing the T current, whereas at 10 microM, AA will prevent Ca2+ entry through voltage-gated Ca2+ channels, both by inducing a K+ conductance and by blocking Ca2+ channels.

Potassium currents and effects of vitamin D-3 metabolites and cyclic GMP in rat osteoblastic cells.

A K+ current (IK1), activated by depolarization above -20 mV, showing voltage-dependent inactivation within a few seconds and reduced by 40% by 1 mM TEA, was observed in all cells. In a few cells, we also observed a progressive K(+)-current increase during cell dialysis. The developing current (IK2) was not sensitive to 1 mM TEA and did not inactivate. It was detectable over the whole voltage range and slowly increased during 10 s depolarizations. 1,25-(OH)2D3 and 24,25-(OH)2D3 did not affect IK1, but induced a small K(+)-current increase in some cells showing no IK2. This effect was not mimicked by cyclic GMP analogs which, on the contrary, induced a K(+)-current decrease. 24,25-(OH)2D3 (even at 10(-11)M, but not 1,25-(OH)2D3, strongly reduced IK2. The results further document the diversity of voltage-gated currents of osteoblastic cells, confirm the existence of immediate effects of vitamin D-3 metabolites, which are independent of classical 1,25-(OH)2D3 receptors.

Chloride current activated by cyclic AMP and parathyroid hormone in rat osteoblasts.

In primary cultures of rat osteoblasts, studied with the whole-cell configuration of the patch-clamp technique, 8-bromo-cyclic AMP (8BrcAMP) forskolin (FS) and 1-34 parathyroid hormone (PTH) were shown to activate a Cl conductance. This conductance shows a pronounced outward rectification, even with symmetrical Cl concentrations. It is blocked partially and reversibly by 4,4'-diisothiocyanatostilbene 2,2'-disulfonic acid (DIDS) or diphenylcarboxylate (DPC). The blockade induced by DIDS is time- and voltage-dependent. The Cl responses to FS and PTH develop slowly, after a delay of several seconds and are very slowly reversible. These responses were observed only in a fraction of the cells tested and their detection was favoured by cell dialysis. This Cl current should be taken into account for studying possible modulations of the voltage-gated Ca currents of osteoblasts. It is suggested that its physiological role may be related to the well-known morphological changes induced by PTH in osteoblasts. The cyclic AMP-sensitivity, the outward rectification and the sensitivity to dialysis of this Cl current are reminiscent of the properties of the cystic fibrosis-sensitive Cl channels of epithelial cells.

Voltage-gated sodium and calcium currents in rat osteoblasts.

1. The whole-cell voltage-clamp mode of the patch-clamp technique was used to investigate the presence of voltage-gated inward currents in osteoblasts from newborn rat calvaria. 2. In K+-free solutions, three kinds of inward currents could be activated by depolarization: a voltage-gated Na+ current and two different types of Ca2+ currents. 3. The Na+ current was activated by depolarization above -40 mV in all the cells. It was reduced by half by 10 nM-TTX (tetrodotoxin). 4. In an isotonic Ba2+ external solution containing TTX, and with a Cs-EGTA internal solution buffered at pCa 8, depolarizing jumps induced both a transient Ba2+ current and a sustained Ba2+ current. The relative proportions of these two currents varied greatly among cells. 5. The transient and sustained Ba2+ currents differ with respect to their time course and their voltage dependence. 6. The depolarization-activated inward currents were also observed under more physiological conditions, in the presence of only 2 mM-external Ca2+ and with a K+ internal solution buffered at pCa 7. 7. A few records obtained in current clamp showed that it is possible to induce action potentials in osteoblasts.

Chloride channels activated by hyperpolarization in Aplysia neurones.

The outside-out configuration of the patch-clamp technique was used in order to characterize the elementary events underlying the previously described hyperpolarization-activated Cl current of Aplysia neurones. A few membrane patches bathed with K-free internal and external solutions showed channels characterized by long duration (up to several seconds) bursts of activity which were clearly activated by hyperpolarization. I-V curves of elementary current obtained for three different values of the Cl equilibrium potential show that these channels are selective for Cl ions. During the bursts, the current fluctuates between three levels: the resting (closed) level, a level of maximum conductance (10-15 pS) and a half-conductance level. This behaviour is similar to that of the Cl channels extracted from Torpedo electroplaques.

Nonselective ionic channels in Aplysia neurones.

Single-channel recordings from outside-out patches of Aplysia neurones in K-free solutions revealed the presence in most membrane patches of ionic channels showing surprising selectivity properties, as deduced from reversal potential measurements. After complete substitution of external NaCl by mannitol (in the presence of internal CsCl), these channels are more permeable to Cl than to Cs, but are also slightly permeable to Cs: PCl/PCs = 4. Furthermore, in the presence of external NaCl, their ability to discriminate cations from anions seems lower than in external mannitol. Substitutions of external Cl by various anions showed that the channels are more permeable to NO3 than to Cl, and that they are appreciably permeable to isethionate, SO4 and methanesulfonate. Their elementary conductance is about 100 pS in 600 mM symmetrical Cl. However, different conductance states (usually 2 or 3) can often be detected in the same membrane patch. By using voltage ramps, we established the I-V curves corresponding to each of these states and found small but significant differences between the reversal potentials of each state.